lliiiii iiP- ■li^li itirwi^iwuy ^^■1 WM M iion By. No. 575^^4 C. V. I-osby ^0. St, Louis, i:o. IMMUNOLOGY IMMUNOLOGY BY NOBLE PIERCE SHERWOOD, Ph.D., M.D., F.A.C.P. Professor of Bacteriology, University of Kansas, and Pathologist to the Lawrence Memorial Hospital, Lawrence, Kansas SECOND EDITION ILLUSTRATED ST. LOUIS THE C. V. MOSBY COMPANY 1941 Copyright, 1935, 1941, by The C. V. Mosby Company (A II rights reserved) Printed in the United States of America Press of The C. V. Mosby Company St. Louis TO MY WIFE THIS BOOK IS MOST AFFECTIONATELY DEDICATED PREFACE TO SECOND EDITION 111 preparing this edition of the book the author has attempted to bring the subject matter as nearly up to date as possible and to rearrange the material so that it can be outlined by the student more readily. This has necessitated rearrangement of the chapters as well as of their contents in many instances. AVhere it has been deemed advisable, material has been deleted. Two new chapters, one on the reticulo-endothelial system and the other on serum reactions, have been added. The chapter on colloids has been placed in the Appendix where it can be referred to by students unfamiliar with the elementary chemistry of colloids. The chapters dealing with the serology of syphilis have been revised in accordance with the procedures approved by the "Committee on the Need of Ad- herence to Conventional Technique in the Performance of Reliable Serologic Tests for Syphilis" appointed by the Surgeon-General of the United States Public Health Service. The author is very appreciative of the helpful suggestions given him by Dr. K. Landsteiner, Dr. W. J. Nungester and others work- ing in the field of immunology. The many constructive criticisms given by reviewers of the first edition have been of considerable help in this revision. As in the preparation of the first edition, a number of colleagues and members of my family have rendered material assistance in Ihe preparation of the manuscript of this revision. Among these, Dr. C. M. Downs, Mr. Harold Nelson, and my Avife have been extremely helpful. Noble Pierce Sherwood. Lawrence, Kansa?. >> /or' ^o\ TL PREFACE TO FIRST EDITION This book is written for medical students and for others who have had training in pathogenic bacteriology, inorganic and or- ganic chemistry and who are interested in the underlying prin- ciples involved in infection, resistance, and diagnostic laboratory tests. It has been used in mimeograph form at the universities of Kansas and Montana, respectively, for the past few years. From this experience we have gained the impression that medical students and college students majoring in bacteriology are able to read and discuss intelligently the subject matter as it is here presented. It is assumed that the text will be supplemented by laboratory experi- ments since it is difficult and perhaps impossible to gain a working knowledge of the subject without observing many of the phe- nomena discussed. The author has attempted to lead the student to correlate some of the teachings of physiology, pharmacology, organic, biological and physical chemistry as well as anatomy, pathology and general biology and apply these teachings to the elucidation of the mys- teries surrounding infection, resistance, and diagnostic procedures. He has attempted to show that clinical, experimental and pre- ventive medicine contribute a great deal to our knowledge of the subject. In fact it is hoped that the student will acquire enough of the underlying philosophy of immunology to enable him to attack, with reasonable success, the practical problems he will encounter when he cjomes into contact with patients and the com- munity at large in his clinical years in medicine. While empirical knowledge may enable one to perform laboratory or other immu- nological techniques, clinical experience and an understanding of the underlying mechanisms and extraneous factors involved is (juitc necessary to a correct interpretation of the results obtained. In view of the fact that the student needs to acquire an ade- quate vocabulary of technical terms used in the text, definitions are given quite freely. A chapter on colloids is introduced just before the chapters that deal with the mechanisms involved in agglutination, opsonification, and complement fixation. This is done for the benefit of those students who are unfamiliar with colloid chemistry. 8 PREFACE y Throughout the book standard techniques are presented, ana- lyzed, and discussed. It is lioped that reference to the practical application of immunological procedures will i)romote a greater in- terest in the subject on the part of the student. The chapters on hypersensitiveness are placed last because an understanding of the material contained in the chapters preceding them is of material value in understanding the present theories pertaining to the complex problems inherent in allergy. A great deal of emphasis is placed upon the subject of specificity since it plays such an important part in diagnostic tests and in passive and active immunization. Since the material in many of the chap- (ei's is rather condensed, the lists of references at the ends of most chapters have been somewhat extended to accommodate the stu- dent who desires to read more extensively about some particular subject. For the convenience of the reader a synopsis of the three chapters on specificity is presented in a separate chapter (XV). A number of colleagues, former students and friends have ren- dered material assistance in the preparation of the manuscript for this book. To all of them the author wishes to express his ap- preciation. There is space for mentioning only a few. Among these, Mr. Harold Clark deserves credit for outlining the chapter on flocculation tests in syphilis, in addition to rendering help in many other ways. Doctors C. M. Downs, H. R. Wahl, and Joel Wahlin have criticized parts of the manuscript. Dr. Ray Brewster and Mr. ]\Iorgan Rarick have rendered a great deal of assistance in the preparation of the chapter on modified antigens. Four other individuals whose services have made possible the completion of this volume are IMiss Rosella Blood, the artist, Miss Letha Lemon and Mr. Harold Nelson, secretaries, and Mr. Louis Forman, who checked the bibliography. The author has drawn quite freely from information contained in various publications. These are listed in the text, and an attempt is made to give due credit to l)oth author and publisher. Noble Pierce Sherwood. Lawrence, Kansas. / i I a o A D .,...„.„.,« CONTENTS CHAPTEE I PAGE InFECTIOX and IXFKCTIOUS AGENTS ___________ 29 Infection; Classification of Parasites; 8ymbiosis, 29; Infection Without Pathological Change ; Infection With Patliological Change ; Types of Pathological Changes; Localized and Generalized Infec- tion; Bacteremia, Septicemia, and Pyemia, 30; Acute and Chronic Infections; Focal Infections; Beneficent Infections; Specific Mi- crobic Association, 31; Metastasis; Secondary Infections; Infec- tious and Contagious Diseases; Toxemic Diseases; Bacterial Toxins, 32; Botulism; Tetanus, 33; Gaseous Gangrene; Diphtheria; Endo- toxins; Phytotoxins and Zootoxins, 34; Antitoxins; Ptomaines, 35; Pathogenicity; Virulence; Definition of Virulence; Measure- ment of Virulence, 36; Factors Altering Virulence; Relation of Capsule Formation to Virulence; Hypothesis of Welch; Correla- tion of Virulence and Colony Type, 37; Labile Antigens and Virulence, 38; Importance of Host Factors in Determining Viru- lence; Aggressins; Nature and Characteristics of Viruses, 39; Chemical Composition of Bacteria, 40; Carbohydrate; Protein; Effect of Environment on Bacteria; Theory of Constancy of Char- acteristics, 41; Evidence of Heterogeneity Within a Culture; Fluctuating Variability; Early Theories of Bacterial Character- istics, 42; Relation of Environment to Size, 43. CHAPTER II Host-Parasite Relationship _____________ 47 Introduction; Portal of Entry, 47; Endemics, Epidemics, Pan- demics; Incubation Period; Environmental, Not Involving Immu- nity; Protection Due to Lack of Contact, 48; Removal of Primary Host; Sanitary Measures, 49; Racial Freedom From Disease May Be Due to Customs Rather Than Immunity; Effect of Customs; Environmental Conditions: Absence or Presence of Factors Af- fecting Immunity Mechanisms; Climate Factors; Resistance and Susceptibility Factors, 50. CHAPTER III Inflammation and Leucocyte Response _________ 54 Introduction; Acute Inflammation; Lobar Pneumonia, 54; Furun- cle, 55; Chronic Inflammation — Formation of ;i Tubercle, 57; Tubercle Phosphatid and Cell Stimulation; Varieties of the Tu- bercle Bacillus; Development of Hypersensitiveness; Tissue Re- sponse to Viruses, 59; Leucocyte Count, 60; Arneth and Schilling Counts, 61; Neutrophilic Myelocytes; Juvenile Neutrophiles, 62; "Stab" or Band or Rod Nuclear Neutrophilic Cells, 63; Leuco- penia, 65; Leucocytosis and Increased Capillary Permeability, 66. 11 12 CONTENTS CHAPTEE IV PAGE Anatomical and Physiological Factors in Infection and Kesistance OF THE Individual _________-_-__- 70 Mechanisms of Infections, 72; Establishment Within the Tissues; Eoutes of Dissemination, 74; Lymphogenous Extension, 75; Hem- atogenous Extension, 76; Direct Extension; Factors Involved in Pulmonary Infection; Lobar Pneumonisi,, 77; Factors Involved in Intestinal Infection, 78; Ulceration of Intestine; Ulcer, Pan- creatitis, Cholecystitis, 79; Appendicitis; Importance of Blood Supply at Lower Orifices, 80; Factors in Genitourinary Tract Infections; Cystitis, 81; Pyelonephritis; Stone in the Ureter or Kinks; Acute Nephritis; Importance of Elimination; Mechanical Factors of Safety; Physiological Mechanisms, 82; Prenatal vs. Postnatal Immunity; Importance of Considering the Body as a Whole; Underlying Principle of Therapy, 84. CHAPTER V The Eeticulo-Endothelial System ___________ 87 Phagocytosis, 95 ; Role of Complement, 96. CHAPTER VI Natural and Acquired Immunity ____-__-_-- 102 Natural Immunity-^Species Differences in Immunity; Racial Dif- ferences in Immunity, 102; Endocrines and Immunity, 103; Age and Resistance; Vitamins and Food Factors in Resistance, 105; Nutrition and Resistance, 106; Neuf eld's Theory of Phases of Heightened and Lessened Resistance; Hereditary Factors in Nat- ural Immunity, 107; Antibodies and Natural Resistance, 109; Acquired Immunity; Immunization Against Viruses and Ricket- tsiae, 110; Immunization With Attenuated Bacteria, 112; Immu- nization With Killed Bacteria, 113; Immunization With Bacterial Polysaccharides, 115; Whooping Cough Immunization; Immuniza- tion With Detoxified Toxins, 116; Immunization Programs, 117; Passive Immunity, 121. CHAPTER VII Immunity Mechanisms in Experimental Infections ______ 128 Experimental Streptococcus Infections in Rabbits, 128; Impor- tance of Antibodies; Passive Immunization With Pleural Exu- - date; Experimental Staphj-lococcus Infections in Guinea Pigs, 129; Local Fixation; Summary of Cannon's Work on Tissue Im- munity to Staphylococci, 130; Immunity to Pneumococcus Infec- tion, 131; Experimental Infection of the Chorio-Allantoic Mem- brane of Chick Embryos, 133; Role of Clasmatocytes in Other Infections, 134; Tissue Resistance to Cysticercus Pisiformis; Re- moval of Bacteria From the Blood Stream, 135; Defensive Mecha- nisms in Peritonitis, 138; Defense Against Viruses; Tolerance, 139. CCntEnts 1^ CHAPTEE VIII PAGE Natural and Immune Antibodies ____________ 143 The Humoral Theory of Immunity, 143; Alpha Lysins; Immune Lysins and Pfeiffer's Phenomena; Pfeiffer's Immunity Unit, 144; Bordet's Explanation of Lytic Mechanism, 145; Bacterial Ambo- ceptor, Immune Body or Sensitizer; Sensitized Cells; Comple- ment; Further Studies on Normal Bacteriolytic Substances; Alpha Lysins, 146; )3 Lysins, Leukins and Lysozyme, 147; Natural and Immune Bacterial Agglutinins; Hemolysins, Hemagglutinins and Other Antibodies, 148; Isohemolysins in Paroxysmal Hemoglo- binuria, 149; Reagins, 150; Lipoidophilie Reagins; Precipitins; Anaphylactic Sensitizers; Antitoxins, 151; Opsonins and Bacteri- otropins; Ablastin; Antiaggressins; Conglutinins, 152; Heter- ophile Antibodies and Heterophile Antigens, 153. CHAPTER IX Complement _________-___----- 157 Nature of Complement, 157; Preservation of Complement, 158; Effect of Heat on Complement; Reversibility of the Reaction; Inactivation by Shaking; Anticomplementary Effects of Inor- ganic and Organic Compounds, 159; Origin of Complement; De- sirable Qualities of a Complement; Complement From Various Animals, 160; Specific Fixation of Complement; Specific Comple- ment Fixation by Precipitates; Time of Occurrence of Fixation, 161; Nonspecific Adsorption; Chemical and Physical Factors in Cell Sensitization, 162; Thiele and Embleton's Observations; Bordet's and Ehrlich's Views on the Mechanism of Sensitization; Visible Phenomena of Cell Lysis, 163; Fixation of Complement by Products of Lysis; Hill and Parker's Physicochemical Inter- pretation; Action of Complement Depends on Concentration, 164; Effect of Temperature on Complement Action; Discussion of Eagle 's Work, 165. CHAPTER X Isohemagglutinins — Blood Groups _______---_ 168 Isohemagglutinins — Discovery, 168; Classification in Use; Classifi- cation Based on Agglutinogen Content ; Landsteiner 's Three Groups, 169; Mechanism Postulated by Landsteiner; Moss's Classification in General Use, 170; Suggested Method for Learning the Classifica- tion, 171; Time of Appearance of Agglutinogen and Agglutinins; Inheritance of Blood Group Factors, 172; Racial Distribution of Groups; Racial Types; Frequency in Different Populations; Higli Incidence of O Group in the American Indian, 173; Distribution of Agglutinogen in Lower Animals, 174; Agglutinogen in Monkeys and Higher Apes, 175; Comparison of Agglutinogen in Human and Monkey Blood; Human A and B Factors in Anthropoid Apes; Medico-Legal Application of Blood Groups, 176; Determination 14 CONTEXTS PAGE of Nonpaternity, 177; Bernstein's Triple Allelomorph Theory, 178; Subgroups; Importance of Quantitative Difference, 179; Presence of Subgroups Does Not Interfere With Eoutine Typing; Irregular Agglutination; Importance of Temperature; M, N, and P Factors of Human Blood, 181; Extra Agglutinin 1; Irregular Isoagglutination at Temperatures Below 37° C. ; Autoagglutinins, 183; Cold Agglutinins; Pseudoagglutination ; Inhibition of Eoule- aux Formation, 184; Nature and Distribution of Red Cell Haptens, 185. CHAPTEE XI Nature, Formation, Action and Measurement of Antibodies _ _ _ 193 Introduction; Antibodies, 193; Antigens, 194; Nature and Origin of Antibodies, 195; Origin of Antibodies, 197; Unitarian Theory of Antibodies, 198; Mechanism of Antigen- Antibodj^ Union, 199; Method of Measuring Antibodies; Antitoxins; Unit of Tetanus Antitoxin, 200; Agglutinins; Precipitins, 201; Bacteriotropins; Antiaggressins; Hemolysins, 202; Method of Titrating Hemolysin, 203; Complement Titration; Unit of Complement, 204. CHAPTEE XII Mechanism of Antigen-Antibody Reactions. Cei^lular A(ioi.utination 208 Period of Discovery and Early Investigations; Grubor's Theory of Agglutination; Bordet's Early Work; Discovery of Precipitins; Dineur's Hypothesis of Agglutination, 208; Bordet's Objections to Current Theories; Bordet's Two-Phase Theory, 209; Bordet's Defi- nition; Bordet's Experiments on Effect of Electrolytes on Cholera Vibrios, 210; Ehrlich's View; Subsequent Lines of Eesearch; Studies on Effect of Dilution on Adsorption, 211; Effect of Frac- tional Addition of Antigen; Bordet's Experiment Explaining Danysz Phenomenon; Heidelberger and Kendall's Theory of Re- action Mechanism, 212; Early Cataphoresis Experiment; Powis' Work and the Critical Potential; Similarity to Denatured Pro- teins; Buchanan's Suggestion, 213; Loeb; Northrop and DeKruif; Shibley's Summary of Northrop and DeKruif 's Work, 214; Effect of Sensitization on Charge, 215; Techniques; Measuring Cohesive Force, 216; Extent of Surface Coating; Interfacial Tension Tech- nique, 217; Behavior of Cells; Change in Surface Molecular Orientation; Antigenic Components and Antibodies; Nonprotein Carriers of Haptens, 218. CHAPTEE XIII Precipitins __________________ 222 Introduction, 222; Early Investigations; Group Reactions; Value in Tracing Biological Eelationship, 223; Nuttall's Conclusions, CONTEXTS 113 PAGE Ol) ■i; Extent of Early Use of Prccipitiu Test; Preparatiou aud Prerequisites of a Satisfactory Immune Serum, 225; Inoculation of Animals; Inoculation of Animals According to Kolmer; Inocu- lation of Animals According to Dean; Methods Used in This Laboratory, 226; When to Bleed Animals; Obtaining Immune Serum; Titration After Uhlenhuth; Technique of Nuttall, 227; Quantitative Technique of Nuttall; Time of Incubation; The King Test, 228; Eange of Specificity; Practical Use of the Test; Adsorption and Agglutination Technique; Optimal Proportion of Immune Serum and Antigen, 229; Proportion of Immune Serum and Antigen Used by Nuttall; Importance of Optimal Proportions of Antigen and Antibody, 230; Coarse Test of Dean and Webb; Optimal Proportions Fine Test, 231; Tube Five Contains Optimal Proportions; Unit Suggested by Dean and Webb; Precipitins Used to Estimate Haptens and Proteins; Eeason for Coexistence of Antigen and Antibody in Blood, 232; Nature of Precipitate; Effect of pH on the Eeaction; Effect of Salts on the Keaction, 233; Suppression Phenomenon of Landsteiner; Haptens, 234; Haptens Yield Precipitates With Homologous Immune Serum; Eeasons for Diluting Immune Serum in Agglutinin and Antigen in Older Precipitation Tests, 235. CHAPTER XIV Toxins and Antitoxins _______-------- 241 Toxins Defined; True Toxins, 241; Etiological Agent of Diph- theria and Tetanus Discovered; Invasive Power of C. Diphtheriae; Effect of Toxin on Lower Animals, 242; Symptoms in Susceptible Animals; Pathology in Lower Animals, 243; Effect on Dogs, Sheep, and Birds; Diphtheria in Man, 244; Symptoms; Pathology in Man; Causes of Death in Diphtheria; Source and Nature of Diphtheria Toxin, 245; Effect of Heat, Drouth, and Chemicals on Toxins, 246; Toxoid or Anatoxin; Method of Measuring Toxin and Antitoxin, 247; L, Dose of Toxin, 249; pH of Toxin; Reli- ability of Ramon Test, 250; Theories of Toxin- Antitoxin Mech- anism; Ehrlich, 251; Arrhenius and Madsen; Bordet's Theory of Adsorption; Zone Phenomenon, 252; Other Examples of the Zone Phenomenon; Danysz Effect, 253; M.R.D., 254; Schick Test for Susceptibility; Variation in Susceptibles in Urban and Rural Pop- ulations, 255; Susceptibility in Young Adults; Susceptibility De- termined by First Dose of T.A.T. ; Active and Passive Immunity, 256; Passive Immunity; Dosage of Antitoxin; Method of Admin- istering Antitoxin, 257; Prophylactic Dose of Antitoxin; Active Immunity; Active Immunization of Horses With T.A.T., 258; Sus- ceptibility Tests in New York; Park's Pioneer Work on Active Immunization; Preparation of T.A.T., 259; Advantages of New 16 CONTENTS PAGE Toxin- Antitoxin Mixture; Kesults Determined by Schick Test, 260; Detoxified Toxin, 261; Toxoid Specifications of U. S. Public Health Service, 262; Inunction as Method of Immunization; Alum Toxoids, 263 ; Active-Passive Immunity ; Eecommendations of New York City Department of Health, 264. CHAPTEE XV Toxins and Antitoxins (Continued). Convalescent and Immune Sera 276 Introduction; Scarlet Fever, 276; Prophylactic Immunization; The Dick Test and Toxin Valency, 278; Measurement of Antitoxin and Toxin ; Scliultz and Charlton Blanching Test ; Potency of Con- valescent Serum, 279; Park's Eecommendations; Early Malignant Cases; Late Septic Cases, 280; Immuno-Transfusion; Erysipelas; Types of Lesions in Erysipelas, 281; Streptococci in Erysipelas; Puerperal Sepsis, 282; Streptococcus Septicemia; Immuno-Trans- fusion in Septicemia, 283; Blood Banks; Pneumococcus Pneumo- nia; Types in Lobar and Bronchopneumonia, 284; History of Serum Treatment of Pneumonia; Discovery of Types; Discovery of Soluble-Specific Substance ; Concentration of Antibody, 285 ; Eesult of Serum Treatment; Enzyme Treatment of Type III Pneu- mococcus Infection, 286 ; Chemotherapy in Pneumococcal and Other Bacterial Infections, 287; Meningococcus Meningitis; Cellular Eo- sponse Due to Serum; Use of Serum in Virus Diseases, 289; Measles; Preparation of Convalescent Serum; Partial Eather Than Complete Protection Eecommended, 290; Importance of Epidemi- ological Factors; Use of Pooled Placental Blood; Poliomyelitis, 291; Mumps; Vaccinia and Variola, 292; Tetanus; Eeason for In- effectiveness of Tetanus Antitoxin in Treatment, 293 ; Gas Gan- grene, 294 ; Infection, Factors Affecting the Germination of Spores ; Importance of Eemoval of Devitalized Tissue, 295 ; Summary of Important Factors Eelative to Gas Gangrene; Incubation Periods for Various Toxins; Tetanus in Wounds; Tissues Affected, 296; Invasive Power; Difference in Pathological Picture; Effect of Toxins on Heart and Adrenals; Therapeutic Value of Antitoxins, 297; Botulism; Tularemia, 298. CHAPTEE XVI Serum Eeactions _________________ 306 Immediate Eeactions, 306; Delayed Eeactions; Serum Sickness in Man, 309; Serum Sickness in the Lower Animals; Mechanism of Serum Sickness, 310. CHAPTEE XVII Biological and Antigenic Specificity __________ 31 7 Immunological Specificity; Biological Specificity; Specificity and Incompatibility of Species; Specificity and Fertilization, 317; CONTENTS 17 T'A(iK Union of Egg and tSperm; 8pecilicity and Genetics; Production of Specific Hormones, 318; Immunological Specificity; Durham's Explanation — Multiple Antigens, 319; Precipitins and Specificity; Precipitins and Species Kelationsliip ; Serological Types Within a Species; Bacterial Types Within a Species, 320; Serum Proteins vs. Cellular Antigens; Properties of an Antigen; Animal Proteins, 321; Red Cell Proteins; Tissue Proteins; Organ Specificity; Lens and Testicular Proteins Show Nonspecies Specificity, 322 ; Keratin, Mucin, etc.. Lack Species Specificity; Thyroglobulin ; Milk Pro- teins; Antigenic Components of the Egg, 323; Biological and Im- munological Relationships Among Ameba; Vegetable Proteins; Algae, 324; Antigens in Fungi; Bacterial Antigens; Structure and Properties of Proteins, 325; Importance of Physical and Chemical Changes in Antigen, 326; Enzyme Action Destroys Antigenic Prop- erty; Relationship of Digestibility and Antigenic Property; Pos- sible Number of Compounds Formed by Amino Acids, 327; Impor- tance of Aromatic Amino Acids in Protein Antigens; Acid and Basic Groups of Amino Acids; Aliphatic and Aromatic Amino Acids, 328; Zozaya's Nonprotein Antigen, 329. CHAPTER XVIII Modified and Conjugated Antigens ___________ 332 Early Work on Modified Antigens; Early Work on Conjugated Antigens, 332; Altered or Modified Antigens; Iodized Protein; lodotryptophane, 333; Acetylated Proteins, 334; Conjugated Anti- gens of Landsteiner-Diazo Compounds; Coupling With a Protein; Arsonic Haptens, 335; Arsonic Protein Compounds; Aliphatic Side Chains Not Diazotizable, 336; Protein Structure, 337; Ni- troso Compounds; Azo Dyes; Cysteine and Cystine, 338; Stereo- isomers or Spatial Relationships, 339; Position Isomers, 340; Linkage With Salt-Forming Groups; Removal of Acid Properties of an Amino Acid, 341; Removal of Basic Properties of an Amino Acid; Other Ways of Salt Formation, 342; Effect of Esterification and Methylation of Protein; Cross Reactions; Specific Reactions, . 343 ; Formalized Rabbit Serum as an Antigen ; Recent Work on Iodized Antigens, 344; Minimum Amount of Iodine to Change Antigenic Property; Specificity for Iodized Proteins; Group Reac- tions Due to Different Haptens in the Same Molecule, 345; Egg Albumen Contains Tyrosine and Histidine. Gelatin Contains Histi- dine; Use of Diazo Dyes for Suppression of Specific Reaction; Tyrosinediazoarsanilic Groups in Protein Molecule, 346; Tyrosine- Like Diazoarsanilic Acid Dye; Histidindiazoarsanilic Acid Groups in Gelatin Molecule; Histidine-Like Diazoarsanilic Acid Dye, 347; Antibodies to Strychnine, 348. 18 CONTEXTS CHAPTEE XIX PAGE Bacterial Antigens axu Specificity ___________ 350 Bacterial Antigens; Complexity; Living Attenuated Antigens; Killed Suspensions as Antigens; Early Eesearch on Antigens; Species Specificity vs. Immunological Specificity, 350; Limitation of Serological Methods; Species Showing Antigenic Homogeneity; Sjtecies Showing a Few Serological Types; Species Showing Anti- genic Heterogeneity, 351; Lack of Standard Procedure, .".52; P^rrors Due to Presence of Extraneous Material in Antigens; Early Work on Antigenic Fractions; Subsequent Investigations; Prop- erties of Tuberculin, 353; Specific Substances in Bacteria, 354; Antigenic Comparisons of Acid-Fast Bacteria; Lipoids; Rehitive Importance of Lipoids, Proteins and Carbohydrates, 355 ; Tuber- eulo-Phosphatids and Fatty Acids; Immuno-Chemical Studies of tlie Pneumococcus ; Properties of Type Specific Polysaccharides, 356; Difference Between Immune Serum From Horses and Rabbits; Possible Explanation of Inhibition Phenomenon, 35S; The "C" Species-Specific But Not Type-Specific Substance of Tillett and Francis; Carbohydrate Fractions Adsorbed on Carbon Particles; Synthetic Carbohydrate Haptens, 359; Friedlander 's Bacillus, 361; Escherichia coli ; E. lypliosa, 3(>.'! ; Shigella dysenteriae; V. cholerae; H. influenzae, 364; H. pertussis; Brucella abortus; Hemolytic Streptococci, 365 ; Other Bacterial Specific Substances, 366. CHAPTER XX Recapitui.ation of Chapters on Specificity ________ 374 Examples of Specificity in Nature ; Antigens ; Properties of Protein Antigens, 374; Species-Specificity and Type-Specificity; Differences Within a Species, 375; Hapten May Dominate a Serological Reac- tion; Suppression Phenomenon of I^andsteiner ; Arsanilic Acid Cou- pled to Histidine and T^TOsine ])y Hooker and Boyd, 376; Haptens Itosponsible for Cross-Reactions; Pneumococfus Type-Specific Poly- saccharide Serum Globulin Antigen; Spatial Relationships and Specificity Stereoisomers as Haptens, 377; Landsteiner 's Method of Producing New Conjugated Antigens ; Suggested Explanation of Drug Allergy; Mosaic Structure of Antigens, 378. CHAPTER XXI The Importance of Antibodies in Diagnosis ________379 Discovery of Role of Agglutinins in Diagnosis — the Widal Test; Scope of Subsequent Investigations; Antigens Used in the Widal, 379; Two Techniques Employed, 380; Incubation, Observation and Interpretation of Results; Microscopic Method, 381; Controls; Variation in Time of Appearance of Agglutinins, 382; Agglutinins in Carriers; Normal Agglutinins and Diagnostic Titers; Flagellar, Somatic and Labile Agglutinins, 383 ; Variations in Agglutination CONTENTS 19 PAGE Titer Following Vaccinatiou, 'dSi; Effect of Allergy on Agglutinin Titer; Experimental Eesults on Agglutinin Variation; Time of Ap- pearance of Maximum Titer, 385; The Use of the Widal in Vac- cinated Individuals ; Importance of ' ' Fine Granular ' ' and ' ! Loose Flocculation " ; Time and Temperature of Incubation, 386; Undu- lant Fever, 387; Tularemia; Agglutinin Eesponse to Infection in Partially Immune Animals, 388; The Effect of Specific Infection on the Titer of Agglutinin for Other Organisms; Felix-Weil Phe- nomenon, 389; Tularemia — Specific and Cross Reactions With Br. Abortus Antigens, 390 ; Antigenic Eelationships Between S. Galli- narum and E. Typhosa, 391; The Agglutination Reaction in Other Specific Infectious and Toxemic Diseases — S. Dysenteriae ; Gland- ers; Asiatic Cholera and a Few Other Diseases; Test for Heter- ophile Antibodies in Acute Infectious Mononucleosis; Identification by Agglutination or Precipitation, 392; Bacterial Types; C. Diph- theriae, 393; CI. Tetani; CI. Botulinum; E. Typhosa; Pneumo- coccus Typing, 394; Technique Neuf eld's Quellung Reaction; Sabin's Description of Neufeld's Method, 395; Typing of Hemo- Ij'tic Streptococci, .'{9(5; Skin Tests for Antibodies; Tlie Francis Skin Test, 397. CHAPTER XXII The Basis of Bactkiual Compj.ement Fixation Technique _ _ _ _ 401 Introduction; The Original Bordet-Gengou Technique, 401; Appli- cation of Complement Fixation, 402; Reagents and Factors In- volved ; Development of Modern Technique, 403 ; No Hemolysis in Tube 5, 409; Complete Hemolysis in Tube 5; Object of Controls; Results WTien Fixation Is Due to Antibodies, 412; Quantitative and Qualitative Tests; Reading the Results; Reporting Results; Corre- lation With Clinical Findings; Discussion, 413; Variation in Titer of Hemolysin; Variation in Agglutinin and Precipitin Titer, 414; Antibody Variation During Infection; Summary of Recommenda- tions, 415. CHAPTER XXIII Complement Fixation in Syphilis ___________ 419 History of Syphilis; Etiology, Incubation and Primary Lesion; Laboratory Procedure Indicated During Primary Stage, 419; Blood Tests of Little Value During Primary Stage, 420; Secondary Stage — Time of Appearance of Symptoms, Laboratory Findings and Duration; Complement Fixation and Precipitin Tests in Untreated Cases and Treated Cases ; Syphilis May Clinically Resemble Almost Any Disease, 421; Syphilis Characterized by Periods of Latency Followed by Periods of Activity; Serology of Umbilical Bloods; Tertiary Sj-philis, 422; Tabes, Paresis, and Cerebrospinal Lues I^ate 20 CONTENTS PAGE Manifestations of Tertiary Stage of Syphilis; Wassermann and Bruck Modification of the Bordet-Gengou Technique for Bacterial Complement Fixation, 423; Total Volume; Bacterial Antigens; Complement; Immune or Patient's Serum; Ked Cell Suspension; Hemolysin; The Application of Complement Fixation to the Diag- nosis of Syphilis, 424; Controls Employed in the Original Wasser- mann Technique, 425; Importance of Citron's Work; First Stage of the Development of the Wassermann Eeaction; Second Stage Be- gan With the Use of Alcoholic Extracts of Guinea Pig Heart, 426; Third Stage Introduced by Cholesterinized Antigen; Fourth Stage: Introduction of Improved Antigen, 427; Fifth Stage: Standard- ization of Wassermann Reaction by Kolmer; Kolmer Test, 428; Titration of Antigen, 429 ; Kilduff e 's Ten Basic Principles in Sero- logical Diagnosis, 431 ; The Provocative Wassermann Reaction ; Wassermann-Fast Cases, 432 ; Effect of Malarial and Diathermy Treatment; Immunity to Syphilis, 4.'^? ; Nonspecific Wassermann Reactions, 434 ; Mechanism of the Wassermann Reaction, 435. CHAPTER XXIV Precipitin Tests in Syphilis _____________ 444 Introduction; Historical Development; Introduction of Cholesterol, 444; Simplicity of Flocculation Method; Status of Flocculation Tests; Principle of Flocculation Tests, 445; Role of Ingredients, 446; Antigens; Synergistic Substances, 447; Role of Cholesterol; Water, 448; Electrolytes; Alcohol, 449; Ratio of Components; Concentration of Lipoids; Cholesterol-Lipoid Ratio, 450; Alcohol- Water Ratio; Miscellaneous Factors; Finished Antigen, 451; Choice of Test; Kahn Precipitation Reaction; Salt Titration, 452; Stand- ardization of Antigen; Inactivation ; Shaking, 453; Time of Read- ing Results; Controls; Choice of Kahn Test, 455; Presumptive Test; Quantitative Test; Spinal Fluid Procedure; Application of Quantitative Tests, 456; Kahn's Verification Test; Description of Kline Test, 457; Sensitivity; Agreement With Other Tests; Tech- nique of Tests; Preparation of Antigen; Reading Test, 458; Elimi- nation Test; Use of Elimination Test; Sources of Error, 460; Prin- ciple of the Meinicke Reaction; Specificity; Antigen; Mixing of Antigen, 461; Serum; Technique of the Test; Interpretation of Results, 462; Hinton Test; Glycerinated Indicator Solution; Tech- nique of Test, 463; Citochol Reaction; Antigen; Serum, 464; Test; Bruck 's Nitric Acid Reaction; Formol Reaction; Sachs-Georgi Re- action; Vernes; Eagle Flocculation Test, 465; Rosenthal Test; Mazzini Microflocculation Test; Importance of Using Both Com- plement Fixation and Precipitation Tests; Hypersensitive Tests of Doubtful Value, 466. CONTENTS 21 CHAPTEK XXV page Hypersensitivkness ________________'±70 Discovery of Anaphylaxis; Early Studies of Anaphylaxis, 470; Eichet and Portier's Work; Arthus Phenomenon; The Theobald Smith Phenomenon; Rosenau's and Anderson's Studies, 471; Dis- covery of Passive Sensitization; Sensitizing Substance, 472; Sensi- tizing Dose of Antigen; Incubation Period in Active Sensitization, 473; Duration of the Hypersensitive State; Shocking Dose of Anti- gen, 474; Passive Sensitization, 475; Desensitization; The Refrac- tory State, 476; Anaphylaxis in the Guinea Pig — Acute Shock, 477; Protracted or Subacute Shock in the Guinea Pig; The Dale Phe- nomenon, 478; Anaphylactic Shock in the Rabbit, 479; Protracted Anaphylactic Shock in the Rabbit; Local Anaphylaxis or Arthus Phenomenon; Anaphylaxis in the Dog, 480; Anaphylaxis in the Cat, 481 ; Anaphylaxis in Eats, 482 ; Anaphylaxis in Frogs ; An- aphylaxis in Turtles, 483; Anaphylaxis in Chickens; Anaphylaxis in Monkeys, 484; Anaphylaxis in Man; Criteria of Anaphylaxis, 485; The Sensitizing Antibody, 486; Specificity of the Reaction; The Changes in Metabolism During Shock, 487; The Nature of Anaphylaxis, 488; Site of Reaction, 489; Tissues Involved in An- aphylaxis, 490 ; Mechanisms of the Reaction, 492 ; Further Discus- sion of the Pliysical Tlicory, 494 ; Further Discussion of the His- tamine Theory, 495. CHAPTER XXVI Hypeksensitiveness Due to Infection __________ 504 Allergy in Tuberculosis; Tuberculosis and Tuberculin Hyperseusi- tiveness; Tuberculin OT, BE, and TR; Objections to OT ; Long's Synthetic Medium, 504 ; Synthetic Medium of the Bureau of Animal Industry; The Active Substance, Its Recovery and Properties, 505; TPT of Seibert and Munday, 506; Seibert's SOT and SOTT or PPD, 507; MA-100 of Funk and Huntoon; Relationship of Lrolecular Weight to Sensitizing Property, 508; Chemical Nature of PPD, 509; Classification of Standardization Methods; Koch's Method; Method Used in the Institute of Experimental Therapy, Frankfurt, 510; Method of U. S. Bureau of Animal Industry; Skin Test Method of Lewis and Aronsor. ; Standardization of Tuberculin by the Precipitation Method of Dreyer and Vollum, 511; Complement Fixation Method of Watson and Heath; Ob- jections to Lethal Tests, 512; Advantages and Disadvantages of the Various Tests, 513; Standardization by Long's Sper- matocyte Reaction; Spermatocyte Tuberculin Unit, 514; Depend- able Methods of Tuberculin Production and Standardization; Diag- nostic Tuberculin Tests; The von Pirquet Test, 515; Moro 's Percu- taneous Test; Wolff -Eisner Test; Patch Test; Mantoux Test, 516; Funk and Huntoon 's Results With OT and MA-100; Dosage Em- ployed; Reading the Reactions, 517; Incidence of Reactions in 22 CONTENTS PAGE Known and Suspected Cases of Tuberculosis; Incidence of Reac- tions in Clinically Nontuberculous Cases; Noncorrelative Reactions; Cases of Tuberculosis That Did Not React; Mariette and Fenger's Results With MA-100 and OT, 518; Slater and Jordan's Studies of the von Pirquet and Mantoux Tests; Survey to Determine Pri- mary Tuberculosis Infection Attack Rate, 520 ; Significance of Skin Reaction in Children; Tuberculin Surveys With PPD, 521; Effect of Age Upon Diluted Tul)erculin; System of Grading Mantoux Tests, 522 ; Incidence of Tuberculosis Determined at Autopsy ; Rob- ertson 's Anatomic Studies, 523 ; Tissue Response to Various Frac- tions of the Tubercle Bacillus; The Necessary Factors for the Development of Tuberculin Allergy, 524; Possible Explanation of Apparent Dissimilarities Between Anaphylaxis and Tuberculin Al- lergy, 526; Similarity Between Immune Response and Tuberculin Allergy, 528; Summary of Evidence Indicating an Antigen-Anti- body Mechanism, 529 ; Questions Raised by Antigen-Antibody Con- cepts, 530; Antigenic Factors Present in Acid-Fast Bacteria; Epi- flielium as the Site of Antibody Production and Antigen-Antibody Reaction, 531; Studies of Corper and Cohn, 532. CHAPTER XXVII The Significance ok Ali.ergy in Tuberculosis and a Few Other Diseases _________________ 538 Immunity in Tuberculosis, 538; Effect of Administering Vaccine Intravenously, 540; Experimental and Clinical Studies of Allergy and Immunity in Tuberculosis, 541; Summary of Theobald Smith's Studies of Spontaneous Bovine Tuberculosis, 542; The Immunizing Value of Nonprogressive Primary Lesions, 543; Summary of Lurie's Studies on Active Immunization; Lurie's Investigation of the Mechanism of Immunity and the Role of Allergy in Tubercu- losis, 544; Cannon's and Hartley's Work on Allergy, and Inmm- nity, 546; Effect of Primary Lesion in Children Upon Subsequent Infection, 547; Different Opinions as to B.C.G. Vaccination, 548; Park's Studies on B.C.G., 549; Other Bacterial Allergies — Brucel- losis, 550; Streptococcus Allergies; Rheumatic Fever; Pneumo- coccus Infection, 551; The Shwartzman Reaction, 552; Frei Test in LATiipliogranuloma Inguinale, 553. CHAPTER XXVIII IlvrKKSEXSITlVENESS ________________ 559 Human Idiosyncrasies; Clinical Allergies; Reactions Occur in Local Shock Organs; Inheritance Factors in Atopy, 559; Reagins Not Demonstrable in All Cases of Allergy; Passive Transfer of Reagin and the P-K Reaction, 560; Sensitization Through Alimentary and Respiratory Tracts and Through Placenta, 561; Effect of Heredity on Age of Onset ; Facts About Reagin, 562 ; Comparison CONTENTS 23 PAGE of Keagiu With Antibodies, 563 ; Antibody liespousible for liuniu- nity; Definition of Hay Fever, oGi; Historical; Blackley's Obser- vations, 565; Dunbar Confirmed Blackley's Work; Perennial Vaso- motor Rhinitis; Asthma, 566; Food Allergy, 567; Drug Allergy; Contact Dermatitis, 56S ; Substances Responsible for Contact Der- matitis, 569; Physical Factors in Allergy; Diagnosis of Allergy; Elimination Tests, 570; ('onjunctival Tests; Scratch Tests; Punc- ture Test; Intradermal Test; Patch Test, 5i71; Biological Test; Leucopenie Index; Fosinophiles and Allergy, 572; Meclianism of the Allergic Response, 573; Committees on Allergy Clinics; Com- mittee Recommendations for Clinic Equipment, Supplies and Per- sonnel, 574; Pollen Extracts — Noon Pollen Unit; Controversy Over Nature of Exciting Agent in Pollen, 575; Preparation of Pollen Extract; Standardizing Pollen Extracts, 576; Character of an Al- lergic Skin Reaction; Recording Skin Reactions; Treatment of Allergy, 577; Correct Breathing; Adrenalin and Ephedrine; Dia- tliermy, 578. APPENDIX Colloids ___________________ 984 Crystalloids and Colloids; True Solution; Colloidal State; Homo- geneous and Heterogeneous Systems, 584; The Tyndall Effect; Sus- pensoids and Emulsoids — Suspensoids; Emulsoids; Transferability of Emulsoid to Suspensoid; Chemical vs. Colloid Reactions, 585; Specific Surface; Surface Tension, 586; Electrical Phenomena and Surface Potentials; Electrical Double Layer, Helmholtz, 587; Mem- brane Potential; Effect of Cations on Colloids; Critical Potential; Factors Governing Stability of Emulsoids; Migration of Charged Particles — Cataphoresis, 588; Adsorption — -Adsorption and Surface Affinities; Positive Adsorption; Surface Concentration of Dissolved Substance; Surface Wetting; Devaux Experiment, 589; Molecular Orientation. Harkins' Theory; Attraction of Colloids for Each Other — Cohesion, Adhesion and Precipitation, 590 ; Fundamental Problem of Colloid Chemistry; Factors in Emulsoid Stability; Pre- cipitation by Electrolytes, 591; Protective Action of Colloids, 592; Autoprotection ; Effect of Speed of Mixing; Solubility of Precip- itates, 593. ILLUSTRATIONS FIG. PAGE 1. Sagittal section of head to show spread of suppuration from infected teeth; and also location of retropharyngeal abscesses _ _ _ _ 73 2. Portals of infection and the most frequent nodes involved in tubercu- losis of the cervical lymph nodes __________ 75 3. Pulmonary pleural, and tracheal lymphatic drainage _____ 76 4. Drainage of periphery of lung ____________ 76 5. The supposed intergenetic relationships and convertibility of cells of the "macrophage (reticulo-endothelial) system" of mammals _ 92 6. Distribution of agglutinins and agglutinogens in human blood groups 171 7. Graph showing distribution of agglutinogens in apes resembling those in man ______________---175 8. Pseudoagglutination or rouleaux formation of red cells _ _ _ _ 185 9. True agglutination of red cells ____-______-185 10. Bed cell before adding immune serum containing hemagglutinin (Color) ______-____-__--- 214 11. Red cell after adding hemagglutinin partly or completely filmed with antibody globulin absorbed from immune serum (Color) _ _ _ 214 12. Measurement of cohesive force of bacteria ________ 216 13. Interfacial tension technique of Mudd _________ 21 7 14. Durham's conception of the multiplicity of cellular antigens _ _ _ 319 15. Positive Widal test _______________ 380 16. Negative Widal test ________-__-_-- 381 17. Graph showing fluctuation in agglutinins in partially immune and im- mune animals during infection with P. tularensis _ _ _ _ 388 IS. Type II pneumococeus in sputum mixed with Type I antiserum (rab- bit) ; no "quellung" _____________ 397 19. Type II pneumococeus in sputum mixed with Type II antiserum (rab- bit) ; "quellung" reaction ___________ 397 20. Photomicrograph of a strongly positive Kline test ______ 459 21. Photomicrograph of a moderately positive Kline test _____ 459 22. Photomicrograph of a weakly positive Kline test ______ 459 23. Photomicrograph of a negative Kline test ________ 459 24. Negatively charged particle surrounded by single layer of positive charges, theory of Helmholtz _________--587 25. Negatively charged particle surrounded by diffuse atmospliere of posi- tively charged particles, theory suggested by Gouy _ _ _ _ 587 26. Orientation of molecules of an alcohol (or an organic acid) at the surface of its aqueous solution __________ 590 27. Factors involved in dispersion and precipitation of colloids _ _ _ 592 24 COLOR PLATES PLATE PAGK I. Blood cells (fixed and stained preparations) _______ 56 II. Cells in peritoneal exudate of white rat. Supra-vital preparation using neutral red and Janus green _________ 58 III. Cells of reticulo-endotlielial system showing phagocytosis of India ink __________________ 90 IV. Titration of hemolytic amboceptor and complement _____ 204 V. Original Bordet-Gengou bacterial complement fixation test _ _ _ 402 VI. Anticomplementary and antigenic titration of antigen _ _ _ _ 400 VII. Qualitative "one tube" bacterial complement fixation completed test __________________ 412 IMMUNOLOGY IMMUNOLOGY CHAPTER I INFECTION AND INFECTIOUS AGENTS Infection. — The phenomenon of infection is usually considered one in which an infectious agent gains entrance to the tissues of a susceptible organism (the host) and finds suitable conditions for growth and development in its new environment. Classification of Parasites. — These agents show wide variation in their habitat, size, shape, chemical composition, and physiological functions. They are usually classified into groups such as (1) animal parasites, (2) pathogenic fungi, (3) pathogenic bacteria, and (4) viruses. These groups are not sharply marked off from each other, but each embraces many organisms of doubtful taxonomic standing. Kendall (1923) divides tlie disease-producing bacteria into two main groups and discusses them under two headings: (1) the life cycle of parasitic bacteria; (2) the life cycle of pathogenic bacteria. The paradtic bacteria in contrast with the pathogenic are deficient in invasive power and in the ability to escape from the tissues. Both types of organisms are able to multiply within the tissues of the host after they gain entrance. Goodpasture (1936) suggests that infectious agents be classified as extracellular, facultative intracellular, and obligate intra- cellular parasites. The immunologist was in reality first interested in one of the virus diseases, smallpox, and many years later in bacterial infections. ]\Iore recently lie has extended his interest to infections caused by members of the first two groups. Symbiosis. — After infection has occurred, it may or may not disturb the tissues of the host to any great extent, and in some instances both parasite and host may be benefited. In the case of root-nodule or nitrogen-fixing bacteria that produce tumorlike gro^\1;hs on the roots of leguminous plants, the host is dependent upon them for simple nitrogen compounds necessary for the life 29 30 IMMUNOLOGY of the plant, while the bacteria are supplied certain food materials by the host. This relationship of mutual helpfulness is spoken of as symbiosis. Infection Without Pathological Change. — A generalized in- fection of the wild rat witli Tnjponosome leivesi or with Leptospira icterohemorrhagiae is not beneficial to tlie host but apparently does not elicit any physiological or anatomical disturbances that can be designated as infectious disease. In both of these examples of infection the organisms enjoy a wide distribution in the body and are present in great numbers in the blood. Infection With PathologicaI: Change. — These examples of infection without pathological change or infectious disease are in nuirkod contrast to what occurs when the Icptospira, mentioned al)ove, gains entrance to tlie tissues of another rodent, the guinea pig, or for that matter into the tissues of man. In either case the presence of generalized infection causes profound pliysiological and structural disturbances, frequently resulting in death. Types of Pathological Changes. — It would seem that if an infectious agent is to produce disease, it must not only gain entrance to the tissues and be able to live and multiply there, but it or its products of growth must be incompatible with the normal physiological functioning of the host and lead to discernible patho- logical (abnormal) changes. These may be local, focal, or general. For example, organisms gaining entrance to the body are fre- quently "filtered out" by the regional lymph glands. This maj^ occur without any symptoms other than a slight increase in size and change in consistency of the glands. The organisms may be destroyed or remain quiescent for long periods of time or until conditions become favorable for their growth and dissemination. Localized and Generalized Infection. — A wound may become infected and show abnormal changes such as redness, swelling, local heat and pain, without general physiological symptoms. As long as the infectious agents remain at the site of injury, it is a localized infection, but when they multiply and invade the blood stream, it becomes a generalized infection. This usually calls forth definite symptoms even before the blood stream is invaded. Bacteriemia, Septicemia, and Pyemia. — If bacteria gain en- trance to the blood stream from some focus but are unable to multiply in the circulation, the condition is spoken of as a INFECTION AND IxXFKCTIOUS AGENTS 31 hactericmia. If multiplication occurs in the blood stream, it is called septicemia unless multiple abscesses develop, when the term pyemia is used. Acute and Chronic Infections. — Infections are often classified according to the duration of the disease. Those that disappear within a few days or at most a few weeks are called acute infections, while those that persist are either subacute, or cJironic infections. Focal Infections. — Chronic infections causing pus pockets around the roots of the teeth and inflammatory processes in the tonsils, sinuses, prostate gland, cervix of the uterus, or elsewhere are called focal infections. The detection and eradication of such foci of infection is regarded by most ph^'sicians as good medical practice. A critical apprai-sal of their importance in systemic dis- ease is given by Reimann and Havens (1940). They seem to feel that an overemphasis has been placed upon the importance of focal infections in systemic disease and tliat surgical interference is fre- quently detrimental to the patient. Beneficent Infections. — There are a few infections in man which, w'hen viewed in one way, may be regarded as beneficent although not without danger in themselves. For example, consider a patient suffering from syphilis, which is caused by Treponema pallidum. He may have extensive infection of the tissues, includ- ing the brain, and develop spastic paralysis and insanity. This condition resists the ordinary treatment of syphilis, but if the patient should become infected with the malarial parasite, a clinical cure is passible. After his symptoms of syphilis have dis- appeared, he still has malaria, which can be treated and usually cured with quinine. Specific Microbic Association. — Until recently it has been thought that each infectious disease was caused hy a single specific infectious agent. Now it appears that swine influenza may be an exception to this concept. Shope (1931) and Lewis and Shope (1931) seem to have shown that this disease is due to simultaneous infection with two agents, one filtrable and a second that belongs to the hemophilic group of bacteria. This would seem to be an example of an infectious disease caused by specific microbic asso- ciation wiiere both microorganisms may be regarded as primary agents. Zinsser and Bayne- Jones (1939) seem to regard these as examples of primary (virus) and secondary (hemophilic bacteria) infections. 32 IMMUNOLOGY Metastasis. — During the period of existence of a focal infection or the course of an infectious disease or, for that matter, following- major or minor operations, there may occur new foci of infection in parts of the body not primarily involved. This extension of organisms to new regions, which is called metastasis, may occur either by way of the blood stream or lymphatics. This explains the development of pneumococcus meningitis, typhoid meningitis, or tuberculous meningitis associated with the respective diseases. It likewise explains bone involvement (osteomyelitis) and inflamma- tion of venous walls (phlebitis) during typhoid fever. Patients operated upon for localized streptococcus infections in the abdomen not infrequently develop phlebitis of the femoral or popliteal veins of the left leg. It is upon this basis that one commonly explains many cases of inflammation of the gall bladder (cholecystitis), kidney (nephritis), pancreas (pancreatitis), etc. Secondary Infections. — Not infrequently a patient suffering from a specific infectious disease may develop a secondary infec- tion due to an entirely different organism. The first disease lowered the patient's resistance and created a favorable soil for secondary invaders. One sees this occasionally in measles, which is apparently caused by a virus. These patients may develop a secondary streptococcus invasion of the lungs, which is called a streptococcus pneumonia. Secondary pneumonias of this type may be due to any of a number of organisms found upon the respiratory mucous membrane of tlie patient. In like manner secondary involvement of other tissues may occur and complicate the primary disease. Infectious and Contagious Diseases. — Any disease which is caused by an infectious agent is called an infectious disease. When it is quite readily communicated from one individual to another, it is classified as a contagious disease. Thus it is apparent that while all contagious diseases are due to infectious agents, not all infectious diseases are contagious. Toxemic Diseases. — In a few diseases such as botulism, tetanus, gaseous gangrene, and diphtheria, infection either does not occur or is very slight. They are in reality toxemic in nature whereas in scarlet fever and in staphylococcus and pneumococcus infections there are both toxemia and severe infections. Bacterial Toxins. — Roux and Yersin (1888) were the first to show that some bacteria secrete toxins having specific actions in the INFECTION AND INFECTIOUS AGENTS 33 body and for which Behring and Kitasato (1890) have shown that antitoxins can be developed. Pathogenic streptococci also produce a substance, leucocidin, toxic for leucocytes. McLeod (1914) and Gay (1931) regard this as an important factor in the pathogenicity of streptococci. It is now generally agreed that each type of bacteria owes its specificity to its chemical constitution and in some cases to specific chemical products secreted by it. All diphtheria bacilli are not only more or less alike chemically and also chemically different from other bacteria, but they likewise all secrete a specific poison called diphtheria toxin. The true toxins differ from other poisons in that antitoxins are formed by the body in response to their presence within tlie tissues. One can then define a true toxin as an antigenic poison. It has been customary to classify bacterial poisons into "exo- toxins" and "endotoxins." The former are highly antigenic and are readily liberated from the bacterial cell while the "endotoxins" either fail to stimulate specific neutralizing substance or at best produce only a feeble response and are liberated with more or less difficulty from the bacterial cell. Eaton (1939) states, in effect, that a sharp line cannot be drawn between ' ' exotoxins ' ' and ' ' endo- toxins" because antigenic poisons exhibit all degrees of anti- genicity and toxicity and also marked differences in the ease with which they can be separated from the bacterial cells. For a more extensive discussion of the subject the student is referred to Eaton's (1939) excellent review of chemical investigations of ])acterial toxins and to the discussion of toxins by Zinsser, Enders, and Fothergill (1939). Botulism. — In botulism the disease is due to a toxin formed ])y the bacteria outside the body, taken in with the food and ab- sorbed through the mucous membrane of the intestine. Tetanus. — In tetanus the organisms gain entrance to a wound or are mechanically carried into the tissues by a foreign body. If anaerobic and other satisfactory conditions prevail, the bacteria grow saprophytically and secrete tetanus toxin. This, according to Abels (1934), is carried to the central nervous system by the blood stream. The evidence, however, seems to indicate that the toxin is absorbed, probably through the end plates of the motor nerves, and reaches the central nervous system along their axis cylinders. When the toxin reaches the central nervous system, there develop certain physiological disturbances called tetanus. 34 IMMUNOLOGY Gaseous Gangrene. — Gaseous gangrene is not an infrequent complication of severely lacerated Avounds. It is caused by CI. welchii and a few other anaerobes and is characterized by necrosis (death) of the tissues infected and the liberation of gas within them. It has been quite definitely established that hemolytic streptococci, growing in gas-bacillus infected wounds, exalt the virulence of the anaerobes. In this wc have another type of bacterial association. Diphtheria. — In diphtheria the organisms do produce a slight infection of the mucous membrane and grow luxuriantly in the inflammatory exudate. The disease is caused by the specific toxin secreted by the bacteria and absorbed into the general circulation. The disturbed physiological state is called diphtheria. Diphtheria toxin causes degenerative changes in the motor nerves, heart muscle, and vascular system. Endotoxins. — By means of extraction methods weakly anti- genic endotoxins from members of the Salmonella, Proteus, and Colon groups have been obtained by Raistrick and Topley (193-4), Boivin and Mesrobenu (1937) and others. These endotoxins seem to be carbohydrate-lipid complexes although a second but weaker endotoxin, apparently a polypeptid. has been described. It has been known that Shiga dysentery bacilli produce a specific exo- toxin. Boivin and Mesrobenu (1937) made a chemical investiga- tion of the toxins of Shiga dysentery bacilli. They report that the exotoxin, which is produced by both R. and S cultures, is a protein and acts on the central nervous system but not on the gastrointestinal tract. They were able to isolate endotoxins of a carbohydrate-lipid nature from both Shiga and Flexner dysentery organisms. These endotoxins produced symptoms of acute gastro- enteritis in animals. Phytotoxins and Zootoxins. — The mycologist and the para- sitologist have been more interested in morphological studies, life histories, and host-parasite reactions and relationships, than in chemical and physiological studies of parasites. A great many important physiological observations, however, have been recorded. Soule (1925) and Salle (1931) are pioneers in the study of the metabolism of protozoa. It is known also that the poisonous mushroom Amanita pludloides produces a soluble toxin whose properties have been studied by Ford (1910-11). Soluble toxins IXFI':CTI()X AXI) IXKl'X'TIOUS AGENTS 35 luive also been found in the seeds of Ricinus communis, Croton iif/Ihnn, and the bark and leaves of the locust tree {Robinis pscudoacacia). In the animal kinodom one finds toxins in the fi'landular secretions of a number of snakes, the ones most studied I)eing' those of the cobra, water moccasin, copperhead, and rattle- snake. Flcxner and Noguchi found the venoms of the first two rich in neurotoxins but attributed the injurious effect of rattle- snake venom to hemorrhagin (vascular toxin). It is not uncommon for a plant or animal to produce several toxins that differ in their affinity for different tissues. Perhaps the most common is a hemotoxin which destroys red blood cells. This type of toxin is found in arachnolysin (from certain spiders), ichthyotoxin (in eel serum), phrynolysine (blood and skin of toads), in tlie poisojious secretions of certain fish {TnicJnyius draco), and in scorpions. These toxins of plant and animal origin are called ''phytotoxins" and ''zootoxins," respectively. For a more comprehensive discussion of the latter, the student is referred to a paper by Do Amarol (1928). Antitoxins. — The antitoxins are specific neutralizing substances produced by the reticulo-endothclial system of the body and given off into the general circulation. Normal rabbit blood will not neutralize diphtheria toxin, but after the animal is given several small injections of toxin, it is found tliat the blood will neutralize the toxin, hence it is said that this blood contains diphtheria anti- toxin. CI. tetani, CI. hotidinum, CI. ivelcMi, and a few other anaerobes produce soluble toxins that show remarkable specificity. Recently Dick and Dick (1924) have shown that scarlet fever streptococci produce a soluble toxin responsible for the rash in scarlet fever. It is possible to purchase specific antitoxins for each of these bacterial toxins. Ptomaines." — Other toxic substances are formed in culture media as the result of bacterial enzymes acting upon the food material. AVhen enzymes act upon amino acids in such a way as to remove the carboxyl group (COOH) the process is called decarhoxylization and the residue is an amine. Toxic amines are called ''ptomaines." Kendall (1931) has made the most extensive investigation of bacterial metabolism and has noted histamine production by a variety of organisms. Ptomaines are not only nonspecific but are ]iroduced by many saprophytes as well as parasites. 36 IMMUNOLOGY Pathogfenicity. — The term pathogenicity is usually applied to the disease-producing property of an infectious agent. Virulence. — The term virulence has been used by many as synonymous with either the invasive power of an organism or its ability to multiply in the tissues or blood stream of a host. This definition seems inadequate for several reasons. In the first place, the term is applied to such toxicogenic organisms as CI. tetani and C. diphtheriae as an index of their ability to form tetanus toxin and diphtheria toxin, respectively. The ''virulence test" of C. diphtheriae is essentially one for toxin production. CI. tetani has no invasive power while C. diphtheriae invades the mucous membrane to a slight degree only. Both produce disease in suitable hosts by means of specific soluble toxins. In the second place, examples have just been given of infection without and also with pathological change. The host must be considered as a factor. Definition of Virulence. — It would seem appropriate to define virulence as the relative pathogenicity of an organism for a particular host. In examples previously cited it will be recalled that Leptospira ictcroheniorrJiafjiae is practically a virulent for the wild rat but highly virulent for guinea pigs and man. The same hosts exhibit similar resistance and susceptibility to diphtheria toxin. Measurement of Virulence. — Virulence is usually expressed in terms of the amount of infectious agent that will, when properly administered, cause certain specified pathological changes in the host within a given time. There has been no uniform standard adopted for the measurement or dosage. Barber (1909), in describing the virulence of the anthrax bacillus for mice, recorded the actual number of organisms inoculated. Others have expressed the dosage of bacteria in terms of milligrams of growth obtained under standard conditions. The virulence of many bacteria has been described in terms of the least volume of a 24-hour broth culture grown at 37° C, or the fraction of growth obtained from a blood agar or plain agar slant grown under similar conditions, that will produce death or specified elianges in the test animal. Some have even measured the dosage in terms of the standard platinum wire loop. INFECTION AND INFECTIOUS AGENTS 37 Factors Altering Virulence. — Virulence may be diminished hy frequent transfers in certain artificial media, culturinj? at temperatures above the optimum, or in some instances by desicca- tion. It may be increased, within limits, by growing the organisms on media containing blood or body fluids that favor capsule forma- lion and also by animal passage. Pasteur showed that the passage of rabies virus through rabbits increased its virulence for the latter but diminished it for certain other animals. He found that exaltation of virulence, by animal ])assage, has a limit beyond wliicli no increase could be obtained. Relation of Capsule Formation to Virulence. — It seems that all pathogenic bacteria produce capsules when growing within the tissues of a host. It has been observed that when cultured outside the body the pneumococcus loses its virulence for white mice simultaneously with its ability to form capsules. Hence capsule formation is regarded as a factor in virulence. Hypothesis of Welch. — Welch offered an interesting hypothesis which bears his name. In this he postulates that the bacterial cell possesses a defensive mechanism which enables it to oppose the defenses of the body. Correlation of Virulence and Colony Type. — Arkwright (1921) called attention to two important colony variations in a member of the colon-typhoid-dysentery group. These were the R or rough, granular colonies, and S or smooth, moist types. Both Arkwright and Baerthlein noted that S colonies might give rise to R colonies but that it was apparently more difficult for the process of dissociation to extend from R to S types. There is a tendency for colony types to remain stable but dissociation may occur spontaneously or may be induced. Intermediate between the R and the S is the 0 colony which has potentialities of both. In the same year that Arkwright called attention to the exist- ence of R, S, and 0 colony types, de Kruif (1921), working with colony variants of Bact. lepisepticwm, found that cultures obtained from S colonies were virulent for rabbits while those obtained from R colonies were not. The phenomenon of colony variation just described is called bacterial dissociation. It is now known that practically aU bacteria may undergo dissociation and that there is a definite correlation between colony type and other biologic characteristics. 38 IMMUNOLOGY Among the pathogenic bacteria the S type is usually capsulated and virulent, while the R type is avirulent. An apparent exception to this occurs with B. anthracis. It should be noted, however, that in this case the R or rough colony is composed of capsulated organ- isms as are the virulent S forms of other bacteria and that the avirulent anthrax, althougli forming smooth S colonies, is said to be noncapsulated. This is another example of the correlation of capsule formation with virulence. Labile Antigens and Virulence. — In 1934 Felix and Pitt reported that virulent strains of E. tijphosa possess a surface anti- gen not possessed by avirulent smooth strains. Because of its role in virulence they named it the ''Vi'' antigen of E. typhosa. It is destroyed by heat and most chemicals although formaldehyde is only slightly injurious to it. Perhaps one of the ways in which it confers virulence upon the bacteria is that it renders them more or less resistant to antibody action. Numerous reports have appeared in the literature stating that the .strains of E. tijphom containing "Vi" antigen are more or less inagglutinable. Craigie (1936) discovered a bacteriopliage specific for inagglutinable strains of E. typhosa. The question of inagglutinability of strains of E. typhosa passess- ing ''Vi" antigen has lieen investigated in this laboratory by Faucett (1940). He confirmed the results of others that the difference in agglutinalnlity of resistant strains depends partly upon the "Vi" antigen content of the individual organisms. The strains of intermediate resistance are mixtures of resistant and sensitive strains. Some doubt as to the correlation of Vi antigens and virulence has been raised by the studies of Robertson and Yu (1936) and of Kauffmann (1936). The former report that its presence is not primarily correlated with virulence of Bacillus typhosus while Kauffmann found that it was present and not correlated with viru- lence in SaJmonelln paratyphi C. Mudd, Pettit, Lackmann, and Morgan (1939) have discovered a partially labile antigen in virulent streptococci. Its relationship to other specific substances and to virulence is not definiteh' determined. These findings suggest that perhaps labile antigens play an important role in bacterial virulence in general. infkctiox and infectious agents 39 Importance of Host Factors in Determining Virulence. — Falk* (1928) has objected to the use of the term virulence "to denote the absolute capacity of a parasite to cause disease." He points out that virulence is not primarily a characteristic of the parasite but "varies reciprocally as resistance or immunity." In other words the relative resistance of the host is a factor. The Leptospira icterohemorrliaqiac is not virulent for the wild rat, which is resistant to it, but is highly virulent for guinea pigs partly because they are very susceptible. Aggressins. — Bail attributed virulence to certain biochemical products formed either within the infected tissue or in cultures. He named these chemical substances "aggressins." It has been shown that if CI. chauvei, the causal agent of blackleg in cattle, is washed with sterile saline until freed of all traces of culture media and products of growth, it loses its virulence. The latter is restored, however, when the washed organisms are mixed with sterile filtrates of the original virulent culture. Scott (1931) has recently reviewed the literature on aggressins quite extensively. Nature and Characteristics of Viruses. — Prior to 1935, very little was known about viruses other than that they are much smaller than bacteria, many being filtrable agents, and that they require living host cells for their reproduction ; they cause specific diseases and recovery from such diseases is usually accompanied by a lasting immunity. From the standpoint of pathology it was well established that in many virus infections characteristic cell inclusion bodies are formed. In 1935 Stanley obtained the virus of tobacco mosaic disease in crystalline form. While Stanley at first thought that the virus protein is a crystalline globulin, Bawden and Pirie (1940) present chemical analyses of six types of viruses which indicate that they are nucleoprotcins. This view is also supported by the work of Rischkaw (1940), Mcintosh (1940) and Daranyi (1940). It is well established that the virus protein is biochemically and anti- genically different from the proteins of the host. In regard to size the viruses appear to exhibit, according to j\IcIntosh, prac- tically a continuous series varying from the smallest 20 mix to the largest which is 250 mu. According to Laidlaw (1938) the size ♦From "A Theory of Microbic Virulence," by I. S. Falk in The Newer Knowl- rdoe of Bacteriology and Immunology edited by E. O. Jordan and L. S. Falk. Reprinted by permission of University of Cliicago Press. 40 IMMUNOLOGY varies from 10 ni/i, for poliomyelitis virus to 250 m/A for psittaco- sis virus. He places the size of vaccine virus at 150 mju,. New instruments and techniques such as the ultracentrifuge, the electron microscope, and x-ray, are beginning to extend our knowledge concerning the size, morphologj^ and structure of both viruses and bacteria. By means of the ultracentrifuge, Bauer and Pickels^ (1940) determined the minimal diameter of yellow fever virus to be 14 m.[x while Gratia- (1940) reports the recovery of polyhedral bodies and of minute granules possessing virulences from serum of diseased silkworms by differential centrifugation. Borries, Ruska and Ruska (1938) describe a new electron microscope witli which they have obtained photographs of several viruses and of bacterial structures previously unrecognized. Ac- cording to Mudd^ (1941), magnification of 50,000 diameters has been obtained with instruments in the RCA laboratories. He mentions a few limitations of the electron microscope such as the in vacuo requirements, the opacity of specimens of Iju, or more in thickness, and the possible effect of drying and electron bombard- ment on the specimen. Bernal (1940) submits x-ray evidence to show that both spherical and rodlike forms exist among the viruses. There has been much discussion concerning the living or non- living nature of viruses. Rivers (1928) suggests three possi- bilities as to the nature of viruses. Some may be miniature bacteria; others may be as yet unrecognized forms of life, while a third group may be inanimate transmissible agents of disease. Green* (1935), Laidlaw (1938) and Gortner (1938) suggest that viruses are living entities that through retrograde changes have lost their enzyme systems and are therefore dependent upon the enzyme system of the host cell. Zinsser and Bayne-Jones (1039) state, however, that tliey regard the bacterial origin of viruses as highly improbable. Chemical Composition of Bacteria. — Bacteria have been investi- gated more extensively from the standpoint of chemical composi- tion and metabolism than any other group of infectious agents. The modern era began with Pasteur's studies in fermentation and his investigation of the germ theory of disease. Koch (1891) obtained from tubercle bacilli a crude group specific protein which iProc. Third International Congress for Microbiology, 1940, p. 287. =Ibid., p. 288. 'J. Bact. 41: 415. 1941; Ibid. 43: 251, 1941. "Science 82: 443, 1935; Bio- dynamics No. 39, 1, 1938. INFECTION AND INFECTIOUS AGENTS 41 he named tuberculin. Seibert crystallized out the active principle, and she and Long studied its properties. They are discussed in Chapter XXVI. Carbohydrate. — Zinsser (1923), Heidelberger and Avery (1923), and others have obtained specific carbohydrates from pneumocoeci and other bacteria. In some instances the exact chemical composition of the polysaccharide has been determined. Protein. — Furth and Landsteiner (1928) isolated from typhoid bacteria specific protein fractions which they designated as Pi and Po. Ando and Ozaki (1930) have shown that scarlet fever streptococci contain nucleoprotein fractions which must be dif- ferentiated in the culture from specific soluble toxins secreted by the organisms. Similar studies on C. diphtheriae have been carried out by Neill and his colleagues (1930). They have apparently shown that all hypersensitive reactions to products of growth in diphtheria cultures are due to a hypersensitiveness to toxin and that no reactions to nucleoproteins occurred. Extensive chemical studies of many other species of bacteria are being reported in the literature and will be referred to in later chapters. Effect of Environment on Bacteria. — The cultivation of bac- teria in artificial media frequently leads to interesting changes. They may lose their ability to form pigment, to liquefy gelatin, to form capsules or to produce infection as well as many other characteristics. When the environment is modified properly, many of these lost characteristics may be restored. The loss of characteristics mentioned above has been spoken of frequently as degeneration phenomena. Occasionally an organism acquires a new character and retains it. This is called mutation. Neisser (1906) described mutation in a strain of B. coli. His observations were soon confirmed by a number of investigators. Barber (1913) and Jordan (1915) have reported mutation arising from the progeny of a single cell. Bacteria are also able to adapt themselves to growth under many favorable conditions. They may become habituated to growth at temperatures normally inhibitory or to develop in the presence of concentrations of chemical agents such as antiseptics or antisera that are destructive to the original culture. Theory of Constancy of Characteristics. — Cohn (1875) and Koch (1877) are credited with the concept that bacterial char- 42 IMMUNOLOGY aeteristics such as morphology, fermentative powers, pigment production, motilitj^, pathogenesis, etc., are constant and inherita- ble and that all organisms in a colony or pure culture are alike. Opposed to this are the extremely radical views of Nageli (1877) who held that theie is no morphological or pliysiological constancy. Evidence of Heterogeneity Within a Culture. — It is now quite generally kno\Mi that among bacteria constituting a pure culture there is definite heterogeneity. The individuals which collectively constitute the culture not only show slight variation in shape, size, and staining reaction but differ in age, physiological stability, pathogenicity and adaptability. Some may be avirulent while others are quite virulent. Fluctuating Variability. — Heterogeneity in a culture may be due to age, fluctuating variability, heredity, true mutation or to the phenomenon of bacterial dissociation. Fluctuating variability is usually illustrated by variations in size or shape which corre- spond to height or weight fluctuations observed in the human being. Any characteristic variation that oscillates around an average type is termed "fluctuating variability." Practically all of the phenomena of bacterial variation are discussed by Hadley* (1927) in his excellent monograph on bacterial dissociation. Many kinds of variation are quite evidently examples of either mutation or fluctuating variability, but other variations are difficult to classify. Early Theories of Bacterial Characteristics. — According to the theory of Cohn (1875), Koch (1877), Migula (1897), and others that bacterial characteristics are constant, one would expect all colonies obtained by plating a pure culture on agar to resemble not only each other, but also the parent colony. That such is not the case w^as noted by numerous workers prior to the extensive study of Baerthlein (1918) in colony variation. The latter investigator not only described in detail various types of colonies obtained from each pure culture but he also attempted to correlate colony variation with other characteristics of the organism. Arkwanght (1921) is credited by Hadley (1927) with being the fii-st to appreciate the significance of Baerthlein 's results. It would seem, however, that Bordet as early as 1909 described bacterial dissociation into what are now called rough and smooth colonies and found that, while both possessed a common antigenic *J. Infect. Dis. 40: 1, 1927. INFECTION AND INFECTIOUS AGENTS 43 factor, the smooth type contained an arlditional factor not pos- sessed by the rough variant. Bordet and Gengou were working A\ith a pure culture of B. pertussis. They succeeded in getting it to grow on plain agar and compared the gro^vth and antigenic properties of the organisms grown upon plain and blood agar respectively. The following quotation from Bordet definitely bears out the above conclusions :* "It [Bacillus pertussis] develops readily in the medium that is rich in defibrinated blood, as already described by Gengou and my- self in our first article on whooping cough. It may be taught to grow on ordinary agar, in which instance it gives a thick and rather coherent layer. The two varieties of organisms obtained in this manner, although coming from a single original colony, give rise on immunizing animals to tAvo different sera. We may consider the scrum of a rabbit that has been imnumized against an organism grown on ordinary agar. It is found that the serum agglutinates these organisms energetically, but has no clumping effect on a culture of whooping cough bacillus grown on the otlier medium containing defibrinated blood. On the other liand, if we test the serum of a rabbit that has been immunized against the organism grown on blood media, we find that it agglutinates both races of bacteria. A careful study of this phenomenon brings out the fact that two definite agglutinins affecting different antigens are present in different proportions. One of these antigens which is present in large amounts in the organism which has developed on blood is not to be found in the organism grown on agar. ' ' Relation of Environment to Size. — In conclusion it would seem advisable to mention a few controversial theories that are being investigated at the present time. It is held by Gotschlich (1927), Kendall (1931), Almquist, Holmes, Enderlein, IMellon and others that filterable forms of bacteria are demonstrable and that these may give rise to the larger forms. Many of these claims have not been confirmed. A brief but excellent review of the subject is given by Zinsser and Bayne- Jones (1939). Entirely aside from the question of filtrable forms of bacteria is the effect of the host's tissues upon the size and perhaps mor- phology of certain organisms. Goodpasture (1937) finds within the walls of the intestinal tract of ty])hoid ])atients coming to autopsy ♦Reprinted by permission from Shtdies in Innnuniti/ by Bordet and Gay, published by John "Wiley and Sons, Inc. 44 IMMUNOLOGY young plasma cells full of very small rods that stain as if they were viable. These are in contrast to larger rods found in the ordinary phagocytes present in the immediate surroundings. In Goodpasture's opinion tliese small rods as well as the large ones are typhoid bacteria differing in size due perhaps to their environ- ment. The small ones have made use of young plasma cells as host cells. After liberation from the host cells they developed into the larger rods found within macrophages. References Abel, John J. : On Poisons and Disease and Some Experiments With the Toxin of the Bacillus Tetani, Science 79: (iZ, 1934. II, Ibid., p. 121. Ando, K., and Ozake, K. : Studies on tlie ' ' Toxins ' ' of Hemolytic Strepto- cocci, V. The Dick Test and Allergic Skin Reactions to Streptococcus Nucleoproteins, J. Immunol. 18: 267, 1930. Arkwright, J. A. : Variation in Bacteria in Relation to Agglutination Botli by Salts and by Specific Serum, J. Path, and Bact. 24: 36, 1921. Baerthlein, K. : Ueber bakterielle variabilitat, insbesondere sogenannte bak- terienmutationen (Orig.), Centralbl. f. Bakt. 81: 369, 1919. Bail, O.r Folio Serol. 4: 129, 1910. (Cited by Scott, J. P.: J. Bact. 22: 323, 1931.) Barber, M. A. : The Effect on Mice of Minute Doses of B. anthracis, J. Infect. Dis. 6: 634, 1909. Barber, M. A.: Philippine J. Sc. 8: 539, 1913. (Cited by Jordan, E. O. : Textbook of General Bacteriology, ed. 12, Philadelphia, 1940, W. B. Saunders Co., p. 50.) Bawden, F. C, and Pirie, N. W. : Some Properties of Purified Preparations of Plant Viruses, Proc. Third International Congress for Microbiology, 1940, p. 279. Behring and Kitasato: See Chapter XIV. Bernal, J. D. : X-ray Evidence on Size and Structure of Plant Virus Prepara- tions, Proc. Third International Congress for Microbiology, 1940, p. 279. Boivin, A., and Mesrobeanu, L. : Cited by Eaton, 1938. Bordet, J.: Studies in Immunity (translated by Gay), New York, 1909, John Wiley and Sons, p. 527. Bronfenbrenner, J.: The Bacteriophage: Present Status of the Question of Its Nature and Mode of Action, Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, University of Chicago Press, 1928, p. 525. Cohn: 1875. Cited by Hadley, 1927. Craigie, J., and Brandon. K. F. : A Bacteriophage Specific for Inaggluti- nable Strains of E. Typhi, Canad. Pub. Health. J. 27: 165, 1936. Daranyi, J.: The Essence of the Ultraviruses Based on Recent Researches, Proc. Third International Congress for Microbiology, 1940, p. 283. DeKruif , P. : Dissociation of Mierobic Species. I. Coexistence of Individuals of Different Degrees of Virulence in Cultures of the Bacillus of Rabbit Septicemia, J. Exper. Med. 33: 773, 1921. Dick, G. F., and Dick, Gladys: A Skin Test for Susceptibility of Scarlet Fever, J. A. M. A. 82: 265, 1924. Do Amarol, Afranio: Venoms and Antivenins, Newer Knowledge of Bac- teriology and Immunology, Jordan and Falk, 1928, University of Chi- cago Press, p. 1066. INFECTION AND INFECTIOUS AGENTS 45 Eaton, Monroe D. : Recent Chemical Investigations of Bacterial Toxins, Bacteriol. Reviews 2: 3, 1938. Falk, I. S. : A Theory of Microbic Virulence, Newer Knowledge of Bac- teriology and Immunology, Jordan and Falk, 1928, University of Chi- cago Press, p. 565. Faucett, R. L.: A Study of the "Vi" Antigen of E. Typhi. Tliesis, Library Univ. of Kans., Lawrence, 1940. Felix, A., Bhatnagar, S. S., and Pitt, R. M. : Observations of the Properties of the "vi" Antigen of B. Typhosus, Brit. J. Exper. Path. 15: 346, 1934. Felix, A., and Olitzki, L.: The Use of Preserved Bacterial Suspensions for the Agglutination Test, J. Hyg. 28: 55, 1928. Felix, A., and Pitt, R. M.: A New Antigen of B. Typhosus, Lancet 227: 186, 19.34. Felix, A., and Pitt, R. M. r Virulence of B. Typhosus and Resistance to *'0" Antibody, J. Patli. and P.act. 38: 409. 1934. Ford, W. W. : Furtlici' Oliservations on the Immunization of Animals to the Poisons in Fungi, .T. Pliarmacol. and Exper. Therap. 2: 145, 1910-11. Furth, J., and Landsteiner, K. : On Precipitable Substances Derived From B. Typhosus and B. Paratyphosus B., J. Exper. Med. 47: 171, 1928. Gay, F. P.: Tissue Resistance and Immunity, J. A. M. A. 97: 1193, 1931. Goodpasture, E. W. : Concerning the Pathogenesis of Typhoid Fever, Am. J. Path. 13: 175, 1937. Goodpasture, E. W. : Intracellular Parasitism and the Cvtotropism of Viruses, South. M. J. 29: 297-303, 1936. Gotschlich: Cited by Ward, 1928, Newer Knowledge of Bacteriology and Immunology, 1928, University of Chicago Press, p. 4. Heidelberger, M., and Avery, O. T. : The Soluble Specific Substance of Pneumococcus, J. Exper. Med. 38: 73, 1923. Jordan, E. O.: Natur. Acad, of Science L: 160, 1915. Cited by Jordan, E. O.: Textbook of General Bacteriology, Ed. 10, 1931, W. B. Saunders Co., p. 130. Kauflfmann, F. : Zur Frage des VI- Antigens der Paratyphus B- und Mause- typhus-Bacillen, Ztschr. f. Hyg. u Infektionskr. 118: 318, 1936. Kendall, A. I. : Bacterial Parasitism, Bacterial Pathogenism, and Resistance to Bacterial Infection, J. Infect. Dis. 32: 341, 1923. Kendall, A. I. : Mediums for the Isolation and Cultivation of Bacteria in the Filterable State, Bull. Northwestern University, Oct. 19, 1931. Koch: 1877. Cited by Hadley, 1927. Laidlaw, Sir Patrick P.: Virus Diseases and Viruses, The Rede Lecture 1938, New York, 1939, The Macmillan Co. Lewis, P. A., and Shope, R. E. : Swine Influenza. II. A Hemophilic Bacillus From the Respiratorv Tract of Infected Swine, J. Exper. Med. 54: 361, 1931. Long, E. R. : Tuberculin and the Tuberculin Reaction, Newer Knowledge of Bacteriology and Immunology, 1928, University of Chicago Press, p. 1016. Mcintosh, J.: The Nature of Viruses: Proc. Third International Congress for Microbiology, 1940, p. 283. Migula: 1897. ated by Hadley, 1927. Mudd, Stuart, Polevitsky, U., and Anderson, T.: Structural Differentiation Within the Bacterial Cell as Shown bv the Electron Microscope, J. Bact. 41: 25, 1941. Mudd, S., Pettit, H., Lackman, D. B., and Morgan, I. M. : Antigenic Struc- ture of Hemolvtic Streptococci of Lancefield Group ''A," J. Immunol. 36: 381, 1939." 46 IMMUXOLOGV Neill, J. M., Fleming, W. L., Sugg, J. Y., Avery, R. C, Riehurdtion, L. V., and Kane, B. E. : Hypeisensitiveness to Diphtheria Bacterial Products. Comparison of Filtrates of Highly and of Weakly Toxicogenic Strains, J. Immunol. 18: 437, 1930. Neisser, M. : Ein Fall von Mutation nach D Vries bei bakterien und andere Demonstrationen, Centralbl. f. Bakt. I., Ref. 38: 98, 190(3. Raistrick, H., and Topley, W. W. C. : Immunizing Fractions Isolated From Bact. Aertrycke, Brit. J. Exper. Path. 15: 113, 1934. Reimann, H. A., and Havens, W. P. : Focal Infection and Systemic Disease. A Critical Appraisal, J. A. M. A. 114: 1, 1940. Richardson, L. V., and Kane, B. E. : Hrpersensitiveness to Diphtheria Bac- terial Products. Comparison of Filtrates of Highly and of Weakly Toxicogenic Strains, J. Immunol. 18: 437, 1930. Rischkow, V. L. : Nature and Mechanism of Virus Action, Proc. Third Inter- national Congress for Microbiology, 1940, p. 282. Rivers, T. M. : Filterable Viruses, Newer Knowledge of Bacteriology and Immunology, 1928, University of Chicago Press, p. 517. Rivers, T. M. : Nature of Viruses, Physiol. Rev. 12: 423, 1932. Further Observation on Cultivation of A^'accine Virus in Lifeless Media, J. Exper. Med. 57: 741, 1933. Robertson, R. C, and Yu, H. : The Vi Antigen of Bacillus Typhosus, J. Path, & Bact. 43: 191, 1936. Roux and Yersin: See Chapter XIV. Salle, A. J.: >Ietabolism of Protozoa; New Solid and Now Liquid Medium for Cultivation of Leishmania donovani, J. Infect. Dis. 49: 473, 1931. Scott, J. P.: Aggressins- — An Outline of the Development of the Theorv and Notes on the Use of These Products, J. Bact. 22: 323, 1931. Shope, R. E. : Swine Influenza, Experimental, Transmission and Pathology, J. Exper. Med. 54: 349, 1931. Swine Influenza; Filtration Experiments and Etiology, Ibid., p. 373. Soule, M. H. : Microbic Respiration; Re.spiration of Trypanosoma Lewisi and Leishmania Tropica, J. Infect. Dis. 36: 245, 1925. Stanley, W. M. : The Isolation and Properties of Tobacco Mosaic and Other "virus Proteins, The Harvev Lectures, 1937-38, Science, 1935, 81: 644; ibid. 1937, 87: 469. Topley, W. W. C, and Wilson, G. A.: The Principles of B.acteriology and Immunology, New York, 1929, Wm. Wood and Co. 2: p. 616. Williams, J. W. : Your Skin Resurfaces Your Body to Protect It From Infec- tion and Injury, J. Bact. 41: 73, 1941. Zinsser, H., and Bayne-Jones, S.: Textbook of Bacteriology, Ed. 8, New York, 1939, d". Appleton-Century Co., pp. 136, 351, 771, Zinsser, H., Enders, J. F., and Fothergill, L. D. : Immunity, Ed. 5, Mac- millan Co., 1939. CHAPTER II HOST-PARASITE RELATIONSHIP Introduction. — The environment of man teems with potential pathogenic agents. The soil frequently harbors ova of animal para- sites, spores of the gas bacillus and of CI. tetani, as well as vegeta- tive forms of staphylococci and other micro-organisms that survive well outside of the body. It is well known that the surface of the body is commonly contaminated. The skin and mucous mem- branes harbor streptococci and staphylococci in great numbers, while anaerobes, facultative aerobes, colon bacilli, streptococci, staphylococci and many other kinds of bacteria are present in enormous numbers in the contents of the intestinal tract. Rivers (1928, 1932, 1933) has even found a pathogenic virus on the skin of rabbits. These organisms have very little invasive power and are able to enter the body only through mechanical or chemical injury or as a result of altered physiological integrity of tlie mucous mem- branes or skin. Theobald Smith has designated such organisms as "opportunists." They are to be contrasted with the group of in- fectious agents producing contagious disease and also with the group of agents transmitted by insects. Portal of Entry. — The portal of entry into the body, as well as the route of exit, is quite important in determining infection of the host as well as the danger of spread of the infectious agent. When the portal of entry is through the alimentary canal and the exit is through the feces, as in typhoid fever, bacillary dysentery and asiatic cholera, then fingers, flics, food, feces and fomites play an important role in disseminating the infectious agents of these respective diseases. In measles and smallpox, where the virus nor- mally enters through the mucous membrane, it appears that infec- tious droplets are chiefly responsible for the spread of both diseases, although fluid from vesicular lesions may be a factor in the spread of smallpox. In diphtheria and scarlet fever, the primary infec- tion occurs in the upper respiratory tract and is spread by direct or indirect contact. 47 48 IMMUNOLOGY Endemics, Epidemics, Pandemics. — When a few cases of an infectious disease occur from year to year in one locality, even though this is quite large, the disease is said to be endemic in that region. When more than the usual number of cases develop in a community, the phenomenon is called epidemic. The disease may spread to adjacent communities and constitute a large epidemic. When it becomes world-wide in nature, it is called a pandemic. A small percentage of the population at large harbors pathogenic organisms known to cause specific infections. These individuals are called earners and are thought to be responsible for endemic dis- ease and for many epidemics. During pandemics, it has been ob- served that the various diseases spread along the lines of travel, but there seem to be other factors involved that arc at present unknown. Incubation Period. — The time interval between infection and the occurrence of symptoms is called the i^icuhation period. This is frequently of importance in the spread of infectious diseases since many are more contagious during the latter part of this period when the patient is unaware of the nature of his infection. The duration of this incubation period is quite variable. In diplitheria it is short, being from twenty-four to forty-eight hours, wliilc in typhoid fever, measles, and smallpox, it is commonly two or moic weeks. The converse of infection is freedom from disease. Large areas of population may be free from certain infectious diseases for reasons which may be illustrated as follows : 1. Environmental, Not Involving- Immunity Protection Due to Lack or Contact. — Euru|>can people did not suffer from syphilis prior to the return of Columbus' sailors from the West Indies where syphilis was i)revalent among the natives. That this freedom was due to lack of contact rather than to great morality or a high degree of immunity is evident by the virulence of the infections which developed and the high mortality that prevailed after it was introduced. It might be noted here that this is the general experience when a new disease is introduced into a population. HOST-PARASITE RELATIONSHIP 49 Measles was unknown in the Faroe Islands from 1781 to 1846, when it was introduced from Copenhagen. During the next year over 75 per cent of the population developed the disease. These two examples indicate that freedom from disease may occur in peoples who are extremely susceptible, merely because they enjoy the freedom from contact. Present quarantine laws have this as one of their objectives. The same line of reasoning is applied in hospitals where certain types of infections are isolated to prevent spread. REMOVAii OF Primary Host. — The attempt of the French to build the Nicaraguan Canal failed because of malaria and yellow fever. The U. S. Government was able to maintain a healthy per- sonnel of labor in the same environment by eliminating mosquitoes, the primary hosts for the infectious agents concerned, rather than by the use of immune individuals in the construction work. Such control may depend upon two possible factors: one is personal prophylaxis against contact with the primary host and the second is the limitation of the habitat of the same. It is obvious that in areas where the primary host cannot survive, a break in the chain of infection will protect the human population. The United States is practically free from bubonic plague. The Public Health Serv- ice has combated this disease by measures designed to eradicate the natural host or rodent rather than the insect carrier. During the World War delousing measures were employed extensively in order to prevent the spread of typhus fever and trench fever. Sanitary Measures. — Mills-Reincke Phenomenon. It has been observed that when a large city water supply which has l)een consistently polluted with sewage is purified by the introduction of filtration and treatment methods, the morbidity and mortality of diseases other than gastrointestinal in origin are noticeably re- duced. This is another example of protection by removal of causes l)y sanitary measures. It is quite generally conceded that our low morbidity rate for typhoid fever today is due largely to sanitary measures relative to water supplies, milk supplies, oyster industry, and also to food handlers, since only a small percentage of our population has typhoid fever or has been immunized. The corollary to this is the relatively high incidence of tyjilioid fever among children who are essentially the milk drinkers of the population. 50 IMMUNOLOGY 2. Racial Freedom from Disease May Be Due to Customs Rather Than Immunity Effect of Customs. — The Hebrew people do not suffer from trichinosis. Since trichina infection is obtained by eating infected or "measly" pork it is obvious that this is not an example of im- munity but that freedom is due to racial taboo of pork. Since cases have been reported among Hebrews who violated the taboo accidentally or intentionally, we have no reason to suspect im- munity. 3. Environmental Conditions: Absence or Presence of Factors Affecting- Immunity Mechanisms Climate Factors. — Dysentery is much less prevalent in the tem- perate and northern climates than in the tropics. It has been sug- gested that this illustrates the devitalizing effect of heat. It should not be assumed, however, that heat is the only factor. Lobar pneumonia and upper respiratory infections are said to be much less prevalent in the tropics than in the colder climates. The inference is made that this may be due to the effect of tempera- ture, humidity, and air currents on the mucous membranes, lower- ing their resistance. The so-called "fog pneumonia" in England and Belgium reported in 1930 is probably an example of this type. Many occupational diseases come under this group. 4. Resistance and Susceptibility Factors It seems quite evident from the experimental studies of Topley, Wilson, and Greenwood in England and Webster and others in America that in so far as mouse typhoid is concerned, the addition of fresh mice to an infected popuhition initiates an epidemic. Webster and Ilodos have shown that the mortality, during an epidemic, is conditioned by the number of highly susceptible con- stituents. When the latter are few in number the deaths will be sporadic; if the number of highly susceptibles is great the mor- tality will be of epidemic proportions; if the susceptibles are de- pleted the mortality subsides. When recruits are added to sucli surviving populations, made up chiefly of resistants, Webster and Ilodes find that mouse typhoid infection spreads to both resistant and susceptible recruits but that the mortality is limited to the susceptible recruits. The innately HOST-PARASlTE RELATIONSHIP 51 resistant recruits remain well unless subjected to conditions that would lower their innate resistance. There are several reasons why great caution and conservatism should be used in translating the results of experimental epi- demiology to human field conditions. These reasons may be sum- marized as follows: 1. There are many species differences between man and mice. While Webster and Hodes have apparently shown that there is no tendency for susceptible mice to become immunized through herd exposure, their experiments do not prove that susceptible humans will react in a like manner. 2. Man is a heterogeneous mixture as far as breeding is con- cerned while the mouse research has been done with inbred strains of known pure lines of mice whose susceptibility and resistance could be determined. 3. The experimental epidemics in mice were carried out under controlled conditions as to recruits, diet, temperature, humidity, etc. This is in great contrast to epidemic disease in man. Such epidemics occur spontaneously in a mixed popula- tion that exhibits great variation in age, history of previous disease, diet, health habits, natural fitness, occupation, previ- ous immunization, etc. The source of the infection, the num- ber of susceptibles, resistants, and carriers, as well as the num- ber and condition of new recruits, are usually unknoAvn. 4. In a mouse population there is no conscious effort on the part of individuals to protect themselves and others against infection such as occurs during epidemics in man. Since there is in every human population great variation in education, knowledge, wisdom, and prejudice, it is not surprising that co- operation of the public in applying what appears to be intelli- gent methods of control is not accomplished. In spite of these reasons for caution it would seem that experi- mental epidemiology is making major contributions to our knowl- edge of infection and host-resistance. In man the carrier state (both temporary and chronic) presents an interesting hast-parasite relationship that in many cases devel- ops into a public health problem. There is a great deal of data bearing upon the incidence of typhoid, diphtheria, meningococcus and pneumococcus carriers in a population but relatively little 52 IMMUNOLOGY information concerning the virulence of the organisms isolated from carriers. Hadfield and Garrod (1938) review the studies in the distribution of pneumococcus types in normal individuals re- ported by Webster and Hughes (1931), Gundel and Linden (1931). They say that the evidence indicates that the less virulent types attack the respiratory tract, set up mild infection, establish them- selves and remain in intimate relationship with the host for long periods of time. This is in contrast with the virulent types which iinist either produce an acute infection or be destroyed. Theobald Smith taught long ago that lymph glands are filters. 1^'ailure to realize that liacteria thus filtered out may survive for long periods of time within the lymph glands is probably tlie rea- son so many different kinds of bacteria have been described as the cause of Hodgkin's disease. The phenomenon of phagocytosis ob- served in various diseases is regarded by Smith as an expression of an affinity possessed by certain infectious agents and cell types of the host for each other. This subject is discussed more fully in Chapters IV, V, and XXVII. References Cinndel, M., and Linden, H. : Cited by Hadfield and Garrod, Pneumonia, Recent Advances in Pathology, P. Blakiston's Son and Co., Phila- delphia, 1938. Hadfield, G., and Garrod, L. P.: Pneumonia, Recent Advances in Pathology, P. Blakiston's Son and Co., Philadelphia, 1938. Rivers, T. M. : Filterable Viruses, Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, 1928, Univ. of Chicago Press, p. 517. Rivers, T. M. : The Nature of Viruses, Physiol. Rev. 12: 423, 1932. -Rivers, T. M. : Further Observations on the Cultivation of Vaccine Virus in Lifeless Media, .T. Exper. Med. 57: 741, 1933. Topley, W. W. C, and Wilson, G. S. : Principles of Bacteriology and Im- munity, Ed. 2, Baltimore, William Wood and Co., 1936. Webster, L, T. : Microbic Virulence and Host Susceptibility in Mouse Typhoid Infection, J. Exper. Med. 37: 231, 1923. Webster, L. T.: Microbic Virulence and Host Susceptibility in Paratyphoid- Enteritidis Infection of White Mice. IV. The Effect of Selective Breed- ing on Host Resistance, J. Ex-per. Med. 39: 879, 1924. Webster, L. T. : Microbic Virulence and Host Susceptibility in Paratyphoid- Enteritidis Infection of White Mice. VIII. The Effect of Selective Breeding on Host Resistance. Further Studies, J. Exper. Med. 42: 1, 1925. Webster, L. T. : Inherited and Acquired Factors in Resistance to Infection; Development of Resistant and Susceptible Lines of Mice Through Selec- tive Breeding, J. Exper. Med. 57: 793, 1933. — : Inherited and Acquired Factors in Resistance to Infection ; Comparison of Mice Inlierently Resistant or Susceptible to Bacillus Enteritidis Infection with Respect to Fertility, Weight, and Susceptibility to Various Routes and Types of Infection, Ibid., p. 819. HOST-PARASITE KKLATIONSHIP 53 Webster, L. T., and Burn, C. G. : Biology of Bacterium Lopiseptii'um. III. Physical, Cultural, and Growth Characteristics of Diffuse and Mucoid Types, and Their Variants, J. Exper. Med. 44: 343, 1026. Webster, L. T., and Burn, C. G.: Biology of Bacterium Lepisepticum. IV. Virulence of Diffuse Mucoid Types and Their Variants, J. Exper. Med. 44: 359, 1926. Webster, L. T., and Hodes, H. L. : The Role of Inborn Resistance Factors in Mouse Populations Infected with Bacillus Enteritidis, J. Exper. Med. 70: 193, 1939. Webster, L. T., and Hughes, T. P. : Epidemiology of Pneumococcus Infec- tion; Incidence and Spread of Pneumococci in Nasal Passages and Throats of Healthy Persons, J. Exper. Med. 53: 535, 1931. Webster, L. T., and Pritchett, I. W. : Microbic Virulence and Host Sus- ceptibility in Paratyphoid-Enteritidis Infection of White Mice. V. The Effect of Diet on Host Resistance, J. Exper. Med. 40: 397, 1924. CHAPTER III INFLAMMATION AND LEUCOCYTE RESPONSE Introduction. — lu regard to the phenomena of infection and its sequelae the older clinicians carefully describe inflammation as the reaction of the tissues to injury. Virchow and succeeding pathologists have reported upon the microscopical changes which occur. Experimental hematologists such as Ehrlich, Sabin, Doan and Cunningham, Maximow, Downiey, Bunting, Pappenheim, and others have introduced methods of staining, identifying and classi- fying the various types of cells ol)served in the blood, inflammatory exudates, and lesions and have attempted to determine their origin (Plates I and II). Arneth, Schilling, and more recently Haden have suggested that in acute infections the activity of the bone marrow (Plate 1,1) is reflected by the relative number of young forms of neutrophiles in the peripheral circulation. They believe that in acute infections a prognosis can be based upon indices or hemograms giving this information and accordingly suggest for- mulae for such indices and liemograms. Acute Inflammation. (1) Lobar Pneumonia. In acute lobar pneumonia, which is usually caused by the pneumococcus, there is an acute inflammation of at least one lobe of tlie lungs accom- panied by systemic disturbance. It is ushered in with a chill, fever, leucocytosis, rapid pulse, and rapid respiration. Obviously there is a disturbance of respiratory function. The inflammatory process in the lung is characterized by several stages. Like all acute inflammatory processes the first stage is one of liyperemia or increase in ])lood supply to the infected lobe. In pneumonia this is called the stage of engorgement. During this stage there results a change in permeability of the vessel walls, a slowing down of the rate of blood flow and a margination of leuco- cytes. This is ako common to all inflammatory processes. Leuco- cytes are attracted to the infected lungs. Fluid, leucocytes, and even some red cells pass through the vessel walls into the alveoli. Tlie second stage is ushered in by the consolidation of the lung due to the clotting, so to speak, of the inflammatory exudate. Tliis 54 INFLAMMATION AND LEUCOCYTE RESPONSE 5o is called the stage of red Juptitizatioh or liverlike ai)i)earaiice and consistency of the lung. The microscopic picture at this stage would show the alveolar walls thickened, the capillaries distended, the alveoli filled with coagulated albuminous fluid, leucocytes, mononuclear cells, bacteria, some red cells, and a great deal of fibrin. The next stage is one described as "gray hepatization." The red cells either lose their hemoglobin or are destroyed while the lung remains otherwise the same. The last stage is that of resolu- tion. Proteolytic enzymes of leucocytic origin (from leucocytes that are destroyed) liquefy the coagulated proteins and the prod- ucts are eliminated in the sputum and urine and the lung returns to normal. The bacteria have been phagocytized by neutrophiles, free mononuclear and fixed mononuclear cells. The phenomenon of crisis which is generally regarded as peculiar to lobar pneumonia in man has been experimentally duplicated by Goodner in his "dermal pneumonia" (intradermal infection) in rabbits. (2) Furuncle. — Another interesting example of acute pyogenic infection in man is the furuncle (boil) due to the staphylococcus. Infection usually occurs around a hair follicle. Here one observes hyperemia, exudation, the accumulation of fluid, leucocytes, red cells, fibrinogen, and bacteria in the area of inflammation. Coagula- tion of the protein occurs. There is a piling up of neutrophiles and mononuclear cells at the outer margin of the spherical zone of coagulation. The small blood vessels and lymphatics are injured and become plugged with thrombi and hyaline plugs, respectively, which tend to prevent the spread of the infection. In the furuncle the coagulated mass of cells dies. Here again proteolytic enzymes of leucocytic origin begin to liquefy the coagu- lated mass which in this case contains dead tivssue. Liquefaction begins at the outer margin and progresses toward the center. The liquefied tissue debris, dead and living leucocytes, bacteria, fibrin, etc., constitutes the pus that one sees while the unliquefied central portion is the "core" of the boil. The cavity thus formed is lined with a membranous-like material made up of fibrin, various types of white cells, tissue cells, etc., and is called the pyogenic membrane. It, with its phagocytes and the hyaline and fibrinous plugs in the lymphatics and blood vessels, acts as a protective barrier to the spread of infection. 56 IMMUNOLOGY Plate I. — Blood Cells (Fixed and Stained Preparations) 1. Section of bone marrow — guinea pig. Bml — Basophilic myelocyte. Nbl— Erytluoblast. Ere — Erythrocyte. Meg — Giant cell. Fc— Fat cell. He — Histiocyte. Ret — Reticular cell. Nml — Neutrophilic myelocyte. Eml — Eosinophilic myelocyte. 2. Erythrocyte. o. Blood platelets. 4. Large lymphocyte. 5. Small lymphocyte. 6. Monocyte. 7. Neutropliile (mature). S. Basophile. 9. Eosinophile. 10. Monocyte (showing azurophilic granules). Plate I, R &I00J ^.SVp explanation on opposite page.) INFLAMMATION AND LEUCOCYTE RESPONSE 57 Menkin, Opie, and others have shown that when one injects dyes, proteins, etc., either locally or intravenously the material is picked up and fixed by areas of inflammation. The final process in recov- ery is the replacement of dead tissue by connective tissue which through changes in the fibers and shrinkage forms scar tissue. It is obvious that if scar tissue forms in the walls of the esophagus, intestines, ureters or urethra, the lumen will be narrowed by a stric- ture and functional impairment will result. The numerous investi- gations relative to the cellular (phagocytic) defense of the body against streptococcus, staphylococcus, and other infectious agents which have been cited all point to the great importance of fixed and free mononuclear cells rather than the neutrophiles in combating the infection. Chronic Inflammation. — Formation of a Tubercle. — In tuber- cular and many chronic inflammations it has been noted that there may be very little hyperemia, local increase in temperature or exudation of fluid. Neutrophiles may appear early, but they are soon supplanted by mononuclear celLs. Pns may be formed as in the ''cold abscesses" of tuberculosis with little systemic disturb- ance. Maximow, as well as Sabin, Cunningham, and Doan, has given excellent descriptions of the formation of the tubercle under experi- mental conditions. When tubercle bacilli set up a primary infec- tion in tissue, there is an early appearance of neutrophiles, mono- nuclear cells, a few red cells, and some fluid. The first inflam- matory reaction subsides and the exudative process disappears. The mononuclear cells then increase in number and collect in a mass around the tubercle bacilli. The mononuclear cells, according to Maximow, gradually undergo transformation. Their cytoplasm becomes more abundant and acquires the capacity to stain with acid dyes. Near the center of the mass one or more "giant cells" appear. These are the giant cells of Langhans. They are described as "protoplasmic masses with the nuclei arranged in a peripheral ring or in an equatorial band. " It is Maximow 's opinion that they are formed by the fusion of the large mononuclear or epithelioid cells and that mitosis of the nuclei follows. Sabin et al. classify the mononuclear cells forming the tubercle as monocyte?. They did not observe fusion of monocytes to form 58 IMMUNOLOGY Plate II. — Cells in Pekitoneal Exudate ok \\'uite Rat. Supra-vital Preparation Using Neutral Red and Janus Green 1. Lymphocyte showing both neutral red granules and mitachondria. 2. Lymphocyte showing only mitachondria. 3. Clasmatocyte showing vacuoles of neutral red and mitachondria. 4. Monocyte showing the rosette and mitachondria. o. Clasmatocyte which has phagocytized a neutrophile (note tliat tlie nucleus of the neutrophile, being dead, stains witli neutral red). -s^. • A* .1 •• • • • ^mTi •«• *• ft # VI' *?# M R Blooci Plate II. ('iSVe explanation on opposite page.) INFLAMMATION AND LEUCOCYTE RESPONSE 59 ,<>iant cells but think tlicy arc formed by mitosis and increase in cytoplasm. There is no blood supply extending into the mass or tubercle. A mantle of granulation tissue due to connective tissue prolifera- tion forms about the mass of mononuclear cells forming the tubercle. Small mononuclear cells resembling lymphocytes tend to collect in and about the outer elements of tlie mantle of granulation tissue. Tubercle Phospflvtid and Cell Stimulation.- — Sabin and Doan (1927) report that Anderson's phosphatid fraction of the tubercle bacillus will cause ''decided proliferation of monocytes, epithelioid cells, and giant cells leading to a massive formation of tubercular tissue." Varieties of the Tubercle Bacillus. — Winn and Petroff* (1932) have obtained four interesting variants which they call S, F.S., R and Ch. dissociated from an avian tubercle bacillus W They state that these variants show different physical and chemical properties and elicit different tissue responses in the host. They summarize their results as follows : ''The leucocyte response in S and F.S. is of an acute type wiiile that produced by R and Ch. variants is indicative of a chronic, healing tuberculosis. ''The tubercle formed Iw S is of an acute toxic type, the F. S. more of a foreign body type and that of R and Ch. relatively benign. "The S variant is by far the most virulent and is closely fol- lowed by the F. S. type. The R and Ch. variants are comparatively avirulent." Dev-elopment of Hypersensitiveness. — Primary infection with the tubercle bacillus renders the animal hypersensitive to tuber- culin. This state of hypersensitiveness is responsible for the vio- lent inflammatory reaction around new and latent foci of infection which develops when reinfection with tubercle bacilli occurs or when a normally nontoxic dose of tuberculin is injected into an infected animal. It is responsible also for many systemic symp- toms which are known to be pathognomonic of tuberculosis. Tissue Response to Viruses. — In regard to virus disease one is dealing with infectious agents that enter the cells of the host and •Winn, W. A., and Petroff, S. A.: J. Expor. Med. 57: 239, 1932. 60 IMMUNOLOGY bring about certain changes which usually result in a secondary inflammation accompanied by mononuclear cell infiltration. Ac- cording to Rivers (1933) if the virus does not act rapidly and explosively and the host cells are capable of multiplication, the primary effect of infections is stimulation leading to cellular hyper- plasia. Following the latter there is usually necrosis and then a secondary inflammation. Rivers states that the balance between stimulative and destructive tendencies of the virus is the determin- ing factor between predominance of hyperplasia and necrosis re- spectively. He cites smallpox lesions as examples of stimulation followed by necrosis and liquefaction of cells, i.e., there is first a hyperplasia of cells followed by vesicle formation. As examples showing an overgrowth of tissue as the prominent feature he cites warts and certain tiunors. When the infected cell ''is incapable of dividing and multiplying as is the case of nerve cells, then the primary pathological changes arc necrobiosis and lysis of cells or the appearance within the cellular elements of inclusion bodies." This latter phenomenon is observed in rabies. Leucocyte Count. — It is. an interesting physiological fact that in a normal individual the red cells, white cells, and the platelets are maintained at a fairly constant level. Walters (1934) has shown that exercise and rest affect the concentration of red cells in the venous circulation. Doan (1927) and others have observed a rhythmical variation in the number of wliite cells. Mueller has shown that the vegetative nervous system exercises some control over the number and distribution of white cells. It is also of interest to note that differential counts indicate that there is to a great extent a constant percentage of each type of white cell in the peripheral circulation. Under normal conditions there are, on an average, approximately 5,000,000 red cells and 7,500 white cells per cubic millimeter of capillary blood. There are a few infectious diseases characterized by a leucopenia (low white count). Among these diseases are measles, malaria, influenza, typhoid fever, and tuberculosis. The differential count of a normal blood shows that there are approximately 60 to 70 per cent neutrophiles, 2 to 8 per cent monocytes, 25 to 30 per cent lymphocytes (large, intermediate and small), 1 to 3 per cent eosino- philcs, and from i/^ to 1 per cent basophiles. In the five infec- INFLAMMATION AND LEUCOCYTE RESPONSE 61 tious diseases just mentioned there are a relative increase in mono- nuclear cells and a relative decrease in granulocytes. In lobar pneumonia, meningitis, and severe septic infection the white count is usually 20,000 to 30,000 with a high percentage (over 90 per cent) of neutrophiles. Occasionally one finds a low total white count with a high percentage of neutrophiles in malig- nant septicemias. In appendicitis great variations are observed. One usually thinks that a patient with a "pus appendix" will have a white count of between 16,000 and 20,000 with 90 to 98 per cent neutrophiles. On the other hand, cases of catarrhal appendicitis usually show white count under 15,000 per cubic millimeter and from 80 to 85 per cent neutrophiles. Exceptions are frequently encountered, hence the surgeon must always regard the laboratory findings as of less importance than his clinical findings and the condition of the patient. The student should become familiar with other conditions (be- sides infection) that affect the white count. There is usually a Icucocytosis following hemorrhage of any consequence; and an increase in leucocytes is observed in pregnancy, during labor, and also during digestion and after cold baths. ' A leucocytosis is fre- quently observed when the patient is moribund and is prol)ably due to a terminal infection. In acute infections, Sondern suggests that the percentage of neutrophiles indicates the degree of toxic absorption while the number of leucocytes per cubic millimeter of capillary blood reflects the power of resistance of the patient. Wilson (1919) has devised a formula to express this information in numerical terms. The student should consult his original papers for a more extensive discussion of the subject. Todd and Sanford (1931, p. 293) incor- ])orate Wilson's formula in a short discussion of his work. Arneth AND Schilling Counts. — In an attempt to obtain addi- tional information that might indicate the severity of an infection and perhaps whether the ])rognosis is good or bad, Arneth and more recently Schilling (1929) and Haden have suggested the use of indices and hemograms 1)ased u])on tlie relative num])er of young and old neutrophiles in the peripheral circulation at different stages of infection. 62 IMMUNOLOGY According to Schilling the percentage of different kinds of white cells in normal blood is : Basophiles 0.5 per cent Eosinophiles 2-4.0 per cent Myelocytes 0.0 per cent Juveniles (Metamyelocytes) 0.0 per cent Stabs .3-5.0 per cent Segmented 51-67.0 per cent Lymphocytes 21-35.0 per cent Mononuclears 4-8.0 per cent The myelocytes and metamyelocytes (juveniles) are bone mar- row cells and give rise to the granulocytes of the peripheral circula- tion. Gradwohl has described the myelocytes, juveniles and stabs as follows: "Neutrophilic Myelocytes. — The cytoplasm of neutrophilic myelocytes varies from very pale blue in the younger stages to pink in the older forms. The nucleus varies in shape ; round, kid- ney-shaped, or oval. It may or may not contain one or more nucleoli, and is trabeculated. In leucemia the granules of the myelocytes are very delicate and difficult to stain. They are pur- ple and pinpoint in size. They are seen usually only here and there; at times they may be entirely absent. In the blood stream in severe infections, myelocytes usually have a pale blue cytoplasm filled with coarse neutrophilic (purple) toxic granulation. The myelocyte of leucemia has a fairly even periphery, while that of infections is fragile, and consequently irregular. "Juvenile Neutrophiles. — Juvenile neutrophiles are found normallj^ in the circulating blood in the percentage of 0 to 1 per 100 leucocytes. They are slightly larger than the mature neutro- philes. The cytoplasm varies from a bluish pink to a definite pink. It is much wider than that of the myelocytes. The nucleus is sausage-shaped to almost bean-shaped. It does not stain intensely, and shows little or no chromatin structure, although it is definitely divided into fields. It often contains one or more nucleoli, usually situated in the end bulbs, which are definitely protruding struc- tures. The granules are sometimes definite and sometimes very fine. When they are coarse, thej^ are strikingly responsive to stain ; when fine, they stain with difficulty. The same variations in .stain- ing and structure- may be noted in leucemia and infections as is seen in the mvelocA-tes. Thev are differentiated from 'stab' cells IXFLAMMATIOX AND LKI'COCVTE RKSrONSE 63 by the pale nucleus without chromatin, tlie nucleoli and the width of the nucleus. 'Stab' cells are older, and consequently stain more deeply, have no nucleoli, show dense chromatin structure, and have a fairly narrow nucleus. "When any reasonable doubt exists as to whether a cell has reached the 'stab' stage or is still a juvenile, Schilling suggests that it be called a 'stab.' " 'Stab' or Band or Kod Nuclear NEUTRorriiLic Cells. — The term 'stab' is a German word wliich refers to the ability of the rod-shaped nucleus to become bent or twisted. Since there is no liandy English equivalent, the German term has been retained. Numerous synonyms are found in the literature; such as, 'staff cells,' 'rod nuclears,' 'nonfilamentous forms,' 'band cells,' etc. 'Stab' cells are neutrophilic cells and are found in the normal cir- culating blood in the percentage of 3 to 5. The cytoplasm is pink or lavender. The nucleus is a definite flexible rod, which may be bent in smearing, so tliat it takes the form of the letters S, T, V, V, or ir. It does not show segmentation, although degeneration forms in which the nucleus is vacuolated or fringed may occur. Any cell which shows even slight segmentation is not considered a 'stab' cell. The niicleus contains definite chromatin structure, which must not be mistaken for nucleoli. It stains more intensely than the nucleus of the juvenile cell, and is slightly narrower. It does not contain nucleoli. "The granules are usually distinct, very fine, purple-staining stippling, distributed less regularly than the granules of the eosino- philes, but more regularly than those of the basophiles. Under conditions of severe infections, they are very coarse and clumped. If there is any reasonable doubt as to whether a cell is a 'stab' or a segmented form. Schilling suggests that it be called a segmented cell. The nucleus of the stab may undergo changes due to mechan- ical pressure in smearing. Unusual forms are to be looked for and recognized. "It is important to remember, too, that there are degenerative forms of the stab cells. The degenerative forms show small band forms of nuclei, irregular, hyperchromatic, pyknotic, and structure- less. They are easily broken up in making the preparation. There is tendency toward vacuolization and diminished resistance are a result of a toxic element or infection. /^ \wS, 64 IMMUNOLOGY The segmented forms constitute the major portion of the neutro- philes. The cytoplasm is mature and filled with neutrophilic granules. The nucleus consists of two to five unequal segments united by fine threads. A Schilling hemogram follows : HEMOGRAM* X •r. la u t-i W ;^ Ph m f o 2; o K o H ci4 p H >* t5 H c/} H < ■-< '"-' m •^ U 12; H C „ o w t>2 p w S M ,-1 1-1 rH ca o rj2 1-i W 1-3 p o w H H Eh w ;?; P K-l a o •< B >J S >< O 1-3 w S Eh o H g 5 O O w < o g o o O o o W ^ pa a s ►^ o w ? M ►H S &H <: Normal 5,000 to Otol 2 to 4 0 otol 3 to 5 51 to 67 21 to 35 4 to 8 limits 8,000 *After Schilling: The Blood Picture, The C. V. Mosby Co., p. 148. 'Per cent. From an inspection of the differential count recorded in the above hemogram it is obvious that the young forms of neutrophiles (stab forms) and their precursors are recorded to the left of the line separating the stab forms from the older or segmented neutro- philes. For various reasons Schilling has found it expedient to place the basophiles and eosinophiles on the left and the lympho- cytes and monocytes on the right. When a blood count is made on a patient suffering from an acute pyogenic infection, the percentages of the various types of cells are recorded in this form of hemogram. When the sum of the per- centages to the left or right of the dividing line between the stabs and segmented nuclears shows an increase above normal, it is re- corded as a shift to the left or right as the case may be. In acute sepsis, appendicitis, etc., there is a high total white count with a regenerative shift to the left. This is due to the appearance of myelocytes and juvenile forms in the peripheral circulation and also to an increase in stab forms. Schilling thinks that repeated counts are of great value in mak- ing a prognosis. A continued or increased shift to the left is re- garded as unfavorable, while a shift to right is indicative of a INFLAMMATION AND LEUCOCYTE RESPONSE 65 favorable prognosis. In two of the diseases, e.g., tuberculosis and typhoid fever, in which a leucopenia is commonly encountered, one may observe a degenerative shift to the left. Juvenile forms and myelocj'tes do not appear in the peripheral circulation, but there is an increase in the stab forms. Schilling interprets the blood pic- ture during the course of an acute infection according to three phases : Phase I. The neutrophile battle phase. Leucocyte count B E M J St. S L. Mono. Count 'increased 0 0 0 16 8 55.5 18 2.5 This phase characterized by neutrophilia with a severe regenera- tive nuclear shift. Lymphopenia, monopenia, and eosinopenia occur at the peak of the infection. Phase II. The phase of monocyte defense. Leucocyte count B E M J St. S L. Mono. High normal 0 2 0 0 7.5 58.5 15 17 Here one knows from the receding neutrophilia and the shift to the right, from the reappearing eosinophiles, the rising number of lymphocytes and the high number of monocytes that the critical terminal period of the infection has arrived when the patient puts up an energetic defense. Phase III. The phase of lymphocyte cure. Leucocyte count B E M J St. S L. Mono. High normal 0 7 0 0 4 .33.5 42.5 13 This stage is characterized by Ivmphocytosis, eosinophilia with no neutrophilic shift. Schilling says that the persistence of a high neutrophilic shift and high monocytosis with few eosinophiles indicates a condition of chronic infection. Leucopenia.* — An excellent discussion of the factors involved in leucopenia is given by La^vrence (1941). He lists the following five major mechanisms which may operate in the production of this condition : 1. Diminished production of leucocytes due to (1) simple in- hibition, (2) arrested maturation, (3) bone marrow aplasia and (4) infiltration of foreign cells into the bone marrow. *A recent paper of interest is by Olitzki. L., Avinery, S. H., and Bendersky, J. : The Leucopenic Action of Different Microorganisms and the Antileucopenic Immunity, J. Immunol. 41: 361, 1941. 66 IMMUNOLOGY 2. Excessive eliiniiialion of leucocytes through such normal chan- nels as the lungs, gastromtestinal tract, liver, or spleen or under pathological conditions when large numbers of cells are poured into an infected area such as empyema. 3. Excessive destruction of white cells due either to abnormality of the white cells or to leucotoxic substances in the blood. 4. Eedistribution of leucocytes in the vascular channels such as occurs from the intravenous injection of hydrophilic colloids. The peripheral leucopenia apparently results from a mobilization of the granulocytes in the internal organs. 5. Eedistribution of leucocytes in the body as a whole. He cites leucopenic phases of leucemia as an example of the breakdown of the mechanism responsible for the distribution of leucocytes be- tween the tissues and vascular channels. In discussing Lawrence's report Haden (1941) suggests that since leucopenia, from the clinical point of view, concerns the poly- morphonuclear cells almost entirely, the term granulopenia seems preferable. Haden also points out that the life of a white cell is probably not over four or five days and that therefore the normal demand for replacement is probably 5 to 10 billion new cells per day. In his opinion almost all clinical cases showing granulopenia belong to Lawrence's first group. Leucocytosis and Increased Capillary Permeability. — Any consideration of leucocytosis associated with inflammation should not only consider the phenomenon of increase in the number and kind of leucocytes but should also consider the phenomenon of cliemical attraction of the neutrophiles to the point of infection (positive chemotaxis) and their passage tlirough tlie vessel walls respectively. According to Menkin (1940) numerous investigators have found that the injection of nucleic acid into animals produces an initial leucopenia followed within a few hours by a leucocytosis. Splenectomy apparently favors the production of a leucocytosis without the initial leucopenic phase. It is reported that the Arneth or Schilling count is deflected (shifted) by the administration of irradiated ergosterol, gelatin, trypsin, nucleic acid, thyroxin, and colchicine. Moon* (1938) re- gards histamine or an H-substance as of importance in the produc- tion of a leucocytosis associated with inflammation. He found that the intravenous injection of 1 or 2 mg. of histamine into cats pro- *Moon, V. H. • Pathology and Mechanism of Anaphylaxis, Ann. Int. Med. 12: 205, 1938. INFLAMMATION AND LEUCOCYTE RESPONSE 67 duced a leueocytosis. Menkin (1940), however, reports tliat the intravenous injection of simihir or slightly higher amounts into dogs failed to alter appreciably the leucocyte count. In connection with his studies on inflammation, Menkin (1936, 1938, 1939, 1940) has investigated experimentally the various phe- nomena associated with phagocytosis mentioned earlier in this sec- tion. His results may be summarized as follows : 1. He has obtained a crystalline nitrogenous substance from in- flammatory exudates which increases capillary permeability and causes the migration of leucocytes through the vessel walls into the tissues. AYhile it exhibits chemotaxic properties it will not pro- duce a leueocytosis in either the dog or the rabbit. This substance is called "leucotaxine." 2. He has found a leiicocytods-promolinfi factor in inflammatory exudates that causes a rise in the level of leucocytes in dogs. It stimulates the bone marroAV causing a *' shift to the left" in the hemogram. Tliis factor is thermolabile (inactivated at 60° C.) and there is some evidence to indicate its being either a globulin or associated with the globulin fraction. 3. Menkin reports that histamine, adenosine, blood serum, sterile broth and cultures of Staphylococcus aureus are all ineffective in causing an increase of the level of leucocytes in the circulation. 4. The leucocyte-promoting factor causes a prompt leueocytosis whereas nucleic acid produces a delayed reaction. It is suggested that the student read a few of the papers bearing upon the Arneth and Schilling counts Avhich are included in the list of supplementary references. From a survey of the literature and some personal experience, one feels warranted in suggesting Ihat tlie student weigh carefully tlie claims of those authors who feel that any method of classification of white cells is infallible iu prognosis. While the Schilling count is often of value, it is also at times misleading. It is well to remember that even accurate lab- oratory findings must be interpreted by a trained, intelligent indi- vidual with considerable clinical experience. References Cunningham, R. S.. Sabiii, F. R., and Doan. C. A. : The Development of LeucocA"te.s, Lymphocytes and Monocytes from a Specific Stem Cell in Adult Tissue.". Carnesrie Inst. Contributions to Embryology 16: 229, 3925. 68 IMMUNOLOGY Cunningham, R. S.: On the Origin of Free Cells of Serous Exudates, Am. J. Physiol. 59: 1, 1922. Cunningham, R. S., Sabin, F. R., Suceyama, S., and Kendwall, J. A.: The Bole of the Monocyte in Tuberculosis, Bull. Johns Hopkins Hosp. 37: 231, 1925. Doan, A. A., and Zerf as, L. G. : Rhythmic Range of the White Blood Cells in Human, Pathological, and Leucopenic and Leucocytic States, with a Study of Thirty-two Human Bone Marrows, J. Exper. Med. 46: 511, 1927. Gradwohl, R. B. H. : Clinical Laboratory Methods and Diagnosis, St. Louis, 1938, The C. V. Mosby Co., p. 340. Haden, R. L. : Discussion of Leukopenia, J. A. M. A. 116: 483, ]941. Lawrence, J. S.: Leukopenia, J. A. M. A. 116: 478, 1941. Maximow, A. : The Role of the Non-granular Blood Leucocytes in the Forma- tion of the Tubercle, J. Infect? Dis. 37: 418, 1925. Maximow, A.: Relation of Blood Cells to Connective Tissue and Endothelium, Physiol. Rev. 4: 533, 1924, Maximow, A.: Development of Non-granular Leucocytes (Lymphocytes and Monocytes) Into Polyblasts (Macrophages) and Fibroblasts in Vitro, Proc. Soc. Exper. Biol, and Med. 24: 570, 1927. Maximow, A.: Morpliology of the Mesenchymal Reactions, Arcli. Patli. and Lab. Med. 4: 557, 1927. Menkin, Valy: Studies on Inflammation, J. Exper. Med. 53: 171, 179, 647, 1931. Menkin, V. : Studies in Inflammation. XII. Meclianism of Increased Capil- lary Permeability, A Critique of the Histamine Hypothesis, J. Exper. Med. 64: 485, 193G. Menkin, V.: Studies in Inflanuiiation. XIV. Isolation of tlie Factor Con- cerned with Increased Capillary Permeability in InjurA^, J. Exper. Med. 67: 129, 1938. Menkin, V.: Studies in Inflanunation. XVIII. On the Mechanism of Leuko- cytosis with Inflammation. Am. J. Path. 16: 13, 1940. Menkin, V. : A Note in tlie Differences Between Histamine and Leukotaxine, Proc. Soc. Exper. Biol, and Med. 40: 103, 1939. Mueller, E. F. : Evidence of Nervous Control of Leucocytic Activity by the Involuntary Nervous System, Arch. Int. Med. 37: 2(i8, 1926. Opie, E. L. : The Fate of Antigen (Protein) in an Animal Immunized Against It, J. Exper. Med. 39: 659, 1924. Opie, E. L.: Inflammation, Arch. Int. Med. 5: 541, 1910. Rivers, T. M. : Viruses in Relation to the Practice of Medicine, Pennsylvania M. J. 36: 489, April, 1933. Sabin, F. R. : Cellular Studies in Tuberculosis, Am. Rev. Tuberc. 25: 153, 1932; also Tubercle 13: 206, 19.32. Sabin, F. R., and Doan, C. A. : Tlie Relation of Monocytes and Clasmatocytes to Early Infection in Rabbits with Bovine Tubercle Bacilli, J. Exper. Med. 46: 627, 1927. Sabin, F. R., and Doan, C. A. : Biological Reactions in Rabbits to Protein and Phosphatide Fractions from Chemical Analysis of the Human Tubercle Bacilli, J. Exper. Med. 46: 645, 1927. Sclulling, v.: The Blood IMcture and Its Clinit-til Significance; Translated by R. B. H. Gradwold, St. Louis, 1929, Tlie C. V. Mosby Co. Todd, J. C, and Sanford, A. II.: Clinical Diagnosis by Laboratory Methods, Philadelphia and London, 1931, W. B. Saunders "Co., p. 293. Walters, O. S.: Normal Erthrocyte, Hemoglobin and Packed Cell Volume Standards in Young Men, J. Lab. and Clin. Med. 19: 851, 1934. INFLAMMATION AND LEUCOCYTE RESPONSE 69 Winn, W. A., and Petioff, S. A. : Biological Studies of the Tubercle Bacillus. II. A New Conception of the Pathology of Experimental Avian Tuber- culosis with Special Reference to the Disease Produced by Dissociated Variants, J. Exper. Med. 57: 239, 1933. Additional References Bredeck, J. F. : The Schilling Differential Count in Tuberculo.sis, Am. Rev. Tuberc. 20: 52, 1928. Bunting, C. H.: The Leucocyte, Physiol. Rev. 2: 505, 1929. Colbert, C. N. : The Arneth Count with Supravital Staining Under Living Blood Conditions, J. Lab. and Clin. Med. 10: 126, 1924. Doan, C. A.: The Neutropenic State, J. A. M. A. 99: 194, 1932. Fitzhugh, Thomas, .Jr.: The Age of the Leucocyte in Relation to Infection, J. Lab. and Clin. Med. 17: 975, 1932. Hunt, E., and Weiskotten, H. G. : Value of the Arneth Count in Determining the Age of Neutrophile Leucocytes (Rabbits), The Action of Benzol VIII, Am. J. Path. 6: 175, 1930. Medlar, E. M. : A Critical Studv of the Polvnuclear Count as Advocated by Schilling, J. Lab. and Clin. Med. 17: l(i9, 1931. Opie, E. L. : The Occurrence of Cells with Eosinophilic Granulations and Their Relation to Nutrition, Am. J. M. Sc, February, 1924. Piney, A. E. : Recent Advances in Hematology, Philadelphia, 1931, P. Blakis- ton's Son and Co., pp. 136, 161. Pinev, A. E. : The Significance of the Polynuclear and Schilling Leucocytic Count, Quart. J. Med. 22: 405, 1928. Pons, C, and Krumbhaar, E. B. : Extreme Neutrophilic Leucocytosis With a Note on a Simplified Arneth Count, J. Lab. and Clin. Med. 10: 123, 1924. Reznikoff, Paul : Immature White Blood Cell Counts in Infectious Diseases, J. A. M. A. 93: 963, 1927. Reznikoff, Paul : Wliite Blood Counts in Convalescence from Infectious Dis- eases, Am. J. M. Sc. 184: 167, 1932. Shaw, A. F. B. : The Diurnal Tides of the Leucocytes of Man, J. Path, and Bact. 30: 1, 1927. Weiss, A.: The Staff Count; Its Importance in Acute Infectious Disease, Arch. Int. Med. 48: 399, 1931. Weiss, A. : Newer Hematological Aspects of the Neutrophils in Infection, Am. J. M. Sc. 174: 45, 1927. Yaguda, A. : Studies on the Schilling Count in Appendicitis, J. Clin. Path. 1: .39, 1931. CHAPTER IV ANATOMICAL AND PHYSIOLOGICAL FACTORS IN INFECTION AND RESISTANCE OF THE INDIA^CDUAL When the opportunity occurs for an infectious agent to come into contact with the animal body, it is said that the animal is exposed to infection. The portal of entry, dosag-e, and virulence of the patliogen, as weU as the anatomical and phj'siological condition of the host are factors determining whether entrance is gained to the body and whether disease results. The significance of many of these factors has been described in the preceding pages. Attention has been called to the presence of myriads of potentially pathogenic micro-organisms present on the body surfaces awaiting an opportunity to invade the tissues. The first line of defense against invasion of the tissues is the epithelial coverings of the body and the various secretions which bathe them. The second line of defense consists of cells of the reticulo-endothelial system present in the skin or mucous mem- branes and in the lymph glands which drain these areas. When one considers the central nervous system, he finds that the choroid plexus seems to act as a barrier between the general circulation and the circulation of the brain and cord. The skin, with its outer insoluble, keratinized epidermis, its multi- ple layers of stratified squamous epithelium, and rich capillary blood supply, offers a substantial barrier to bacterial invasion. Williams (1941) says that the nature and low^ pH of the outer layer discourage growth and invasion of bacteria. In his opinion the resurfacing mechanism of the body is important in defense. While tlie mucous membrane of the buccal cavity, as well as those of the nose and throat are knoAvn to harbor a rich bacterial flora, they are protected to a certain extent from contact with bac- teria by a film of mucus. Secretions of the submaxillary and parotid glands enter the buccal cavity through their respective ducts while secretions from the bronchi and trachea are carried upward to the mouth by cilia of the trachea. The phj^siologic vigor and integrity of the mucous membrane are dependent, to a large 70 ANATOMICAL AND PHYSIOLOGICAL TACTORS 71 extent, upon the rich blood and nerve supply with which it is endowed. The coughing- rotlex, depending as it does upon a nerv- ous mechanism, aids at times in the elimination of contaminated material. Bloomfield* (1922) suggests that there are six factors of })ossiblc importance in the elimination of bacteria from the respiratory tract. These factors he enumerates as follows: "(a) anatomic conditions; (b) the flushing mechanisms; (c) 1)actericidal action of the secretions; (d) reactions of secretions: (e) the antagonistic action of the indigenous flora toward invaders; and (f) phago- cytosis." He calls attention to the importance of the swallowing reflex as a protective mechanism and cites experiments showing that the tonsils are subjected to very little exposure to bacteria con- tained in the food or fluids entering the mouth. On the other hand, air currents entering the nose impinge imme- diately upon the mucous membranes. A large number of bacteria present in dust particles or in infectious droplets are arrested very near the anterior nares. Those that succeed in passing this bar- rier are caught in the mucous film covering the nasal membranes and swept toward the pliarynx and esophagus by the cilia of the epithelium. Located in the upper respiratory tract is also the tonsillar ring consisting of the faucial tonsils, lingual tonsils, and adenoid tissue. The latter is located in the dome of the nasophar^Tix and is struc- turally lymphoid tissue. Since the tonsils contain many crypts in which bacteria may find lodgment, it is not surprising that they are frequently the site of acute or clu^onic inflammation. Like all lymphoid tissue they contain along with lymphocytes other white cells that are phagocytic. Directly associated witli the upper respiratory tract and com- municating with it are certain otlier structures, for the most part lined with the same kind of pseudostratified epithelium as that covering the nose. These sti-uctures include the ethmoid, sphenoid, frontal, and maxillary sinuses, as well as the middle ear and con- junctiva. The lacrimal secretions are mildly germicidal and also aid in the mechanical removal of bacteria. Should bacteria enter the sinuses they would become entangled in the mucous film which is propelled to the opening of the ducts •Bloomfield, A. L. : Am. J. M. Sc. 104: S.^4, 1922. 72 IMMUNOLOGY by cilia of the epithelium. Undoubtedly the mucous secretions, drainage, rich blood supply, and type of epithelium together with certain phagocytic cells constitute the defensive mechanism of this region. It should be apparent that any interference with the drainage of the sinuses would impair an important defensive mechanism. The paralysis of the cilia by improper treatment or the occlusion of the ducts by swelling of the nasal mucous mem- brane or occlusion by a deviated nasal septum can impair sinus drainage. This region of the paranasal sinuses and upper lip is called the "danger area" because the veins draining into the blood sinuses of the head are for the most part without valves and the spread of infection to the blood sinuses would mean the development of meningitis. The proximity of the middle ear to both the mastoid and the lateral sinuses is exceedingly important in the spread of infections. Since the roots of the upper teeth (Fig. 1) are in close proximity to the floors of the maxillary sinuses, apical infections may lead to sinus involvement. Mechanisms of Infections. — Just how infectious agents are able to penetrate surface barriers is not always clear. Several pos- sibilities are usually suggested as explanations. It is obvious that chemical injury or trauma (mechanical injury) will permit en- trance. Micro-organisms might be taken up by leucocytes present on the surface and these leucocytes might enter the tissues by diapedesis (somewhat as a drop of mercury passes through gela- tin). Yoffey and Sullivan (1939) describe the fixation of vaccine virus by the lymphocytes and the spread of the virus via the cervical lymph ducts to the blood stream. If bacteria are able to kill the leucocyte after entering or they escape through disengorgement, it Avould seem logical to assume that the bacteria might be able to grow and develop once they get established. A third possibility is that bacteria may colonize upon the surface and produce substances that kill the epithelial cells adjacent and thus directly find themselves in contact with under- lying structures. Such a condition can be readily visualized as occurring when pathogenic organisms are sealed off within a tonsillar crypt or a hair follicle. Undoubtedly the bacterial protein or the products of bacterial growth frequently affect the perme- ability of the cell membrane. ANATOMICAL AND PHYSIOLOGICAL FACTORS 73 Mudd (1924) suggests that perhaps an eleetrocapillaiy mech- anism plays a role in the penetration of the epithelium by virulent bacteria. That physiological factors of the host are of paramount importance in infectious processes is evident from the fact that in insulin deficiency, such as occurs in diabetes, a state of acidosis mav occur and coincident with it the surface resistance against Fig-. 1. — Sagittal section of head to show spread of suppuration from infected teeth ; and also location of retropharyngeal abscesses. (After Eisendrath, Sur- gical Diagnosis, p. 128, by permission of W. B. Saunders Company.) SA, Subperiosteal abscess of upper jaw. lA, Subperiosteal abscess of lower jaw. LA, Infection in submaxillary subcutaneous tissue. EA, Infection around roots of bicuspid and molar teeth. RA, Retropharyngeal abscesses. "opportunists" is lowered as evidenced by the common occurrence of sinus infection and skin infections (boils and carbuncles) in these patients. Likewise frequent attacks of rhinitis are common occurrences in patients suffering from tuberculosis or other debilitating diseases. 74 IMMUNOLOGY EsTABiJSHMENT WiTiiiN TiiE TissuES. — Aftci' the iiivasioii of the bodj^ surface (in trauma), it is necessary for the organisms to establish themselves preparatory to dissemination elsewhere in the body. Just how the pathogen establishes itself is un- known. Wherry (1927) thinks that the bacteria or their prod- ucts of growth cause hydration of the tissue and that this en- ables them to grow and multiply. He calls attention to the fact that there are found in filtrates of certain bacteria (e.g., C. diphtheriae, Str. scarlatinac, Str. erysipilatis, CI. ivelchii, etc.) substances which produce local congestion and edema (water in the tissues). He suggests that the edematogenic substances may be amines. His experimental approach is exceedingly interesting and suggests that in the liydration of proteins the bacteria may have a mechanism that enables them to grow and multiply. That the mere production of edema is not always sufficient is suggested by the lack of serious infection in many cases of extreme edema of the lower extremities in women where the edema is caused by the mechanical pressure of a large ovarian cyst in the abdomen. This does not invalidate Wherry's conclusions that the hydration of tissues by infectious agents aids in their groM^th and development. There are certain kinds of infectious agents such as the viruses, rickettesia, and B. leprae that are intracellular parasites and re- quire the presence of susceptible cells in which to multipl.y. Theo- bald Smith (1933), and Goodpasture and Anderson (1937) have emphasized the importance of hast-cell-parasite relationship in cer- tain disea.ses. In their studies of experimental infections of the chorio-allantoic membrane of chick embryos, Goodpasture and Anderson (1937) found that many pathogenic bacteria find either epithelial or mesodermal cells are both favorable and perliaps nece.ssary media for the invasions of the living host. Their work will be discussed more extensively in Chapter VII. Routes of Dissemination. — After infection is established there are certain avenues of spread or routes of dissemination of the infectious agent to be considered. The four avenues commonly mentioned are surface spread, dissemination by way of the lym- phatics (lympJiogenous) , blood stream {liematogenous) and direct extension to adjacent tissues. The first of these can be illustrated by infection of the nasopharynx. The infection may extend down- ward over the mucous membrane to the tonsil, trachea, and lungs ANATOMICAL AND PHYSIOLOGICAL FACTORS /O or be forced through the Eustachian tube (auditory canal) to the middle ear or to any of the adjacent stiuctures covered by exten- sion of the mucous membrane of the nose and throat. Lymphogenous Extension. — All of the structures just men- tioned, as well as all of the tissues of the body, are extensively supplied with lymphatics which drain into regional lymph glands (see Figs. 2, 3, and 4). The spread of vaccine virus by the lym- phatics described by Yoffey and Sullivan (1939) has been men- Fig. '2. — I'ortals of infection and the most frequent nodes involved in tubei"- tiilo.sis of the cervical lymph nodes. (After Ki.sendrath, Surgical Diagnosis, V>. 171, by permission of Sv. B. Saunders Company.) M, Uppermost node along internal jugular vein. T, Tonsillar gland. S, Sub- maxillary nodes. V, Internal jugular vein. C-P-J, and other lymph nodes in cei'vical region. tioned earlier in this chapter. Infections of the tonsil drain into the tonsillar gland ; infections of the floor of tlie mouth drain into the submental glands; from the adenoids into both the tonsillar gland and the internal jugular group, the latter also receiving lymphatic drainage from the ear. These various lymph glands communicate with others in the cervical region and in some cases 76 IMMUNOLOGY with the supraclavicular group. It is commonly taught that lym- phatic drainage in the lung is from the periphery to the hilus, al- though Neff (1933) has described a number of cases of central pneumonia in children where extension from the hilus toward the periphery occurred. Winternitz (1920) definitely established that Pyjimonafy Pleird fv/iscecal) dnd o-f Luntf Polrnondt'Y Mo- Hilus 4^ 4^ Inf ei-ior TrdcVieo- broncViial Nodes Lateral TracV^eo broncViial Nocles -> Suprd- clavicuiar tSodes Broncho medi a sfindl Trunk Fig. 3. — Pulmonary pleural, and tracheal lymphatic drainage. Peri pViery of LunjJ Pig. 4. — Drainage of periphery of lung. infection of the upper respiratory tract may extend down the lymphatics of the tracheal wall and lead to acute pulmonary in- flammation called pneumonia. Hematogenous Extension.— An excellent example of hematog- enous spread of infection is seen in miliary tuberculosis. Here some focus of infection ruptures into the blood stream and the ANATOMICAL, AND t»HYSIOLOGiCAL FACTORS 77 organisms are showered throughout various tissues. In endo- carditis and also in cases of thrombosis associated with infection, mycotic emboli are occasionally broken off and carried by the blood stream to other parts of the body. The new foci of infection are called metastatic foci. Direct Extension. — This method of spread of infection is seen in gas gangrene where the process extends from one muscle cell to another. While in abscess formation the initial infection is fre- quently of metastatic origin yet the enlargement of the abscess is due largely to direct extension. Because fascia and tendon sheaths act as barriers to direct exten- sion through them, abscesses take the course of least resistance and spread along fascial planes and tendon sheaths. Direct extension of a retropharyngeal abscess along the prevertebral fascia and of the spread of infection by direct extension in the submaxillary sub- cutaneous tissue is illustrated in Fig. 1. Factors Involved in Pulmonary Infection. — It is obvious that infection of the lungs may result from aspiration of contaminated food material such as raw milk down the trachea, by extension along the surface or within the walls of the trachea, through the lymphatics from the cervical region, through the blood stream, from contact of the pleura with an infected pericardium or dia- phragm, by direct trauma such as occurs in chest injuries or by direct extension of infection from the chest wall as might occur in an infected malignancy of the breast that had eroded into the pleural cavity. Small abscesses of the lung, located at the surface, may rupture into the pleural cavity and give rise to empyema or pus in the pleural cavity. It should be remembered that destruc- tion of surface barriers by malignant growths offers excellent por- tals of entry for infection. Lobar Pneumonia. — Hadfield and Garrod (1938) give an excel- lent summary of important research bearing upon lobar pneumonia. They state that Blake and Cecil (1920) definitely settled the route of infection as being via the respiratory tract and not by hematog- enous or lymphogenous routes. Their results and those of Still- man (1930) make it seem improbable that a downward spread on the mucous membrane occurs but that instead the organisms may be carried in by small inhaled droplets which penetrate the lung deeply before coming to pest during inspiration. Hadfield and 78 IMMUNOLOGY Garrod call attention to the accumulation of evidence showing tliat individuals developing lobar pneumonia liave a pre-existing hu- moral immunity and perhaps an allergy for the pneumocoecus. This may seem paradoxical but would fit in with the allergic hypothesis. The conclusion of Blake and Cecil that the primary focus occurs at the hilus of the lung and that the infection spreads from the pri- mary focus outward along the perivascular and peribronchial lym- phatics has been challenged by Loescheke (1931), Terrell, Robert- son, and Coggeshall (1933) and Gunn and Nungester (1936). The newer work indicates that the primary focus is usually near the periphery of the lung and that the infection spreads from the periphery tow^ard the hilus in a wave of edema fluid that is swarm- ing witli pneumoeocci and later invaded by leucocytes. Appar- ently in the dermal pneumonia, as described by Goodner, edema fluid gravitates down the flank of the ral)bit. In producing experimental pneumonia, Gunn and Nungester .suspended the pneumoeocci in mucin for intrabronehial injection. Tlie mucin provides a nidus in wliicli the pneumoeocci can begin to multiply. The mucin holds them together and protects them from phagocytes. These experimental results fit in ^vith one concept of lobar pneumonia which postulates the existence of a nidus in which the pneumoeocci can begin to multiply. Lindau suggests that such a nidus consists of accumulated secretion which may be caused by chilling of the body or other factors. It has l)een shown experi- mentally that the application of cold to the skin causes hyperemia of the respiratory tract and also increased mucous secretion. Since juieumonia occurs quite frequently after operation on the upper abdomen, it is suggested by Coryllos and Birnbaum (1928) that a plug of mucous secretion in a bronchus is the nidus that aids the bacteria to initiate the pneumonia. Factors Involved in Intestinal Infection. — Infections in the nose and tln-oat may extend not only to the lungs, but also the sw^allow- ing of pus, whether it be coughed up from the lungs or come as a discharge from a sinus, etc., may lead to infections of the intes- tinal tract. This occurs quite frequently in tuberculosis where tuberculous sputum is swallowed. As material passes from the region of the tonsillar ring along the intestinal tract, it is interesting to note that it encounters an- ANATOMICAL AND PI 1 VSIULUCilCAL rACTOKS 79 other ring oi" lymphoid tissue near the ileocecal valve. Lymplioid tissue is scattered along- the mucoiLs membrane of the entire intes- tinal ti-act, but opposite tlie attachment of the mesentery and at the lower end of the ileum arc rather extensive areas of it calh^d •'Peyer's patches." Peyer's patches are commonly the site of ex- tensive ulceration in typlioid fever and in intestinal tuberculosis as well as in ileocolitis. Ik'Iow the ileocecal valve is a lymphoid vestigial oi-gan, the vermiform appendix. Ulceration of Intestixe. — In some cases of tularcmui, (((frfuudo- cytic anc/hta. and in melioidosis, there occurs extensive ulceration of the entire alimentary canal. Davis (1928) has reviewed his own work and that of others on the lymphatics of the respiratory and intestinal ti-act. He observed extensive infiltration of plasma cells in the upper respiratory mucous membrane under the epi- thelium of the crypts and along strands of connective tissue. In- filtrations of similar cells were observed by Aschoff in his studies of the appendix. Davis seems to feel that these plasma cells are indicative of either chronic or at least frequent inflannnation. CTOodpasture's (1937) work suggests that the young plasma cell may function as a host cell for certain bacteria. The walls of the intestine may become infected directly through the mucous membrane by specific pathogenic organisms such as E. typhosa or the tubercle bacillus, or the infection may be due to the pyogenic organisms present, and the portal of entry may be through erosions from various causes as suggested by Ivy (1920, 1925). The infections may be hematogenous or lympliogenous in origin. Malignancy also is responsible for secondary infections. Direct extension from an abscess existing in some organ or mass to whicli the intestine has become attached by inflammatory adhe- sions may lead to invasion from the peritoneal side. Ulcer, Pancreatitis, Cholecystitis. — The anatomical relation- ships near the pylorus of the stomach are quite important since ulcer commonly involves the cap or the walls of the duodenum just below the ca]\ The presence of ulcer interferes with the normal functioning of tlie gastrointestinal tract in various ways, is a source of mild or severe hemorrhage and is a defect in the intestinal wall that may lead to perforation and peritonitis. It should be remem- bered that the secretions from the liver and pancreas enter the duo- denum as a rule through the ampulla of Vater. A stone lodging in 80 IMMUNOLOGY the latter may traumatize the tissue and infection result. Opie, many years ago, showed that acute pancreatitis (inflammation of the pan- creas) may result from mechanical blocking, infection and the forc- ing of bile up into the pancreas. Inflammation of the gall bladder is called cholecystitis. This is usually due to extension of infection from some focus in the body and may in some instances result in multiple abscesses of the liver. A knowledge of the regional lymphatics and of the anastomoses of tlie portal circulation is of considerable value in visualizing the possible complications of infections of tissue drained by them. Appendicitis. — Inflammation of the appendix is of rather fre- quent occurrence. While theoretically the route of infection may be hematogenous or lymphogenous, an examination of sections from inflamed appendices suggests that the infection frequently starts from injury to the mucosa. Fecaliths and small foreign bodies are found quite frequently and there are numerous reports of finding animal parasites within the lumen. Since the appendix is a blind vestigial organ, stasis of fecal material within it must be quite common. Thus one can see there is abundant opportunity for both mechanical and chemical injury to the mucosa while the feces in the region of the caecum is notoriously rich in bacteria. There is also the added possibility that vasomotor disturbances might cause vascular changes in the wall of the appendix and to some extent lower the resistance of the tissue. The complication most feared by the physician is the rupture of an infected appendix. This permits pus from the appendix as well as fecal material with its rich bacterial flora to enter the peritoneal cavity and cause peritonitis. INIeasures usually em- ployed to avoid such a catastrophe are surgical interference or bed rest with symptomatic treatm.ent designed to keep the intestine at rest and to get the patient ready for operation if such is decided upon. By the term ''get the patient ready for operation" is meant to employ measures that will tend to restore his physiological mechanisms to as near normal as possible. If he is dehydrated, he will be given fluids; if anemic, he might be transfused, etc. Importance of Blood Supply at Lower Orifices. — At the lower end of the intestinal tract and also in the genitourinary tract there is a rich blood supply that is an important factor in defense against infection since trauma is not uncommon, and infectious ANATOMICAL AND PHYSIOLOGICAL FACTORS 81 agents are numerous. Abscesses in the region of the rectum do occur and may be exceedingly dangerous. Factors in Genitourinary Tract Infections. — The female genito- urinary tract is not uncommonly the seat of infections. These may be exogenous (from without), hematogenous, lymphogenous, or by direct extension. The cervix of the uterus and the vaginal walls are in contact Avith an extensive bacterial flora. The reaction of the secretions from the cervix and vagina may be irritating and the opportunity for fecal contamination is ever present. Dur- ing childbirth the patient becomes exceedingly fatigued, there is always blood loss, the resistance of the tis.sues of the uterine walls and cervix is markedly lowered and the latter frequently lacerated. Thus there are many predisposing factors for infection in such conditions. Infection of the uterus may originate, and frequently does, from some focus within the body carried to the uterus by the blood stream or lymphatics. Septic infection that complicates or follows delivery is called puerperal sepsis. Oliver Wendell Plolmes called attention to its spread by the hands of the physician who goes from a case of septic infection to care for a woman in labor. Not infrequently one observes uterine infection in women who have attempted abortion. Such infections lead to pelvic peritonitis, general peritonitis or septicemia. Cystitis, or inflammation of the bladder, is said to be more common in girl babies than in males of the same age. Since the colon bacillus is found frequently associated with this condition and since the female urethra is shorter than that of the male and commonly contaminated with fecal material in the diapers, it has been suggested that these factors account for the more frequent occurrence of cystitis in the female babies. The development of cystitis in older age groups is commonly associated with conditions that prevent the normal, free emptying of the bladder. This may be due to a cystoeele in the female or to a hypertropliied prostate in the male or to tumor masses in either. Cabot says that the so-called "catheter cystitis^ ^ is always caused by urinary retention with resulting congestion and edema of the bladder mucous membrane and the preparation of a "soiV for bacterial growth and development. In his opinion, proper catheterization prevents infection. 82 IMMUNOLOGY Pyelonephritis. — In a like manner anytliing which prevents the normal function of tlie ureters as conductors of urine from the kidney to the bladder will result sooner or later in damming back the urine into the pelvis of the kidney and tlie development of a pyelonephriiis. This may be unilateral or biUiteral. When the latter occurs, uremic complications are possi))le. Cord changes such as are seen in multiple sclerosis and certain types of syphilis can cause paralysis of both ureters. Stone in the ureter or kinks due to any one of many causes are commonly responsible for ureteral insufficiency and the prep- aration of a proper "soil" for tlie growtli of infectious agents. These latter may come from the blood stream through the kidney or by some other route. Acute Nephritis. — Acute infections of tlie upper respiratory tract are sometimes followed by inflammation of the glomeruli of the kidney (acute glomerular nephritis) as well as infections of the heart or other tissues of the body. It is obviously quite serious for the genitourinary system, heart, central nervous system, or lungs to be impaired by acute infections. In fact, there are many delicate mechanisms that are of great importance in enabling the organism, as a whole, to maintain life in the presence of severe infections. Importance of Elimination. — Clinical experience has shown the great importance and imperative necessity of maintaining elimination by kidney, bowel, skin, and lung. It lias also shown that confined pus disturbs the pliysiological balances in the body and that drainage or removal of such products of suppuration is necessary for recovery. Mechanical Factors of Safety.— Mel tzer (1906-07) called atten- tion to many mechanical factors of safety in the animal organism. A large amount of lung, liver, and kidney may be rendered func- tionless or destroyed by infection or the spleen may be entirely removed without fatal termination. Undoubtedly there are many more islands of Langerhans present in the pancreas than are needed for adequate insulin production. There is also more thyroid, ovarian, testicular, or adrenal tissue than is necessary for minimum requirements of the individual. Physiological Mechanisms. — Some of the physiological mecha- nisms that are important in resistance to infection are those .\\AT():\ric.\i; AND piivsior.odiCAr, iwctors 83 regulating the body temperature, production and distribution of blood, and the nieclianisms regulating the acid-base and water balance. Locke (1937, 1939) has worked out indices of fitness to resist infection of the respiratory tract based upon the relative efficiency of the temperature control mechanism in rabbits and of the capac- ity of oxygen replacement during exercise in man. He defines fit- ness as ability to support a forced performance of work at an effective rate of speed. In his opinion lack of fitness is a predis- posing factor in such infections as pneumonia and the common cold. It is quite evident that both the central and vegetative nervous systems are of considerable importance in immunological phe- nomena since functional change is dependent upon nervous as well as hormonal and direct stimulation. Pain of itself may lower resistance through resulting fatigue of the patient or on the other hand may protect a patient through forcing the immo- bilization of jiart or all of the body, decent investigations involv- ing the use of diathermy and also of llie malarial parasite in the treatment of paresis suggest that fever favors phagocytosis of spirochetes by cells of the reticulo-endothelial system. The use of hot wet dressings is still one of the important measures employed in the treatment of aciite septic infections. When such an infection occurs upon an extremity, the physician shows his interest in the local blood supply and buffer mechanism of the blood by elevating the extremity, thus permitting of removal of venous blood by gravity and at the same time favoring an increased arterial supply. The latter supplies oxygen, leucocytes, antibodies, and an excollent group of buffer substances consisting of hemoglobin, the serum proteins, and alkalies. The physiologist considers hemoglobin tlie most important buffer of all. When the physician examines a patient suffering from severe septic infection, he makes a note of whether the skin shows normal turgor or whether there is evidence of dehydration, and he fre- quently determines tlie COo combining power of the patient's blood to ascertain the condition of the alkali reserve. Both dehydration and lessened alkali reserve favor infection and are combated by the administration of fluids and alkalies or some substitute that helps restore the normal alkali reserve. The state of lowered alkali reserve is spoken of as acidosis wliile the presence 84 iMMUNOLOGt of an excess of alkalies is called alkalosis. Neither is desirable and hence both are to be avoided. Finally it should be recalled that the liver, in addition to other things, is quite potent as a detoxifier of proteins ; the spleen and lymph glands are efficient filters for pathogenic bacteria because they form part of the reticulo-endothelial system. The bone marrow supplies red cells and white cells, the former endowed with a potent oxygen carrier that is also a good buffer and the white cells are phagocytic in nature. The vascular system and the vasomotor mechani.sms aid in maintaining a normal distribu- tion of blood, and tliese and many otlier factors help maintain the noi'mal integrity of every tissue coll in the body. Prenatal vs. Postnatal Immunity.^ — The importance of normal physiological factors in resistance is empliasizcd by the discovery of Woolpert and his associates (1938, 1940, 1941) that fetal susceptibility of guinea pigs to influenza virus is changed at birth to resistance. This suggests that in some unknown way the immunity is associated with postnatal circulatory and respiratory readjustments. Importance of Considering the Body as a Whole. — Since the cells of tlie body are protoplasmic in nature and since protoplasm is an intricate mass of dynamic colloidal SA^stems, it is very essential to consider the "body as a whole" in attempting to understand infection and resistance. It would seem obvious that while circulating antibodies are of value in the body's defense against infection, except for antitoxins their role and relative importance have been considerably exaggerated. In diagnosis, however, they have won for themselves a place of great importance. Underlying Principle of Therapy. — When a patient with a beginning pyogenic infection is treated by a physician, the idea behind the therapy is to maintain the local tissue mechanisms of defense at an optimum and thus encourage localization or prevent extension by any of the possible routes of dissemination. In the case of pelvic peritonitis the head of the bed may be elevated, thus enlisting the forces of gravity while other measures are being instituted. Generalized peritonitis is exceedingly dangerous owing to the large area of peritoneal surface through which absorption of toxic substances and infectious agents occurs and also because of the danger of bowel paralysis and complete suppression of urine ANATOMICAL AND PHYSIOLOGICAL FACTORS 85 that may result. The i)hysi('ian Tavoi-.s the body's defense hy measures designed to prevent the l)ody temperature from going either too high or too low, establishing drainage, encouraging- elimination of toxic substances through natural channels, main- taining an adequate water balance, blood volume and alkali reserve, since these are essential physiological mechanisms of defense. A quiet environment is also necessary since psychic stimulation may lead to expenditures of energy that is needed for recovery. References Adami, J. G.: Inflammation, New York, 1899, The Macmillan Co. Blake, F. G., and Cecil, R. L. : Studies on Experimental Pneumonia ; Produc- tion of Pneumococcus Lobar Pneumonia in Monkeys, J. Exper. Med. 31: 403, 1920. Pathology and Pathogenesis of Pneumococcus Lobar Pneumonia in Monkeys, J. Exper. Med. 31: 445, 1920. Bloomfield, A. L. : The Mechanism of the Elimination of Bacteria From the Respiratory Tract, Am. .J. M. Sc. 164: 854, 1922. Cannon, W. B.: The Wisdom of tlio Body, New York, 1932, W. W. Norton and Co. Coryllos, P. H., and Birnbaum, G. L. : Cited by Hadfield and Garrod. Councilman, W. T. : Changes in the Lymphoid Tissue in Certain of the Infec- tious Diseases, Harvey Lectures. 1906-07, Philadelphia, 1908, J. B. Lip- pincott Co., Vol. 2: pp. 2(58-287. Davis, D. J. : Bacteria of the Respiratory Tract, Newer Knowledge of Bac- teriology and Immunology, Jordan and Falk, 1928, University of Chi- cago Press, p. 650. Dettwiler, H. A., Hudson, N. P., and Woolpert, O. C. : The Comparative Sus- ceptibility of Fetal and Postnatal Guinea Pigs to the Virus of Epidemic Influenza, J. Exper. Med. 72: ()23, 1940. Eisendrath, D. N. : Surgical Diagnosis, Philadelphia, 1909, W. B. Saunders Co. Goodner, K. : The Development and Localization of the Dermal Pneumococcic Lesion in the Rabbit, J. Exper. Med. 54: 847, 1931. Goodpasture, E. W. : Concerning the Pathogenesis of Tvphoid Fever, Am. J. Path. 13: 175, 1937. Goodpasture, E. W., and Anderson, K. : The Problem of Infection as Pre- sented by Bacterial Invasion of the Chorio-allantoic Membrane of Chick Embryos, Am. J. Path. 13: 149, 1937. Gunn, F. D., and Nungester, W. J. : Pathogenesis and Histopathology of Experimental Pneumonia in Rats, Arcli. Path. 21: 8]3, 1936. Hadfield, G., and Garrod, L. P.: Pneumonia, Recent Advances in Pathology, Philadelphia, 1938. P. Blakiston's Son & Co., pp. 187-199. Herter, C. A.: The Common Bacterial Infections of the Digestive Tract and the Intoxications Arising Therefrom, Harvey Lectures, 1900-1907, Vol. 2: pp. 64-98. Hertzler, A. E.: The Peritoneum, St. Louis, 1919, The C. V. Mosby Co. Ivy, A. C : Contributions to the Physiolog-v of the Stomach. Studies on Gastric Ulcer, Arch. Int. Med. 25: (;-31, 1920. Ivy, A. C, and Shapiro, P. F.: Studies on Gastric Ulcer. III. The Experi- mental Production of Gastric Ulcer liv Local AllergT: Preliminary Report, ,T. A. M. A. 85: 113L 1925. Kanavel. W. B. : Infections of the Hand. Pliiladelphia. 1916, Lea and Febiger. Lindau, A. : Cited by Hadfield and Garrod. 86 IMMU^■OLOGV Locke, A.: Lack of Fituess as the Predisposing Factor in Infections of the Type Encountered in Pneumonia and in Common Cold, J. Infect. Dis, 60: 106, 1937. Locke, A.: Non-Specific Factors in Eesi.stauce. I. Capacity to Sustain Effec- tive Circulation, J. Immunol. 36: 159, 1939. Locke, A., and Main, E. R. : Non-Specific Factors in Resistance. II. Ability to Withstand Shock, J. Immunol. 36: '173, 1939. Loescheke, II.: Cited by Hadfield and Garrod. Meltzer, S. J.: The Factors of Safety in Animal Structure and Animal Econ- omy, Harvev Lectures, 1906-07, Philadelphia, 1908, J. B. Lippincott Co., Yoh 2: pp. 1.39-170. Ncff, F. C., and Schwartz, E. J.: Lobar Pneumonia Beginning in the Hilus, Bull. Univ. Kansas School Med. 3: 3, 1933. Opie, E. L. : Lesions Peculiar to the Pancreas and Tlieir Clinical Aspect, Med. News. New York 84: 961-967, May 21, 1904. Opie, E. L.: Inflammation, Arch. Int. Med. 5: 541-568, 1910. Porter, W. T. : Vasomotor Relations, Harvey Lectures, 1906, Vol. 2: pp. 98-117. Smith, T. : Focal Cell Reactions in Tuberculosis and Allied Diseases, Bull. Johns Hopkins Hosp. 53: 197, 1933. Stillman, E. G.: Persistence of Inspired Bacteria in llio Lungs of Alcoholized Mice, J. Exper. Med. 40: 353, 1924. Terrell, E. E., Robertson, O. H., and Coggesliall, L. T.: Cited by Hadfield and Garrod. Wherry, W. B. : Tissue Hydration and Its Relation to Susceptibility and Immunitv: As Showu bv Skin Tests in Asthma, Chronic Sinusitis and Other Infections, J. Infect. Dis. 41: 177-189, 1927. Winternitz, M. C, Smith, G. H.. and Robinson, E. S. : An Unrecognized Path- wav for Bacterial In\asion of tlie Respiratory Tract, Bull. Johns Hop- kins Hosp. 31: 63-66, No. 349, March, 1920. Williams, J. W. : Your Skin Resurfaces Your Body to Protect It From Infec- tion and Injury, J. Bact. 41: 73, 1941. Woolpert, O. C, Gallagher, F. W., Rubinstein, L., and Hudson, N. P.: Propa- gation of the Virus of Human Influenza in the Guinea Pig Fetus, J. Exper. Med. 68: 313, 1938. Yoffey, J. M., and Sullivan, E. R. : The Lymphatic Pathway From the Nose and Pharynx, The Dissemination of Nasally Instilled Vaccinia Virus, J. Exper. Med. 69: 133, 1939. CPIAPTER V THE 1?ETTCUL0-END0THELTAT. SYSTEAF It is interest iiio" to note tliat; the concepts we now hold as to celhilar immunity liave their roots buried deeply in experimental biology, chemistry, and pathology. It was the zoologist, Metch- nikotf, who about 1883-84 extended the then existing knowledge of the distribution and functions of amoeboid cells throughout the body and classified them into two groups which he named mac- roplmges and micvoplwges, respectively. He grouped together the fixed phagocytes which he found in the liver, spleen, lymph nodes, and in the central nervous system and the large free mononuclear phagocytic cells of the circulating blood and named them mac- rophages, while the neutrophilic leucocytes of the blood he called microplwges. It was his opinion that the macrophages were concerned with the phagocytosis and digestion of dead and foreign animal cells and debris while the function of the microphages was to engulf and de- stroy bacteria. The cellular enzymes which carried out the diges- tion of the phagocytized material he named macrocytase and microcytase, respectively, to indicate the type of phagocyte which produced them. These enzymes have been studied rather ex- tensively by Opie (1906-1910). He found that the macrophages contain an enzyme that resembles pepsin in that it acts best in an acid medium while the microphages contain a trypsin-like enzyme that works best in an alkaline medium. Before Metchnikoff began his work on phagocytosis, a great deal had been learned about the manufacture of dyes and their use in histology. According to Conn (1933) it is well established that Hill employed carmine in his histological studies of plant tissues as early as 1770 and there are claims made that either Sarrabat (1733) or E^ichel (1758) were the first to use stains in histological work. About 1854 William Perkin prepared, for the first time, an anilin dye. The discovery of various other dyes now used in biology quickly followed. According to Jaffe (1938), it was Ehrlich's (1879) studies on the chemical constitution and cellular affinity of dyes that were re- 87 88 IMMUNOLOGY sponsible for the great i:)i"ogress tliat lias been made in our knowl- edge of the phagocytic cells of the body. Some of these important contributions may be listed as follows : 1. He was probably the first to recognize the distinction between acid and basic dyes. 2. He used them to differentiate between acidophilic and baso- philic leucocytes. 3. He prepared what he called "neutral stains" with Avhich he demonstrated the existence of neutrophiles. 4. He prepared benzidine dyes such as trypan blue and triphenyl methane dyes such as pyrrhol blue and isamine blue that were later used by Bouffard (1906) and Goldmann (1909) in their important works on vital staining and are now employed ex- tensively in experimental hematology. 5. He prepared neutral red Avhich has been used by Metchnikoff, Sabin and others in supravital studies of amoeboid cells. By means of the acid colloidal dyes of Ehrlich, Goldmann (1909) demonstrated the significance of vital staining. In 1913 Aschoff employed lithium carmine, trypan blue, pyrrhol blue, and other dyes in a study of the extensive system of phagocytic cells scattered throughout the body. Purely upon the basis of the capacity of the various cells to take these acid colloidal dyes and store them, he divided the system, which he named reticulo-endothelial, into six groups. In the following arrangement in which the order given by Mann and Higgins is reversed, group one contains tlie most actively pliagocytic cells while tlie cells of group six are almost devoid of phagocytic powers. 1. The monocytes of the blood (Plates I and II) 2. Wandering cells of the connective tissue called clasmatocyte by Ranvier, polyblast by Maximow, and macrophage by Metchnikoff (Plate II) 3. The living cells of the blood sinuses of the spleen, the lymph sinus of the lymph glands, the blood sinuses of the liver, bone marrow, suprarenal cortex, and hypophyses (Plate III) 4. The reticular cells of the splenic pulp and cords of Ijonph nodes f). Fibrocytes 6. The ordinary endothelial cells of the lymphatic and vascular system THE RKTICULO-KNDOTHELIAL SYSTEM 89 Maim and Higgins suggest that the reactions to vital dyes of the fibrocytes and ordinary vascular and lymphatic endothelium are so slight that these two groups could well be eliminated. Jaffe reviews the various theories that have been offered to explain vital staining. It is evident from his discussion that there is great dif- ference of opinion. A concept that is held by Hadfield and Garrod and many others is that vital staining consists of phagocytosis and concentration of the colloidal dye particles and that it differs from simple staining in this respect and also in that the process of os- mosis is not involved as it is in simple staining. It should also be added that the living nucleus is not stained in vital staining. According to Hadfield and Oarrod (1938) the discovery that the cells of the reticulo-endothelial system come from undifferentiated mesenchyme and that there is actually present in the tissues of the adult, ancestral cells of the embryonic mesenchyme having toto- potentialities should be credited to Maximow. The undifferentiated mesenchyme in adult tissues is described as a cell syncytium in which it is difficult to make out cell boundaries. The cytoplasm of the syncytium is pale and slightly neutrophilic. The nuclei are pale, but there is usually no nucleolus. Hadfield and Garrod say that in the unstimulated organ the cell syncytia are inconspicuous and lie on the basement membrane of the lymph or blood sinuses. When the cells are stimulated, they swell and de- velop a cell outline. Such cells are called reticulum cells (Plate III, Fig. 4), and it can be established that they can be divided into tAvo types only, the so-called littoral cell that does not contain ar- gyrophil fibrils in the resting state while the other does. Both cells may free themselves from the sinus walls and become motile histiocytes (Plate III). These have been called polyblasts by ]\[aximow, macrophages hy Metchnikoff, and clasmatocytes by Ranvier and others. Downey's Handbook of Hematology'^ contains splendid chapters on the fibroblasts and macrophages and on the fixed s^-stem of histiocytes in the liver by Bloom (1938) and Mann and Higgins (1938), respectively, while Jaffe gives an ex- cellent discussion of the reticulo-endothelial system. The supra- vital method of studying blood cells is well presented by Cunning- ham and Tompkins (1938). It should be remembered tliat there are conflicting theories held regarding the origin of cells found in the peripheral circulation ♦Paul B. Hoeber, Inc., Xew York, 1938. 90 IMMUNOLOGY Plate III. — Cells of Eeticulo-Endothelial System Showing Phagocytosis OF India Ink 1. Omentum of rat injected with India ink. H — Histiocyte. 2. Alveolar wall of lung of guinea pig after inhalation of coal dust. S — Septal cell. 3. Bone marrow of guinea pig after India ink injection. R — Reticular cells. 4. Reticulum cells of the spleen of a dog (not injected with India ink). 5. Sinusoid of the liver of a dog after India ink injection showing, K — ■ Kupffer cell containing India ink; E — Endothelium of sinusoid; L— Lymphocyte ; S — Sinusoid ; Er — Erythrocyte. -i'*^'^. ^=*d^ A A- # ^^ ^': , > . ^ ^..©« w Jy p. Oioo^ Plate ITT. (See explanation on opposite page.) TFIE RKTICULO-KNDOTHELIAL SYSTE^E 91 ill health and disease as well as tlic phagocytic cells of the tissues and the varioiLs types of cells observed in inflammatory exudates. The monopkijletic theory assumes that the embryonic mesenchyme gives rise to a stem cell, called l)y Papi)enheim the lymphoidocyte, which is endowed with in toto-potentialities. The environment in which it finds itself determines the type of cell which develops from it. In contrast to this is the pohjphyletic theory which assumes that the embryonic mesenchyme gives rise to stem cells endoAved with different potentialities : one is destined to give rise to gran- ulocytes, another to lymphocytes, etc., regardless of the environ- ment. The adherents of both theories agree that the stem cells originate from cells that are of mesenchymal origin and that the adult mononuclear phagocytic cell of perhaps major importance in the body's defense has a peculiar affinity for trypan blue. The ma- jority of workers are willing to call this cell a clasmatocyte or histiocyte. Sabin et al. formerly (1925) divided the large mononu- clear phagocytes into two groups. One group which has an af- finity for trypan blue she called clasmatocytes. These, she thought, originate intravascularly in the bone marrow, liver, spleen, and lymph glands and migrate into the tissues, to become the tissue phagocytes. The second group, which has little affinity for trypan blue, she called the monocyte. These, she thought, originate ex- travascularly from reticular cells in the same tissues. They enter the blood stream and constitute the monocytes of the peripheral circulation although they too are found in the tissues. More re- cently Sabin (1932) has come to regard the monocyte and clas- matocyte as one and the same cell exhibiting difference in appear- ance as a result of the kind of material it has ingested. The various types of cells found in tlie blood and tissues are illustrated in Plates I, II, and III. These show the monocytes and clasmatocytes of Sabin as they appear in supravital preparations where neutral red is used to demonstrate granules and vacuoles and Janus green to stain any mitochondria present. It will l)e observed that the monocyte has a kidney-shaped nucleus with a rosette of neutral red granules in the cytoplasm within the hof of the nucleus. These granules sur- round the centrosphere. The vacuoles containing neutral red are more or less peripherally arranged. The clasmatocyte has an oval, 92 IMMUNOLOGY round, or sometimes a kidney-shaped nucleus but never possesses a rosette, and, when material is phagoeytized, it appears in vacuoles near the nucleus. In Plate II, Fig. 5, a clasmatoeyte is shown con- taining a neutrophile leucocyte within a vacuole. The neutrophile is apparently dead since its nucleus is stained with neutral red. The concentration of neutral red used in this preparation will not stain a living nucleus. Gay (1931) has illustrated the various types of cells and in- dicated their possible origin and function as described by various authorities. His portrayal of these facts is reproduced in Fig. 5. Fig. 5. — The supposed intergenetic relationships and convertibility of cells of the "macrophage (reticulo-endothelial) system" of mammals. The authorities mainly responsible for the supposed changes are given on the arrowed lines. Heavy lines indicate more generally accepted type cell changes; dotted lines show less authenticated or more recent conceptions. Prom Tissue Resistance and Immunity, by F. P. Gay, v. 27, p. 1195, 1931, Journal American Medical Association. By permission of F. P. Gay and Journal American Medical Asso- ciation. It will be observed from an inspection of Gay's illustration that all are agreed that the large mononuclear phagocytes are produced for the most part in the spleen, liver, and lymph glands. While a few are produced by the bone marrow, it seems that its function is largely that of producing red cells, platelets, and granulocytes. THE RETICULO-ENDOTHELIAL SYSTEM 93 The functions of the cells of the rcticulo-cndothelial system may be summarized as follows : — 1. The macrophages engulf senile and injured red cells and erythrocyte fragments and thus participate in their removal from the circulation, from areas of infection, and from the tissues where extravasation has occurred. 2. They phagocytize and destroy dead and dying leucocytes and injured tissue cells. 3. They phagocytize inert foreign material such as deeply in- haled dust and also carbon particles, India ink, and acid colloidal dyes gaining entrance to the blood stream, thus removing them from the circulation. 4. In inflammatory and degenerative conditions of the nervous system phagocytic cells, according to Hadficld and Garrod, remove the free myelin and its disintegration products. 5. According to Bunting (1938), in certain chronic infections the macrophages form the epithelioid cell which in addition to its property of phagocytosis is able to secrete a precollagenous reticu- lum (Miller) that aids in localizing infection. 6. The macropliages may fuse and form the foreign body giant cell and thus be able to interpose a mass of protoplasm Ijetween the tissue cells and a body too large to be engulfed. 7. According to Mann and Higgins (1938) and Hadfield and Garrod (1938) one of the functions of the fixed histiocytes in the liver is the metabolism of the blood pigments leading to the re- tention and return to the bone marrow of the iron liberated from the disintegrating red cells and the excretion of the iron-free por- tion as bile pigment. 8. These same authors also state that the liver histiocytes to- gether with the liver cell seem to act as a functional unit in lipid metabolism, 9. According to Sabin (1939) and Bunting (1938), there is some evidence suggesting that the cells of the reticulo-endothelial system help in maintaining the normal composition of the plasma pro- teins. 10. There is a great deal of evidence indicating that antibodies are produced perhaps entirely by cells of the reticulo-endothelial system. This view was held by Metchnikoff. 94 IMMUNOLOGV When one injects a cellular antigen into the animal body, the antigen is rapidly removed from the point of injection and ap- pears in the blood stream. From the latter it is removed by phagocytic cells of the reticiilo-endothelial system. If one uses an antigen such as avian red cells which can be readily identified microscopically, one can trace these to the Kupffer cells of the liver and fixed tissue cells of the spleen, lymph glands, etc., by studying properly prepared sections of these tissues. After an interval of several days, specific antibodies for avian red cells, or the particu- lar antigen employed, appear in the blood. ]\rany attempts have been made to determine experimentally where the antibodies are produced. One of the most successful methods of investigation has involved an attempted blockade of the cells of tlic rcticulo-endothelial system by injecting inert particu- late matter such as India ink. Au excellent review of the subject is given by Howell (1928). She concludes that while the evidence points to the reticulo-endothelial system as tlie source of anti- bodies, the question is not definitely settled. Since the publication of Howell's ])aper Cannon, Baer, Sullivan, Webster (1929), Kroo and Jancs(3 (1931) have published results that quite definitely implicate the reticulo-endothelial system as an important source of hemolysin and perhaps other antibodies. Cannon et al. give a critical review of previous work and point out many sources of error. They find that the manner of administra- tion and the amount of antigen injected affects the liberation of hemolysin following blockade. They also determined that an in- complete blockade increases the amount of hemolysin production while a complete blockade inhibits antibody production. Their work is carefully controlled and apparently supports the theory that antibodies are produced by the reticulo-endothelial system. Plate III shows fixed phagocytic cells in various tissues. They have taken up India ink injected intravenously to produce a ])lock- ade of the reticulo-endothelial system. 11. The cells of the reticulo-endothelial system form the main defense mechanism of the body against infectious agents after the latter have once gained entrance. This is discussed more ex- tensively in Chapter VII. Bunting (1938) says that there is sug- gestive evidence that they may aid in preventing invasion of the body. THK Ri:TirT"I.()-KNT)()TnKrJ.\r. SYSTE^L 95 Phagocytosis. — Botli iiiaerophagcs and niicropliagcs have to ])c considered in any discussion of pliagocytosis. From the stand- point of the defense of the body against disease the engnlfment of bacteria or other infectious agents by amoeboid cells raises the following three important questions: (1) How do the phagocytes and the bacteria get together? (2) What is the mechanism of engulfment? (3) What disposition is made of the engulfed organisms ? It would seem evident that tlie fixed histiocytes, as e.g., the Kupffer cells of the liver, are concerned with the removal of bac- teria and other substances brought to them by the circulation while the free histioc^'tes and neutrophiles can he mobilized wherever needed. The mechanism of mohilization has been investigated quite extensively. It is known that many substances, among them bacterial protein, attract phagocytic cells. This is called positive chemotaxis. When phagocytes are repelled it is called negative chemotaxis. Attention is called in Chapter III to Menkin's dis- covery of a leucocyte-attracting .substance in inflammatory ex- udates. He has obtained this nitrogenous substance in crystalline form and named it "leukotaxine." It is thought that amoeboid cells move toward the source of a chemotactic substance because of the change that the latter substance effects in the surface tension of the phagocyte. Among other theories offered in explanation of the observed attractions of phagocytes are that osmotic forces govern their movements, that the difference in the surface potential between the amoeboid cell and the bacteria is the important factor in chemotaxis. IMenkin seems to have shown that the hydrogen ion concentration is an important factor in determining the prevailing type of cell in an area of inflammation although it is not the chemotaxie factor. Apparently there is no theory that explains adequately the mechanism of attraction or tropism of free amoeboid phagocytic cells in the body. In an interesting report by Mallery and Mc- Cutcheon (1940) they say that the motility of leucocytes obtained from acutely ill individuals may not only be diminished but the leucocytes show a less direct approach to the bacteria. In regard to the factors that play a role in the engulfment of bacteria by phagocytic cells Mudd (1927), as well as others, thinks that the presence of a film of denatured globulin on bacteria or 96 IMMUNOLOGY particulate matter makes it possible for leucocytes to engulf them more readily. As will be shown in another chapter there is a great deal of evidence indicating that when immune serum is mixed with homologous bacteria the latter specifically adsorb antibody protein (globulin) on their surfaces and that this film of globulin becomes insoluble in salt solution (denatured). This probably constitutes the process called opsonification or preparation of the cell for phagocytosis. Natural antibody acting in this manner is called opsonin while similarly acting antibody in an immune animal is called ' ' bacteriotropin. ' ' Some of the earliest investigations of the role that antibodies play in the process of phagocytosis were carried out by Denys and Leclef (1895), Mennes (1897), and Marchand (1898). They showed that immune serum increased phagocytosis and thought it was due probably to some action on the bacteria. According to Muir (1931) particulate matter such as charcoal, flour, powdered albumin, milk globules, and other materials, adsorb serum proteins and become nonspecifically opsonized. In 1903, Wright and Douglas carried out an extensive investiga- tion of the whole question and concluded that certain immune bodies which they called opsonins acted upon the bacteria and rendered them more susceptible to phagocytosis. In this work they used a modification of Leishmann's (1902) technique of study- ing phagocytosis. They concluded that normal opsonins are in- activated at 60° C. for fifteen minutes. In 1904, Neufeld and Rimpau discovered that immune opsonins are heat stable. They consider this an important difference between them and normal opsonins. Bullock and Western (1906) and Hektoen (1908) showed that normal opsonins could be specifically adsorbed and Chapin and Cowie (1907) showed that inactivated opsonin could be reactivated. This suggested that it was quite similar to Bordet 's sensitizer or Ehrlich's amboceptor. Role of Complement. — Dean (1905, 1907) and others have shown that even the activity of immune opsonins (named bac- teriotropins by Neufeld) is materially increased by normal serum containing complement. Sleeswijk (1908) concludes that both normal and immune opsonins have dual structiires in that the activity of both is increased by complement. He suggests that THE RETICULO-ENDOTHEIJAI. SYSTEM 97 perhaps the reason complementary actions dominate the picture in the ease of normal opsonins is the low concentration of the latter while the high concentration in immune sera reduces the activating action of complement to a mere enhancement of an effect which takes place in its absence. Apparently surface forces play an important role in phago- cytosis. For those interested in certain theories involving the free surface energy at the interfaces of bacteria and phagocytes, the papers of Fenn (1922, 1928) and Mudd, McCutcheon and Lucke (1934) are recommended as of interest. In regard to the third question concerning the fate of ingested infectious agents a number of possibilities need be considered: 1. The cellular matter may be taken in and then ejected without apparent change. 2. After ingestion, living cells may be killed and digested, and any remaining residue ejected. 3. The ingested infectious agent may be transported within the phagocyte to some depot and destroyed immediately or after an indefinite period of time. This accounts for the presence of viable organisms in cultures from the spleen, lymph nodes, and other tissues when blood cultures are negative. 4. Goodpasture's work on the pathogenesis of typhoid fever suggests that young plasma cells may function as host cells for E. tj/phosa. T). The phagocyte may be destroyed by the ingested organisms. References Arkin, A.: The Influence of Strychnin, Caft'ein, Chloral, Antipyrin, Cholesterol, and Lactic Acid on Phagocytosis, J. Infect. Dis. 13: 408, 1913. AshofP, L.: 1913. Cited by Mann and Higgins, p. 979. Ashoff, L.: Das Eeticulo-Endothieliale S.ysteni, Ergebn. d. inn. Med. u. Kinderh. 26: 1, 1924. Bloom, W. : Fibroblasts and Histiocytes, Downey's Handbook of Hematology II, 1938, Paul B. Hoeber, New York, p. 1335. Bordet, J., and Gengou, O. : Sur 1 'existence de substances sensibilisatrices dans la plupart des serums antimicrobiens, Ann. de I'Inst. Pasteur 15: 289, 1901. Bouffard: 1906. Cited by Jaffe, p. 979. Brand, E.: Ueber das verhalten der kompleniente bei der dialvse, Berl. klin. Wchnschr. 44: 1075, 1907. Brooks, S. C. : Tlie Regeneration of Cotiiiilemont After Radiation or Heating, J. Med. Res. 41: 411, 1919. 98 IMMUNOLOGY Bunting, C. H.: Functions of the Leucocytes, Downey's Handbook of Hematology I, 1938, Paul B. Hoeber, New York, p. 437. Buchner, E. : Cited in Metchnikoff, Resistance to Infectious Diseases, Cam- bridge University Press, London, 1907, p. 193. Bullock, W., and Western, G.: Cited by Topley and Wilson, Principles of Bacteriolog>' and Tnimunitv, New Yoik, William Wood & Co. 1: 175, 1929. Bunting, C. H.: Functions of the Leucocytes, Downey's Handbook of Hematology, Paul B. Hoeber, Inc., N. Y.l: 437, 1938. Cannon, P. R., Baer. R. B., Sullivan, F. L., and Webster, J. E.: The Effect of Blockade of the Reticulo-endothelial System on the Production of Antibodies, J. Immunol. 17: 441, 1929. Chapin, W. S., and Cowie, D. M. : Experiments in Favor of the Amboceptor Complement Structure of the Opsonin of Normal Human Serum for the Staphylococcus Albus, J. Med. Res. 17: 95, 1907. Conn, H. J.: The History of Staining, Book Service of the Biol. Stain Commission, Geneva, New York, 1933. Cowie, D. M., and Chapin, W. S.: On the Reactivation of Heated Normal Human Opsonic Serum With Fresh Diluted Serum: A Contribution to the Study of the Structure of Opsonins, J. Med. Res. 17: 57, 1907. Cunningham, R. S., and Tompkins, E. H. : Tlie Supravital Method of Study- ing Blood Cells, Downey's Handbook of Hematology, New York, 1938, Paul B. Hoeber 1: pp. 553-584, 1938. (See also article on Blood in Pathological Conditions, Ibid. p. 585.) Dean, G. : An Experimental Enquiry Into the Nature of the Substance in Serum Which Influences Phagocytosis, Proc. Roy. Soc, London, s. B. 79: 399, 1907. Dean, H. R.: The Relation Between the Fixation of Complement and the Formation of a Precipitate, Ztschr. f. Immunitatsf. 13: 84, 1912. Dean, G.: The Toxin of Diphtheria Bacillus. The Bacteriology of Diph- theria, Graham-Smith, Cambridge Univ. Press, London, 1913, p. 460. Denys, J., and Leclef, J.: 1895. Cited by Topley and Wilson, Principles of Bacteriology and Immunity, New York, William Wood and Co. 1: 173, 1929. Dick, G. F.: On the Origin and Action of Hemolytic Complement, J. Infect. Dis. 12: 111, 1913. Eagle, H.: The Mechanism of Complement Fixation, J. Gen. Physiol. 12: 825, 1929. Ehrlich, P.: 1879. Cited by Jaffe, p. 979. Ehrlich, P.: Studies in Immunity, New York, 1910, John Wiley and Sons. Fenn, W. O. : The Mechanism of Phagocytosis, Newer Knowledge of Bac- teriology and Immunology, University of Chicago Press, p. 517, 1928. Ferrata, A.: Die Unwirksamheit der komplexen Hamolysine in salzfreien Losungen und ihre Ursache, Berl. klin. Wchnschr. 44: 366, 1907. Freundlich, H.: Kapillarchemie. Millard Physical Chemistry for Colleges, New York, 1931, McGraw-Hill Book Co., "p. 133. Gay F. P.: The Fixation of Alexines by Specific Serum Precipitates, ' Centralbl. f. Bakt., 1905, Orig. 93, 603. ' Gay, F. P.: Tissue Resistance and Immunity, J. A. M. A. 97: 1193, 1931. Gay, F. P., and Clark, A. R.: Furtlier Notes on the Relative Protection by Polymorphonuclear and Mononuclear Cells in Local Streptococcus In- fections, Proc. Soc. Exper. Biol. & Med. 27: 995, 1930. Gay, P. P., and Clark, A. R.: A Comparison of Indifferent Substances and Specific Antigen in Production of Local Streptococcus Immunity, Proc. Soc. Exper. Biol. & Med. 24: 20, 1926. Gay, F. P., and Clark, A. R. : Enhanced Passive Immunity to Streptococcus Infection in Rabbits, J. Exper. Med. 52: 95, 1930. TiiK kkti('i;lo-em)otjielial system 99 Gay, F. P., Linton, E. W., and Clark, A. E. : Transpleural Mobilization of Clasmatocytes With Coincident Streptococcus Protection, Proc. Soc. Exper. Biol. & Med. 24: 23, 1926. Gay, F. P., and Oram, F. : Streptococcus Leucocidin and the Eesistance of Clasmatocytes, Proc. Soc. Exper. Biol. & Med. 28: 850, 1931. Gengou, O.: Sur les sensibilisatrices des serums actifs contre les substances albuminoides, Ann. de I'lnst. Pasteur. 16: 734, 1902. Goldmann, E.: 1909. Cited by Jaffe, p. 979. Goodpasture, E. W. : Concerning the Pathogenesis of Tvphoid Fever, Am. J. Path. 13: 175, 1937. Gramenitski, M. : Ueber die Eegeneratiou des Komplemeuts, Biochem. Ztschr. 38: 501, 1912. Hadfield, G., and Garrod, L. P.: The Eeticulo-Endothelial System. Eecent Advances in Pathology, Philadelphia, P. Blakiston's Son and Co., pp. 15-34, 1938. Hankins, E. H. : Ueber den Ursprung und Vorkommen von Alexinen im Organismus, Centralbl. f. Bakt. 12: 777, 1892. Ibid. 12: 809, 1892. Hektoen, L.: The Action of Certain Ions Upon the Lysins in Human Serum, Trans. Chicago Pathol. Soc, 5: 303, 1901-03. Hektoen, L.: On the Specificity of Opsonins in Normal Serum, J. Infect. Dis. 5: 249, 1908. Hill, A., Parker, G., and McKinstry, E. N. : Observations of the Deviation of Complement in the Wassermann Test, J. Path, and Bact..28: 47, 1925. Howell. K. M. : Origin of Antibodies, Newer Knowledge of Bacteriology and Immunology, Univ. of Chicago Press, p. 1035, 1928. Jacoby, J., and Schiitze, A. : Ueber die Inaktivierung der Komplemente durch Schiitteln, Ztschr. f. Immunitatsf. 4: 730, 1910. Jaffe, E. H. : The Eeticulo-Endothelial System, Downey's Handbook of Hematology, Paul B. Hoeber, Inc., New York 2: 973, 1938. Kolmer, J. A., Matsunami, T., and Trist, M. E. : Studies in the Standardiza- tion of the Wassermann Eeaction. IV. A General Study of the Com- plements of Various Animals With Special Eeference to Human and Guinea Pig Complements and Methods of Collection, Am. J. Syph. 3: 407, 1919. Kroo, H., and Jancso, N. V. : The Significance of the Eeticulo-Endothelial Cells in Immunity and Chemotherapy, Ztschr. Hyg. u. Infekt. 112 (1): 182, 1931. Landsteiner, K., and von Eisler: Cited by Zinsser in Eesistance to Infectious Diseases, The Macmillan Co., 1931, p. 201. Ivcishmann, W. B. : Note on a Method of Quantitatively Estimating the Phagocytic Power of the Leucocytes of the Blood, Brit. M. J, 1: 73, 1902. Liefmann, H., and Cohii, M.: Die Wirkung des Komplementes auf die ambozeptorbeladenen Blutkorperchen, Ztschr. f. Immunitatsf. 7: 669, 1910. (II Teil.) Ztschr. f. Immunitatsf. 8: 58, 1911. Mallery, O. T., and McCutcheon, M. : Motility and Chemotaxis of Leukocvtes in Health and Disease, Am. J. M. Sc. 200: 394, 1940. Mann, F. C, and Higgins, G. M. : The System of Fixed Histiocytes in the Liver, Downey's Handbook of Hematology, Paul B. Hoeber, Inc., New York 2: 1377, 1938. Manwaring, W. H. : On the Destruction of Complement by Heat, Trans. Chicago Path. Soc. 6: 425, 1906. Manwaring, W. H.: The Action of Certain Salts on the Complement in Immune Serum, J. Infect. Dis. 1: 112, 1904. Marchand, F. : Cited by Topley and Wilson. Principles of Bacteriology and Immunity, New York, Wm. Wood & Co., p. 173, 1929. 100 IMMUNOLOGY Maximow, A. : Eelation of Blood Cells to Connective Tissue and Endothelium, Physiol. Kev. 4: 533, 1924. Maximow, A.: The Development of Non-Granular Leucocytes Into Poly- blasts in Vitro, Proc. Soc. Exper. Biol. & Med. 24: 570, 1927. Maximow, A.: Morphologv of the Mesenchymal Reactions, Arch. Path. 4: 551, 1927. Menkin, V. : Studies on Inflammation. XVIII. On the Mechanism of Leuko- cytosis With Inflammation, Am. J. Path. 16: 13, 1940. Mennes, F. : Das Antipneumokokken-Serum und der Mekaniswies der Im- munitat des Kaninchens gegen des Pneumo-kokkus, Ztschr. f. Hyg. 25: 413, 1897. MetchnikoflF, E.: Immunity in Infectious Diseases, Translated by F. G. Binnie, Cambridge Univ. Press, London, 1907. Moreschi, C. : (1905.) Cited by Zinsser. Resistance to Infectious Diseases, New York, 1931, The Macinillan Co., p. 197. Mudd, S., and Mudd, E. B. H. : The Surface Composition of the Tubercle Bacillus and Other Acid-Fast Bacteria, J. Exper. Med. 46: 167, 1927. Mudd, S., McCutcheon, M., and Lucke, B. : Phagocytosis, Physiol. Rev. 14: 210, 1934. Muir, R.r Opsonic Action, a Svstem of Bacteriology 6: 364, Med. Res. Council, London, 1931. Neufeld, L., and Rimpau, W. : Ueber die Antikorper des Streptokokken — und Pneumokokken — Immunserums, Deutsclie med. Wchnschr. 30: 1458, 1904. Nolf, P.: De I'origine du compliment hemolytique et dc la nature de ' I'hemolyse par les serums, Bull, de I'Acad. de Science de Belg., 1908, Classe des Sc, p. 748. Nolf, P.: Le Mecanismc de la globulolyse, Ann. de 1 'Inst. Pasteur 14: 656, 1900. Opic, E. L. : The Enzymes in Phagocytic Cells of Inflammatory I^xudates, .1. Exper. Med. 8: 410, 1906. Opie, E. L.: Inflammation, Arch. Int. Mod. 5: 541, 1910. Perkins, W.: Cited by Conn, 1033, p. 59. Reichel, G. C: 1758,' Cited by Conn, 1933, p. 9. Sabin, F. R.: Cellular Reactions to a Dye-Protein With a Concept of the Mechanism of Antibody Formation, J. Exper. Med. 70: 67, 1939. Sabin, F. R.: Cellular Studies in Tuberculosis, Am. Rev. Tuberc. 25: 153, 1932. Sabin, F. R.: Bone Marrow, Physiol. Rev. 8: 191, 1928. Sabin, F. R., Doan, C. A., and Cunningham, R. S. : The Discrimination of Two Types of Phagocytic Cells in the Connective Tissues by the Supravital Technique, Carnegie Inst. Contributions to Embryology 16: No. 82, 125, 1925. Sabin, F. R., Cunningham, R. S., Doan, C. A., and Kendwall, J. A.: The Normal Rhythm of the Wiiite Blood Cells, Bull. Johns Hopkins Hosp. 37: 14, 1925. Sabin, F. R., Doan, C. A., and Cunningham, R. S. : The Development of Leucocytes, Lymphocytes, and Monocytes From a Specific Stem Cell in Adult Tissues, Carnegie Inst. Contributions to Embryology 16: 227, 1925. Sabin, R. F., and Doan, C. A.: The Relation of Monocytes and Clasmato- cytes to Early Infection in Rabbits With Bovine Tubercle Bacilli, J. Exper. Med. 46: 627, 1927. Sarrabat: 1733. Cited by Conn, 1933, p. 9. Schilling, V.: The Blood Picture and Its Clinical Signiflcance ; Translated by R. B. H. Gradwohl, St. Louis, The C. V. Mosby Co., 1929. THE RETICULO-ENDOTHELIAL SYSTEM 101 Sherwood, N. P. : The Effect of Various Chemical Substances on the Hemolytic Eeaction, J. Infect. Dis. 20: 185, 1917. Sherwood, N. P., Smith, C, and West, R.: The Complement Content of Eck- Fistula Dogs, J. Infect. Dis. 19: 682, 1916. Sleeswijk: (1908.) Cited in Topley and Wilson, Principles of Bacteriology and Immunity, New York, William Wood & Co. 1: 176, 1929. Topley, W. W. C, and Wilson, G. S. : Principles of Bacteriology and Immunity, New York, 1929, William Wood and Co., p. 1. Thiele, F. H., and Embleton, D. : The Mechanism of Antibody Action, J. Path, and Bact. 19: 372, 1914-15. Wright, A. E., and Douglas, S. R.: Does the Substitution of Another Medium for the (Citrated) Blood Plasma Wliich Bathes the Corpuscles Exert an Influence on Phagocytosis? Proc. Roy. Sec, London 72: 362, 1903. Wilde, M. : Ueber die Absorption der Alexine durch abgetodtete Bacterien, Berl. klin. Wchnsehr. 38: 878, 1901. Zinsser, H., Enders, J. F., and Fotliergill, L. I).: Tmmuuitv. The Macmillan Co., N. Y., p. 28^, 1939. Supplementary Reference Hanks, J. H. : Quantitative Aspects of Phagocytosis as Influenced by the Number of Bacteria and Leucocytes, J. Immunol. 38: 159, 1940. CHAPTER VI NATURAL AND ACQUIRED IMMUNITY Natural Immunity. — Species Differences in Immunity. — The two infectious agents that caiLse cattle plague and chicken cholera respectively are unable, under natural conditions, to produce dis- ease in man. Conversely, the infectious agents that cause syphilis, gonorrhea, Asiatic cholera, measles and a few other diseases pe- culiar to man, never, under natural conditions, produce these dis- eases in the lower animals. Dogs, cats and rats are relatively re- sistant to ])neumococcus infection whereas man and guinea pigs are quite susceptible. Rats can ingest relatively large doses of botulinus toxin with apparent impunity whereas man and guinea pigs succumb following the eating of exceedingly small amounts. Coriell and Sherwood* found that cats possess almost solid im- munity to .strains of salmonella oi-ganisms highly virulent for mice as well as to .strains of B. anthracis, P. tuJarensis and a strain of vaccine virus that is exceedingly virulent for rabbits. On the other hand they found cats moderately susceptible to trichina in- fections, and Leasure (1934) observed cases of fatal feline virus enteritis. Racial Differences in Immunity. — Occasionally susceptibility and resistance are found to correlate with racial differences. Algerian sheep are said to show a much higher resistance to anthrax than our domestic sheep and various races of mice differ in their resistance to a number of infectious agents. Bay-Schmith (1929) reports that diphtheria does not occur among the Eskimos although the Schick test indicates that the normal percentage of susceptibles exists among them. Sherwood, Nigg and Baumgartner (1926) ob- served a similar phenomenon as regards scarlet fever among full- blooded American Indians. They rarely have the disease although the Dick test indicates a high percentage of susceptibles in the age group of five to fifteen years. Toyoda, Moriwaki and Futagi (1930) have compared the percentage of positive reactors to scarlet fever toxin among comparable groups of adult Chinese and Japanese. The ratio of susceptibles within the two races is as 1 to 2, while the morbidity statistics show equal susceptibility to *Unpublished work. 102 NATURAL AND ACQUIRED IMMUNITY 103 scarlet fever at birth and a ratio of 1 to 45 among adults. Scarlet fever is said to be unknown among the Eskimos, and it is interest- ing to note that Heinbecker and Irvine- Jones (1928) found no positive reactors to scarlatinae toxin among 53 Eskimos tested by them. These investigations seem to offer examples of racial im- munity to diphtheria and scarlet fever respectively. There is a great deal of controversy over tlie interpretation of the results and more than one explanation of the phenomena. Wells* (1933) made an extensive study of tlie bacterial flora of the tliroat of a fairly large series of Eskimos. He noted the presence of nonvirulent corynebacteria quite frequently in throat cultures. In one instance a transient virulence was observed. From his serological, epidemiological and bacteriological studies he feels warranted in concluding that the immunity to diphtheria observed among the Eskimos ''is specific rather than the result of maturation phenomena." He admits, however, that his evidence is not conclusive. Endocrines AND IMMUNITY. — There is a growing interest in de- velopmental physiology and anatomy. Research in these fields may ultimately result in a better understanding of the defensive mechanisms of the individual at different periods in his life cycle. Scammon (1925) and others have carried out extensive work in developmental anatomy. They have worked out interesting growth curves but have not studied the corresponding cellular and bio- chemical changes. Recent work suggests that the suprarenal cortex regulates, by its secretions, the permeability of vessel walls. In other Avords this secretion determines what substances stay witliin tlie walls and what escape into the tissues. When one turns to the develop- mental curve for the suprarenals, it is interesting to note that Scammon (1925) and others have shown that the suprarenals in- crease rapidly during fetal life, weighing about 7 grams at birth. During the first two years of postnatal life they undergo a rapid diminution in size to about a third of the natal weight. This re- duction in weight is due to an involution of the middle and inner cortical zones. From about tlie second year they gain very slowly in weight until middle or later childhood when accelerated growth is noted for a period and then a gradual growth until maturity. •Wells. J. R. : Am. J. Hyg. 18: 629, 1933. 104 IMMUNOLOGY 111 view of the recent developments in the field of endocrinology and especially the work of Britton and Silvette, Swingle, Hartman and others on the function of the suprarenal cortex, one is en- couraged to hope that correlative studies may result in an enhance- ment of our immunological and pathological concepts. This possibility is suggested by the work of Lewis (1928), Jaffe and Marine (1924), Jaffc* (1926), Scott (1924) and Belding and Wyman (1926). Lewis reports that a dose of 200 M.L.D. of diph- tlieria toxin for a guinea pig lias no effect upon normal rats but is lethal for suprarenalectoiiiized rats. Jaffe calls attention to the conclusions of Stewart and Rogoff (1922), Rogoff and DeNeeker (1926) and RogofP and Ecker (1926) that suprarenalectomized rats do not show increased susceptibility to toxins if tested after having fully recovered. Jaffe's conclusions are summarized as follows : ' ' ( 1 ) Recently suprarenalectomized rats, fully recov- ered from immediate operative effects are highly susceptible to small doses of typhoid vaccine ; (2) that as late as five months after operation suprarenalectomized rats having no gross suprarenal accessory tissue are still susceptible to vaccine; (3) that supra- renalectomized animals when compensated as regards resistance, invariably possess gross cortical accessory tissue; (4) that in the absence of gross accessories, autoplastic suprarenal transplants will protect suprarenalectomized rats against typhoid vaccine." He also states that "suprarenalectomized rats show a lowered re- sistance to natural infections." Belding and Wyman (1926) confirmed and extended the work of Lewis. They report that suprarenalectomized rats are 2.5 times as susceptible to diphtheria toxin as normal rats. They conclude that suprarenal deficiency apparently renders less effective the normal mechanism of tlic rat for tlie elimination or destruction of diphtheria toxin. Jungeblut, Meyer and Engle (1984) report that poliomyelitis virus is inactivated in vitro by biological products containing anterior pituitary-like principles and by cortical and medullary adrenal cortex hormone and that pregnancy urine preparation and adrenal cortex hormone exert the same effect on diphtheria toxin. It would seem that these findings warrant further investigation since Molomutt (1939) cites considerable evidence indicating that *JafE6, H. L. : Am. J. Path. 3: 421, 1926. tMolomut: J. Immunol. 37: 113, 1939. NATURAL AND ACQUIRED IMMUNITY 105 removal of either the adrenals or the hypophysis does not affect antibody formation. He did find that anaphylactic shock could be produced in hypophysectomized cats but not in the normal con- trols. Age AND Resistance. — It is generally recognized that in the clinical entity known as "status lymphaticus" where the thymus gland does not undergo its normal involutionary changes, the child is quite susceptible to intercurrent infections. Clinical experience has also established the fact that at both extremes of life the in- dividual possesses relatively little capacity to combat the majority of infectious diseases. The relationship of age to immunological reactions is discussed at some length by Baumgartner (1934), but her paper refers only to the humoral elements (antibodies). In this connection the work of Sutliff and Finland (1932) is very suggestive. They investigated the bloods of individuals falling into various age groups relative to the content of agglutinating, mouse protecting and bactericidal antibodies for various strains of pneumococci. In regard to mouse protecting and pneumococ- cidal antibodies Finland and Sutliff (1932) observed that they are relatively rare in the blood of children, relatively frequent in adults, and less frequent in elderly individuals. They regard the frequency curve for pneumococcidal power as probably similar for all types, showing a peak in adult life. That the process of aging is associated with increasing resistance to certain viruses is indicated by the work of Olitsky, Sabin and Cox (1936), King (1940) and Casals (1940). Of added interest are the reports of Culbertson and Kessler (1939), Morgan (1939), and Casals and Webster with rabies infection in mice. In later work Casals (1940) finds that in the case of rabies, the influence of age in immunizability is evident only under definite sets of con- ditions. Warthin (1929) discusses the anatomical changes that take place from conception to old age and death, but does not attempt to correlate structural and functional change with the relative in- tegrity of the defensive mechanisms of the body. In view of the apparent increase in interest in the relationship of the aging process to immunity these publications are timely and suggestive. Vitamins and Food Factors in Resistance. — The importance of vitamin A in the defense of the body against infectious agents is 106 IMMUNOT.OGY another subject over which there is a great deal of controversy. As regards the experimental animal there is abundant evidence in- dicating that this vitamin plays an important role in maintaining in a normal healthy state the epithelial coverings of the body. When animals are fed upon a diet deficient in vitamin A, there results marked pathological changes in the cutaneous system, epithelium of the eyeballs, paraocular glands and the respiratory epithelium. These changes, among them being extensive keratiniza- tion of the ocular and respiratory epithelium, are associated w^ith a definite lowered resistance to invasion by pyogenic bacteria. After infection is established, the feeding of vitamin A does not result in a cure. Thus it would appear that its function is pre- ventive in that it aids in maintaining a normal healthy epithelium which is resistant to invasion. Whether vitamin A plays a similar role in man has been much debated. In an interesting discussion of this subject Eusterman and Wilbur (1932) present evidence bearing upon both sides of the controversy. They are quite conservative and state that further research is desired, yet they seem to think that it may play a pro- phylactic role in man similar to the one it plays in the experimental animal. For a more extensive discussion and a comprehensive bibliography of the subject the student is referred to an excellent symposiimi on vitamins (1932), to a discussion by Szent-Gyorgyi (1939), a report by the Council on Pharmacy and Chemistry and Council on Foods (1939) and to a paper by Maekie, Eddy and Mills (1940). While these papers present many new facts about vitamins, they throw very little light upon their role in immunity. Nutrition and Resistance. — Anderson and Fraser (1934) have studied the influence of nutrition on the natural immunity re- actions of the blood and skin to bacterial toxins. They find that a caloric deficient diet decreases both the hemolytic activity of sheep serum toward rabbit erythrocytes and its complementing powers but it increases the agglutinating power of serum for Br. abortus (porcine). They find that intradermal toxin tests are not af- fected either by a deficiency diet or by a diet which includes cod- liver oil and calcium. In regard to the effect of diet on inborn resistance in mice in- fected with B. enteritidis Webster and Hodes (1930, 1939) report that altering the diet did not affect the spread of infection in a NATURAL AND ACQUIRED IMMUNITY 107 mouse population but tlieir resistance measured in terms of the killing potency of the organism was altered. They conclude, how- ever, that in exposed or infected populations of mice the mortality is conditioned, primarily, by the number of highly susceptible constituents. These discussions of hereditary and nutritional factors are in- cluded in this chapter since they play a role in determining both the morbidity and mortality in large groups as well as in individual cases. Neufeld's Theory of Phases of Heightened and Lessened Resistance. — Neufeld and his colleagues, Etinger-Tulczynska (1933) and Kuhn (1933), hold to somewhat different views. They explain the apparent immunity observed among survivors of experimentally infected mice as due to the testing of the animals during a highly resistant period. They seemed to think that there are periods of increased and decreased natural resistance. During an epidemic some individuals will be in the phase of heightened while others will be in a phase of lowered resistance. The former constitute the group of survivors whose immunity is ordinarily at- tributed to previous infection but which Neufeld regards as due to the fortuitous circumstance that they are in the phase of heightened natural resistance, whatever that may mean. Undoubtedly many factors operate. Hirszfeld (1927) thinks that he has demonstrated hereditary factors in natural immunity to diphtheria. His con- clusions have been severely criticized bj^ Snyder (1927), Rosling (1928) and others. Hereditary Factors in Natural Immunity. — ^Webster and his colleagues, Burn and Pritehett, conclude from their studies of microbic virulence and host susceptibility that hereditary factors, microbic distribution, microbic virulence and dietary factors are all quite important in determining whether infection occurs, the severity, the development of the carrier state and also the dura- tion and complications of the disease in mice and rabbits. Lambert (1932) reported that by selective breeding he could produce in- dividuals more resistant to the organisms of fowl typhoid than those in the unselected stock. Since the latter paper appeared, Irwin (1929, 1933) has pub- lished the results of four years' studv of the role of inheritance 108 IMMUNOLOGY in resistance of various inbred litters of rats to S. enteritidvi in- fection. Schott (1932) has also studied the effect of selection on the resistance of mice to S. aertrycke and Monreas (1932) of rab- bits to Br. abortus, var. suis. These have all shown that under experimental conditions, inheritance is important both in resistance and susceptibility of mice and rabbits to infection. Similar con- clusions have been drawn by Lewis* (1928) from an extensive study of experimental tuberculosis in guinea pigs. He says that his "observations agree very well with the older conception of an inlierited predisposing constitutional diathesis as a significant factor in the incidence of tuberculosis. ' ' These views are somewhat at variance with those of Topley, Wilson, Lewis and Greenwood (Topley, and Wilson, 1929, 1936) who conclude from a study of statistical data on host survival during epidemics that active immunity due to either recognized or subclinical infection is a more important factor in survival than selection of innately resistant animals. Similar conclusions have been drawn from Armstrong's studies on mice surviving nasal in- stillation of the St. Louis encephalitis virus. He found them rel- atively resistant to a second instillation and concluded they had become immunized. Webster and Hodcs (1940) point out certain sources of error in the experiments cited above and present very convincing ex- perimental evidence in support of their previous conclusions that inheritance factors play an important role in survival. Their re- sults cast grave doubts upon the conclusions of Topley et al. as well as those of Armstrong. They found that survivors were almost exclusively individuals known to be innately resistant and that there was no tendency for known susceptibles to become immunized through exposure before or during epidemic times. They grant the possibility that future research may show that survivors of an epidemic may develop through infections, what Webster and Hodes call a luxury iynmunity. They^were unable to immunize susceptibles by giving them sublethal doses of mouse typhoid bacilli or St. Louis encephalitis virus by a natural route. These observations may not invalidate the contention of Topley and Wilson that under field conditions the possession of an active ♦Lewis, Paul A., Loomis, D. : J. Exper. Med. 47: 1437. 1928. NATURAL AND ACQUIRED IMMUNITY 109 immunity in man due to previous specific infection influences the survival of many individuals during an epidemic. Antibodies and Natural Resistance. — Antibodies are specific biochemical substances which may or may not be present in the blood. They can be detected by the effect they have upon the specific chemical su])stances (antigens) for which they have an affinity. The antibodies, called antitoxins, neutralize their specific toxins and under proper conditions the toxin-antitoxin mixture may form a precipitate; the union of specific hemagglutinins to homologous red cells in the presence of electrolytes results in the agglutination oi- cliiiiiping of the cells. Likewise the union of antibody with homologous bnctei'ia in the presence of electrolytes results in the agglutination of tlie bacteria. When red cells or bacteria are united with their specific anti- body, they become susceptible to dissolution or lysis by a normal enzyme-like constituent of blood called complement. A more ex- tensive discussion of antibodies is given in subsequent chapters. Because of the specific relationship that exists between antibody and its bacterial antigen, antibodies are regarded as specific im- munity factors. Webster and Hodes (1939) conclude from their studies of mouse typhoid that specific immunity factors play a major role in infections with a liigh morbidity and a low mortality rate and a minor role where the infection gains access to a herd and s])reads for the first time or when the infection is associated with a high mortality rate. Irwin and Hughes (1933) in their study of inheritance im- munity in rats noted that death of infected animals seemed to be correlated with absence of germicidal power in their blood. While this suggests a possible correlation between immunity and anti- body, yet many exceptions have been reported in the literature. There is some evidence that the passive transfer of antibodies from mother to offspring through mammary secretions or through the placenta may confer temporary protection to the latter. This is supported hj the folloAving research: Theobald Smith (1907) demonstrated that in guinea pigs transient immunity can be trans- mitted from mother to offspring through the milk. He has also shown that the newborn calf acquires antibodies through the colostrum from the mother. 110 IMMUNOLOGY Kuttner and Katner (1923) confirmed the observation of other investigators who found that diphtheria antitoxin passes through the human placenta from mother to child but that it is not present in the colostrum or breast milk of the mother. Kuttner and Ratner call attention to the anatomical variations of the placenta in dif- ferent animals. There are four main types of plncentas: those with one, two, three, or iowr cell partitions or layers between the fetal and maternal blood. In man and rodents there is only one connective tissue layer while ruminants have three and other an- imals such as swine have four layers. Acquired Immunity. — It is now generally recognized that indi- viduals recovering from certain acute infectious diseases such as typhoid fever, scarlet fever, measles, mumps, poliomyelitis and smallpox develop a lasting specific immunity as a result of the in- fection. To a certain extent this is true also of tuberculosis. In the case of syphilis the patient is immune to reinfection with Treponema pallidum so long as he lias syphilis, but loses his im- munity after he has been cured of the disease. Individuals who recover from influenza or pneumonia or from various pyogenic in- fections owe their recovery to the operation of immunity mech- anisms but their increased resistance is of short duration. The exact mechanism of acquired immunity is not clearly under- stood. While antibodies are of some importance in resistance, it has been established that a correlation between antibody titer and resistance does not always exist. Hadfield and Garrod (1939) state that the development of lobar pneumonia seems to re- quire a pre-existing humoral immunity ; Coriell and Sher- Avood (1940) found no virucidal antibodies in cats that were immune to vaccine virus highly virulent for rabbits ; Kessel and Stimpect (1941) studied poliomyelitis in a large series of monkeys and compared immunity with antibody titer. They found a very definite lack of correlation since it was present in only 50 per cent of their convalescent monkeys. Further consideration of im- munity mechanisms will be presented in later chapters. Immunization Against Viruses and Rickettsiae. — The phe- nomenon of acquired resistance or active immunity to infection was perhaps first observed by the Chinese. They practiced nasal inoculation of smallpox virus and the production of the disease NATURAL AJSD ACQUIRED EVIMUNITY 111 in individuals in order to render them immune. A procedure of protective inoculation (into skin) was introduced into England in 1717 by Lady Montagu who had learned of it while in Con- stantinople. In 1798, Jenner published his brilliant discovery that inocula- tion of the human being with vaccinia or cowpox viriLs leads to the development of a la^sting active immunity to the virus of smallpox. Thus was introduced the term vaccination as well as the principle of immunization with an infectious agent of low virulence. While at first, vaccination against smallpox encountered great opposition and was fraught with the danger of secondary infec- tion, it has, nevertheless, achieved brilliant results. This is in- dicated by Turgensen's statistics for Sweden, cited by Zinsser and Bayne- Jones (1939). He states that during the 25 years pre- ceding the introduction of smallpox vaccination (1776-1801) the death rate due to this disease was 2,050 per million inhabitants. During the transitional period (1801-1810) it was 680, and after vaccination became compulsory in 1810, it dropped to 169 per mil- lion inhabitants. In the Kansas City epidemic of 1921, there were 1,090 cases with 222 deaths. The deaths occurred among non- vaccinated individuals only. Great progress has been made in the preparation of a satisfac- tory vaccine. Calves have been used almost exclusively in its preparation although Rivers (1933-1939) states that it is now pos- sible to prepare a vaccine of more assured bacterial sterility by cultural methods. Gallagher and Wolpert (1940) describe a meth- od of vaccine preparation in the rabbit fetus. A comprehensive dis- cussion of methods of vaccine preparation and immunization against smallpox, rabies, and rickettsiae is given by Zinsser and Bayne- Jones (1939) and by Zinsser, Robinson, and other contributors to , the Harvard symposium on virus and rickettsial diseases (1940). While it is generally recognized that living, attenuated virus vac- cines are the most potent ones, yet there are many, according to Zins.ser (1940), who feel that killed virus and killed rickettsiae have .some hnmunizing value providing sufficiently large doses are em- ployed. Veintemillas (1939) reports that the formalin-killed sus- pension of ^Mexican rickettsiae prepared in accordance with the methods of Zhisser and Castaneda (1931) has been found of prac- 112 IMMUNOLOGY tical value for large scale immunization against typhus fever. The subject of immunization against the virus of poliomyelitis is dis- cussed in a later chapter. Burnett, Andrews and others have shown that immunity can be induced in animals and man by the inoculation of influenza virus by various means. There is some evidence (Burnett, 1937, 1938) that cultivation of influenza virus on chick embryos alters its virulence without impairing its immunizing properties. Re- cently Horsfall (1940-41) has prepared a complex virus vaccine using influenza A virus and canine distemper virus X. The anti- body response in ferrets and man has been much more adequate than that from any of the other preparations previously used. If this vaccine proves to be as successful in man it will be possible to immunize both military and civil populations against this very serious disease. Since there are a number of strains of influenza virus (Smith and Andrews, 1938) it would seem that for general use a polyvalent vaccine or one with broad antigenic powers should be required. Jenner and his colleagues apparently failed to recognize the underlying principles in the use of attenuated smallpox virus as an immunizing agent. These principles and their broad biological significance Avere disclosed by Pasteur in his studies made a century after Jenner in 1879-80. Immunization With Attenuated Bacteria. — By accident or de- sign Pasteur discovered that chickens inoculated with old or at- tenuated cultures of the organisms of chicken cholera became im- mune to subsequent infection with virulent strains. He had previously noticed that animals recovering from experimental anthrax were immune. It is quite likely that Jenner 's discovery was a factor in enabling Pasteur to grasp the broad fundamental principles involved and to establish the science of preventive in- oculation or immunization upon a firm foundation. His success- ful and spectacular immunization against anthrax and rabies, using attenuated bacteria and virus respectively, placed protective vac- cination upon a firm foundation. Since Pasteur's time the French school has clung tenaciously to the principle of immunization with living attenuated organisms as evidenced by the present use of the B.C.G. (Bacillus of Calmette NATURAL AND ACQUIRED IMMUNITY 113 Hiul Guei'in) vaccine in their campaign against tuberculosis. Al- tention is called in Chapter XXVII to the results of studies by Park and his colleagues on immunization against tuberculosis with B.C.G vaccine. They seemed to find the vaccine of little value in human immunization. It is possible that a new acid-fast organism causing tuberculosis in wild voles (small rodents) in England may be superioi' to B.C.G. The organism originally isolated by Wells (1937) has been cultured on Dorset's egg medium and employed by Wells and Brooks (1940) to immunize guinea pigs against virulent human tubercle bacilli. In their experiments they employed vaccines made from 2 strains of vole acid-fast bacteria, to immunize two groups of guinea pigs; a third group was immunized with B.C.G. vaccine while Group 4 con- sisted of un vaccinated controls. Five months after vaccination. Group 1 was injected with virulent human tubercle bacilli and Group 2 with virulent bovine tubercle bacilli. Group 3 was divided into 2 lots, one receiving virulent human and the second virulent bovine tubercle bacilli. Similar treatment was given to Group 4. The experiments were terminated after 11 weeks because of the war. The results indicate that the vole vaccine gave almost com- plete protection for Groups 1 and 2, that the pigs vaccinated with B.C.G. all showed moderately progressive tuberculosis while the un- vaccinated controls all showed extensive tuberculosis. It is interesting to note that the pigs vaccinated with the vole vaccine did not react to 1 :1,000 dilution of O.T. while those vac- cinated with B.C.G. were positive. The vaccinated animals, how- ever, did react to lower dilutions of O.T. This work warrants further investigation. Immunization With Killed Bacteria. — While it is generally admitted that suspensions of living, attenuated organisms are more potent immunizing agents than suspensions of killed bacteria, the desire for safety has led to the widespread use of the latter. If the organisms used in its preparation are obtained from the pa- tient, the vaccine is described as an '' autogenous" one. When it is prepared from a stock culture it is said to be a "stock vaccine." If more than one kind of bacteria is included in the suspension, it is called a "mixed vaccine." Since the term "vaccine" originally signified a preparation of virus from vaccinia or cowpox, it is evident that its use to desig- 114 IMMUNOLOGY nate an immunizing agent consisting of bacteria is not logical. Usage, however, has led to the policy of applying the terra "vac- cine" to practically all agents employed to produce active im- munity. Agents used to produce passive immunity represent anti- bodies present in or obtained from blood serum and are therefore called sera, antisera or antibody solutions. Bacterial vaccines are employed for many specific purposes which ma}' be enumerated as follows : 1. Bacterial vaccines, especially typlioid vaccine, as well as sterile milk have been used in norisi^ecific protein therapy in such diseases as chronic gonorrheal rheumatism, various other types of arthritis, psoriasis and some other conditions. They are injected to produce a physiological reaction or protein shock in the patient. 2. Specific vaccine therapy has been employed in certain recur- rent or chronic diseases such as acne vulgaris, gonorrheal arthritis, undulant fever, etc. The results reported are not very encourag- ing. Since bacteria have not been incriminated as the cause of the common cold one cannot regard the so-called "cold vaccines" as specific. It is interesting to note that when these "cold vaccines" have been tried out on a fairly large scale and under at least partly controlled conditions, the results have been disappointing. 3. Specific vaccines are injected into animals such as the horse, ox, goat and rabbit to stimulate the production of specific humoral antibodies. The animals are bled and the serum containing anti- bodies is separated from the cellular elements. In the commercial preparation of therapeutic and certain diagnostic sera the globulin fraction of the serimi, containing antibodies, is precipitated, redis- solved and concentrated. The most frequently used antibacterial sera are those for the various t^-pes of pneumococci, meningococci and streptococci. Antisera used to identify E. typhosa, the Sal- vionell€^, various strains of dysentery and other bacteria are pro- duced commercially, but they are frequently produced privately and used without being concentrated. 4. Bacterial vaccines are used quite extensively in prophylactic immunizations against the organisms causing t}T)hoid fever and whooping cough. Typhoid fever is one of the preventable diseases that has decimated armies in the past. According to Jordan (1931) 60 per cent of the German mortality during the Franco- NATURAL AND ACQUIRED IMMUNITY 115 Prussian War (1870-71) was due to typhoid fever. During the Spanish- American War, there Avas one case of typhoid to approx- imately every five men. Largely through the efforts of Wright in England and Russell in the United States, prophylactic inoculation against typhoid and paratyphoid fever was introduced into the British and United States armies, respectively. It was made compulsory in the United States Army in 1911. It should also be added that excellent san- itary precautions have been introduced and enforced. The in- cidence of typhoid fever among the troops of the United States Army during the World War shows quite definitely the impor- tance of these measures. There was only one case for every 3,756 men, which is in marked contrast to one for every five men during the Spanish-American War. After the last war, according to Simmons (1941), the triple vaccine (against typhoid and paratyphosus A and B) was replaced first by typhoid-paratyphoid A and later by a typhoid vaccine only. Simmons states that the army has now gone back to the triple vac- cine. He also mentions that pneumococcus polysaccharide solu- tion is giving encouraging results in experimental immunization against pneumonia. Immunization With Bacterial Polysaccharides. — The employ- ment by the army, as reported by Simmons, of pneumococcus poly- saccharide is justified by the discovery of Francis and Tillett (1930) that Type I pneumococcus polysaccharide injected into human beings gave rise to mouse protecting antibodies. Accord- ing to Horsfall and Goodner (1936) it has been shown that anti- body response to the injection of Type I polysaccharide can be obtained in man, mouse, horse, cat and dog but not in the rabbit, rat, guinea pig and sheep. Boivin (1935, 1936, 1937) has ap- parently isolated carbohydrate-phosphatide compounds from S. enteritidis and 8. aertrycke which stimulated the formation of specific antibodies in rabbits. Similar antigenic polysaccharide- phosphatide complexes have been isolated by Raistrick and Topley. The latter and his associates (1937) obtained antigenic fractions from E. typhosa which contained a large proportion of poly- saccharide. It should be remembered, however, that not all type specific pneumococcus polysaccharides have been shown to stimulate 116 IMMUNOLOGY antibodies when freed from protein, yet all will react with anti- bodies to form precipitates, fix complement, etc. It is always diffi- cult to exclude traces of protein in the polysaccharide preparation a.s important factors in antibody stimulation. WhoopinCx Cough Immunization. — While whooping cough im- munization has been practiced for many years, it is only since Sauer (1933) pul)lished his results using vaccine composed of several freshly isolated smooth hemolytic strains of H. pertussis grown on human l)lood, that its value seems to be established by clinical experience. Since Bradford and Slavin (1937) have reported the isolation of ten atypical strains of //. pertussis like organisms from cases presenting clinical evidence of wliooping cough, it would suggest the existence of more than one serological type or strain of //. pertussis. These atypical strains have been called '^ parapertussis" since they darken the cough plate medium, show early pleo- morphism and to a slight degree are said to resemble B. hronchi- septicus. Sauer (1939) does not approve of combining them with H. pertussis in a vaccine as he thinks this would "weaken the vaccine. ' ' Sauer 's vaccine contains from 15 to 20 billion organisms per cubic centimeter. He recommends giving from 70 to 80 billion bacteria in divided weekly doses over a period of approximately 3 or 4 weeks. In his protocols he mentions giving young nonim- mune children simultaneous injections of 10 to 15 billion in each arm for 3 successive weeks. In Sauer 's hands, the results re- ported are very encouraging since he records failure of the vaccine to protect in only 1.3 per cent of 4,200 cases whereas the incidence in a control group was 13 times as great. Immunization With Detoxified Toxins. — According to Sim- mons (1941) immunization with tetanus toxoid (detoxified toxin) is now employed by practically all armies. The United States Army attempts to establish an initial immunity to tetanus by ad- ministering three 1 c.c. doses of toxoid given three weeks apart. In order to maintain this immunity, an injection of 1 c.c. of toxoid is given at the end of one year, also at the time of departure for active duty in a theater of war if six months has elapsed since the last dose or whenever the soldier is wounded or exposed to tetanus infection. NATURAL AND ACQUIRED IMMUNITY 117 In private practice, tetanus and diphtheria toxoids are employed quite extensively to immunize young children. Since diphtheria toxoid may give severe reactions in older individuals, it is cus- tomary to use toxin-antitoxin in individuals over 8 to 10 years of age. A more extensive discussion of immunization against diphtheria and other toxemic diseases is given in later chapters. Immunization Programs. — It is common practice among pedi- atricians and many general practitioners to follow a definite pro- gram of immunization against the common contagious diseases of childhood. While all physicians do not follow the same pro- gram, a fairly typical one is that suggested hy Neff (1941). He advises that (1) ''smallpox vaccination may be done at the time of birth, either on the delivery table or when coming home from the hospital, but there is sufficient immunity at that time so that the site of vaccination may not take and may have to be repeated at about three months of age. It is well in individual cases to make it a rule to vaccinate at three months of age and repeat each week if a take has not been obtained from the previous attempts. Vac- cination against smallpox should l)e arranged for in any physical examination of a person who has never been successfully vac- cinated. This holds true of all scliool children npon admission. Il is well to inspect the site of vaccination two days following the application of the virus in order to see if there is a reaction of im- munity. In such a case the explanation is furnished for a failure to take in the next few days. It is probable that more successful takes occur from using multiple sites at the time of vaccination. (2) "Whooping cough vaccine may be given at any age in child- hood but as a routine it may be planned for at the age of six months with weekly doses for the required number. There will be fewer reactions if the total amount of vaccine to be used in the individual case is divided up into five graduated doses. It is possible tliat by the use of pertussis antigen, beginning immediately after exposure of the child to a case of whooping cough, the child may be im- munized successfully, or if unsuccessful that the disease when developed will be milder than would otherwise have happened. (3) "Since most infants have relative diphtheria immunity until about nine montlis of age it is well to begin active immunization at that time, using three doses of regular diphtheria toxoid at three- week intervals or alum precipitate toxoid, three doses at two- 118 IMMUNOLOGY month intervals. Two months after either of these immunization programs the Schick test should be done as a routine in order to see that immunity has actually developed. In the infant who has actually received the immunization at that period of life and a Schick test found negative, the Schick test should be repeated once yearly until the age of five years to see that the immiuiity is being held. It is not rare for a child from two to five years to lose im- munity, especially if he has had only one dose of alum toxoid, or two doses of regular toxoid. A check each year is of protective value. (4) "Active immunization against tetanus may be obtained by the use of tetanus toxoid given at two-month intervals and may be combined with the diphtheria toxoid at the first and third doses of that toxoid. It may be prepared separately or combined with the diphtheria toxoid. It is well to use two such doses of toxoid following a passive immunization against tetanus Avith the tetanus antitoxin in which case, however, immunity is probably more complete. Therefore, whenever a protective dose of tetanus anti- toxin is given in cases of an accident the individual should be given a dose of tetanus toxoid within two weeks and follow this two months later with a second tetanus toxoid. (5) ''Scarlet fever active immunization at the second or third year of age is advisable. Most children are susceptible at that age. One objection to the Dick toxin active immunization has been the reactions accompanying the rapidly increasing repeated doses recommended by Dick. This objection can be overcome by using half of the ampule at the injection, thus making ten doses rather than the regular five. There is practically no reaction from this attenuated dose. It is well to do a Dick test first and if negative it will be unnecessary to go ahead with immunization. The Dick test should be repeated two months after the series of injections so that one can see that there is a reversal of the previous positive test. (6) "Measles Immune Globulin will produce passive immunity if injected within a day or two after exposure to measles. This should be done in frail, delicate, young children. But a modifica- tion of the disease may be obtained by one or two doses of this preparation given from six to eight days after a known exposure. NATURAL AND ACQUIRED IMMUNITY 119 Fifteen c.c. of i)urciital whole blood will accomplish much of the same effect given intramuscularly. ' ' There are a number of reasons why such a program is desirable : (1) experience has shown that severe reactions are least likely to occur when immunization i.s carried out early in life; (2) it gives reasonable assurance of protection before exposure usually occurs; (3) it will probably lead to a larger proportion of the population being immune; (4) it spreads the injections of foreign protein out over a reasonable period of time. The question of whether the child has developed immunity in response to vaccination often arises. There is usually little doubt as to whether the vaccination against smallpox is successful and the Schick test is a fairly accurate test for immunity to diphtheria. Many regard the Dick test as a good criterion for scarlet fever im- munity, but this is much more in dispute than the results of the Schick test. In regard to immunity to tetanus, it is possible to determine the amount of antitoxin in the child's blood, but this involves too much time and expense to be made a routine test; hence it is assumed that immunity develops following the injection of tetanus toxoid. There is a great deal of clinical and laboratory data to support this assumption. There is, at present, no way of measuring a child's immunity to H. pertussis although his humoral (antibody) immunity can be measured. Clinical data, however, such as those reported by Sauer (1933) and others support the assumption that immunity results from the administration of his vaccine. It should be borne in mind that the local or systemic re- action to the injection of a bacterial vaccine is no index of either susceptibility or immunity. The question is often asked as to why several instead of one in- jection of vaccine is given. There are probabl}^ several reasons for this. Dean and others seem to have shown that in developing humoral immunity, the first one or two injections are the primary stimulus and that to obtain best results, a secondary stimulus should be given about the time the titer reaches its peak, the ob- ject being to let some immunity develop before giving the sec- ondary' stimulus. With bacterial antigens the antibody titer usu- ally rises for 7 to 10 days after an injection while the antibody titer following toxoid is much slower in reaching a peak. Ex- l)erience has shown that a spacing of one to two months is de- 120 IMMUNOLOGY sirable, although for convenience shorter intervals are often used. After an immunity is once established, the question of how soon is it necessary to revaecinate arises. In the case of smallpox, the Kansas Citj^ epidemic showed that no one successfully vaccinated within six months of exposure con- tracted the disease. Ordinarily it would seem wise to revaecinate whenever exposure occurs. If one is immune, the onlj- reaction will be the immune reaction described by Jenner and this is not ob- jectionable. In the case of typhoid immunization, many advise revaccination every 2 years. With whooping cough and tetanus it is possible that immunity will last for several years although frequently revaccination may be indicated when exposure occurs. The time for revaccination against diphtheria can be determined by means of the Schick test. An interesting phenomenon that may be of importance is the anmnnestic reaction which is the basis for the timing of the sec- ondary stimulus in vaccination. While it takes days or weeks for antibodies to develop following the one or more doses constituting the primary stimulus, after immunity is established, a secondary stimulus or injection of vaccine will lead to a rapid formation of antibody. It has been noted that in children whose Schick test was negative following immunization and a few years later be- came positive the amount of toxin used in the positive Schick test caused a rapid rise in their antitoxin titer so that in retesting they were negative. This is an example of the anamnestic reaction. It is because of this ''hair-trigger" mechanism (anamnestic re- action) that revaccination following exposure is substituted. The revaccination dose is a secondary stimulus and causes a rapid rise in antibody titer. It is well established that the secondary stimulus does not have to be a large dose of antigen. It should be remembered that, granting a good antigen and cor- rect method of administering a vaccine, some individuals do not develop immunity. Furthermore, killed bacterial antigens (e.g., E. typhosa and H. pertussis) do not stimulate as great an im- munity as results from the disease. An explanation of this is, in part at least, to be found in the discoveries of Felix and Pitt (1934) and others studying the NATURAL AND ACQUIRKD IMMUNITY 121 antigenic components of E. typhosa and Mudd, Pcttit, and Lack- man and Morgan (1939) studying the antigenic components of virulent streptococci. This work has been mentioned under viru- lence in Chapter I. It will be recalled that virulence is associated with capsule formation and that each kind of bacterial cell is made up of a number of antigenic components, any one of which may give rise to antibodies, depending perhaps upon its location in the cell. It ha.s been emphasized by Landsteiner, Nicollc, Zinsser and others that there is a "mosaic" of antigens in a bacterial cell. The position and the way the antigens are oriented relative to the surface are important factors. The surface antigens may act as a barrier to antigenic components beneath the surface. These are im- portant concepts when one is considering the factors that determine the immunizing value of a vaccine. It is known, e.g., that E. typhosa can dissociate into smooth motile, smooth nonmotile, rough motile and rough nonmotile variants. It was thought, until 1934, that the best typhoid vaccine should consist of any smooth motile strain because it would contain 0 (somatic) and H (flagellar) antigens. Felix and Pitt, however, discovered that an additional antigenic factor determining viru- lence is important. They named this factor the "Vi" antigen.* It is apparently a surface antigen and is destroyed by lieat and many chemicals. More recently Mudd and his associates (1938, 1939) have shown that virulent streptococci contain a partial heat labile antigen obtained by physical means of disintegration. It is prob- able that many and perhaps all virulent bacteria contain labile antigens important in immunization. It is perhaps because of the heat lability of these antigens and the ease with which they are destroj^ed by most chemicals that killed bacteria are not as good immunizing agents as virulent living ones. Apparently formaldehyde can be used to kill bacteria with only partial destruction of the "Vi" antigen. This may be the reason formalized vaccines have been reported as superior to heat killed ones. Passive Immunity. — When a serum containing antibodies is in- jected into a patient, the latter is being passively immunized. His own tissue cells did not produce these antibodies. In other words, ♦Topley and associates have isolated two carbohydrate-lipid complexes cor- responding to O and Vi antigens respectively. Lancet 2.S2: 252, 193 7. 122 IMMUNOLOGY the patient is passively accepting antibodies produced by another animal. The administration of antitoxin to prevent either tetanus or diphtheria is conferring passive immunity to the recipient. References Anderson, E. J. M., and Fraser, A. H. H. : The Influence of Nutrition on the Natural Jiniimnity Eeactions of tlie Blood and on Skin Reactions to Bacterial Toxins, J. Immunol. 27: 1, 1934. Aycock, W. L.: Immunity in Virus Diseases, J. A. M. A. 97: 1199, 1931. Baumgartner, L.: The Eelationship of Age to Immunological Eeactions, Yale J. Biol. & Med. 6: 403, 1933. Baumgartner, L. : Age and Antibody Production. I. Qualitative Changes in Antisera Associated With Age, J. Immunol. 27: 407, 1934. Baumgartner, L.: Age and Antibody Production. II. Further Observations on Qualitative Changes in Antisera Associated With Age, J. Immunol. 27: 417, 1934. Baumgartner, L. : Age and Antibody Production. 111. Quantitative Studies on the Precipitin-Eeaction With Antisera Produced in Young and Adult Rabbits, J. Immunol. 33: 477, 1937. Bay-Schmith, E.: S'ersuche iiber die Schicksche Reaktion bei Eskimos in Gronland, Klin. Wclmschr. 8: 974, 1929. Belding, D. L., and Wyman, L. C: The Role of the Suprarenal Gland in the Natural Resistance of the Rat to Diphtheria Toxin, Am. J. Physiol. 78: 50, 19:^6. Boivin, A., and Mesrobeanu, L. (193oj. Cited by Zinsser, H., Enders, J. F., and Fothergill, L. D. : Immunity, ed. 5, Resistance to Infectious Dis- eases, New York, 1939, The MacmiUan Co., p. 75. Boivin, A., and Mesrobeanu, L. (1930). Ibid. Boivin, A., and Mesrobeanu, L. (1937; . Ibid. Britten, S. W., and Silvette, H. : On the Function of the Adrenal Cortex — General, Carbohydrate, and Circulatory Theories, Am. J. Physiol. 107: 190, 1934. Burnett, F. M. : Intiuenza Virus on the Developing Egg. The Pathogenicity and Immunizing Power of Egg Virus for Ferrets and Mice, Brit. J. Exper. Path. 18: 37, 1937. Burnett, F. M. : The Specificity of Active Immunity in Mice Against In- fluenza Virus, Ibid, 19: 388, 1938. Casals, J.: Influence of Age Factors on Susceptibility of Mice to Rabies Virus, J. Exper. Med. 72: 445, 1940. Casals, J.: Influence of Age Factors on Immunizability of Mice to Rabies Virus, J. Exper. Med. 72: 453, 1940. Casals- Ariet, J., and Webster, L. T.: Age Factors in Susceptibility and Immunizability of Mice to Rabies Virus, J. Bact. 39: 66, 1940. Council in Pharmacy and Chemistry and Council in Foods: The Status of Certain Questions Concerning Vitamins Based on the Recommenda- tions of the Cooperative Committee on Vitamins, J. A. M. A, 113: 589, 1939. Culbertson, J. T., and Kessler, W. R.: Studies on Age Resistance Against Trypanosome Infections, Am. J. Hyg. 29: 33, 1939. Editorial: Panimmunity, J. A. M. A. 96: 775, 1931. Editorial: Phylogenetic Immunologic Recapitulation, J. A. M. A. 96: 950, 1931. Editorial: A Poliomyelitis Vaccine, J. A. M. A. 103: 264, 1934. Editorial: Upsetting Immunologic Tenets, J. A. M. A. 96: 1:232, 1931. NATURAL AND ACQUIRED IMMUNITY 123 Eusterman, G. B., and Wilbur, D. L. : Chicago, 1932, American Medical Association. The Vitamins, p, 21. Felix, A., and Pitt, R. M. : A New Antigen of B. typhosus, Lancet 2: 186, 1934. Finland, M., and Sutliff, W. D. : Immunity Eeactions of Human Subjects to Strains of Pneumococci Other Than Types I, II, and III, J. Exper. Med. 57: 95, 1933. Francis, T., and Tillett, \V. S. : Cutaneous Eeactions in Pneumonia; Develop- ment of Antibodies Following Intradermal Injection of Type-Specific Polysaccharide, J. Exper. Med. 52: 573, 1930. Gallagher, F. W., and Woolpert, O. C. : Propagation of Vaccine Virus in the Eabbit Fetus, J. Exper. Med. 72: 99, 1940. Hartman, F. A., Brownell, K., and Lockwood, J. E. : Cortin as a General Tissue Hormone, Am. J. Physiol. 101: 50, 1932. Hartman, F. A., Brownell, K., and Lockwood, J. E.: Studies Indicating the Function of Cortin, Endocrinology 16: 521, 1932. Heinbecker, P., and Irvine- Jones, E. I. M.: Susceptibility of Eskimos to the Common Cold and a Study of Their Natural Immunity to Diphtheria, Scarlet Fever, and Bacterial Filtrates, J. Immunol. 15: 395, 1928. Hirayama, T. : Ueber die Eesistenz tuberculos infizierter Tiere gegen andere nachtragliche experimentelle Infektionen: 1. Einfluss der Infektion mit Tuberkel-bazillen auf den Verlauf von Milzbrand, Ztschr, f. Im- munitatsforsch. u. exper. Therap. 68: 218, 1930. 2. Einfluss der ex- perimentellen Tuberkulose auf Htreptokokkeninfektion oder auf Diph- therieintoxikation. Ibid., p. 2;^0. Hirszfeld, H., and Hirszfeld, L. : Weitere Untersuchungen iiber die Vererbung der Empfanglichkeit fiir Infektionskrankheiten, Ztschr. f. Immunitats- forseh. u. exper. Therap. 54: 81, 1927. Hirszfeld, H., Hirszfeld, L., and Brokman, H. : On the Susceptibility to Diphtheria (Schick Test Positive) With Reference to the Inlieritance of Blood Groups, J. Immunol. 9: 571, 1924. Hirszfeld, H., Hirszfeld, L., and Brokman, H. : Untersuchungen iiber Vererbung der Disposition bei Infektionskrankheiten, speziell bei Diphtheria, Klin, W^chnschr. 3: 1308, 1924. Hirszfeld, H.: Die Konstitutionslehre im Lichte serologischer Forschung, Klin. Wchnschr. 3: 1180, 1924. Horsfall, F. L., Jr., and Goodner, K.: Lipids and Immunological Eeactions; Further Experiments on Eelation of Lipids to Type-Specific Eeactions of Anti-pneumococcus Sera, J. Immunol. 31: 135, 1936, Horsfall, F. L., Jr., and Lennette, E. H.: The Synergism of Human In- fluenza and Canine Distemper Virus in Ferrets, J. Exper, Med. 72: 247, 1940. Horsfall, F, L., Jr., Lennette, E. H., and Eickard, E. E. : A Complex Vaccine Against Influenza A Virus. A Quantitative Analysis of the Antibody Eesponse Produced in Man, J. Exper. Med. 73: 335, 1941. Irwin, M. E. : The Inheritance of Eesistance to the Danysz Bacillus in the Eat, Genetics 14: 337, 1929. Irwin, M, E. : Inlieritance as a Factor in Resistance to an Infectious Disease. I. The Uniform Eeaction of an Inbred Strain of Animals, J, Immunol, 24: 285, 1933, Irwin, M, E., and Hughes, T. P.: Inheritance as a Factor in Eesistance to an Infectious Disease. VI. The Correlation Between Eesistance and the Bactericidal Power of the VThole Blood, J. Immunol. 24: 343, 1933. Jaffe, H. L. : On Diminished Eesistance Following Suprarenalectomy in the Eat and Protection Afforded bv Autoplastic Transplants, Am. J. Path. 2: 421, 1926, 124 IMMUNOLOGY Jaff6, H. L., and Marine, D. : Effect of Suprarenalectomy in Eats on Agglutinin Formation, J. Infect. Dis. 35: 334, 1924. Jenner: Vaccination, by George Dock, Osier's Modern Medicine, New York, 1907, Lea Brothers & Co., Chap. X, p. 301. Jordan, E. O.: Textbook of Bacteriology, Philadelphia, 1938, W. B. Saunders Co., p. 330. Jungeblut, C. W., Meyer, K., and Engle, E. T.: Inactivation of Polio- myelitis Virus and of Diphtheria Toxin by Various Endocrine Prin- ciples, J. Immunol. 27: 43, 1934. Kessel, J. F., and Stimpert, F. D.: Immunity in Monkeys Recovered From Paralytic Attacks of Poliomyelitis, J. Immunol. 40: 61, 1941. See also editorial : Immunity vs. Antibody Titer in Poliomyelitis, J. A. M. A. 116: 1146, 1941. King, L. S. : Studies on Eastern Equine Encephalomyelitis, ,T. Exper. Med. 71: 95, 1940. Kuttner, A., and Ratner, B. : Importance of Colostrum to the Newborn In- fant, Am. .1. Dis. Child. 25: 41?,, 192.*^. Lambert, W. V.: Natural Resistance to Disease in the Chicken. I. The Effect of Selective Breeding on Natural Resistance to Fowl Typhoid, J. Immunol. 23: 229, 19.'',2. Leasure, E. E. : Feline Infectious Enteritis, North Amer. Vet. 15: 30, 193-1. Lewis, Paul A., and Loorais, Dorothy: Allergic Irritability. IV. Tiie Capacity of Guinea Pigs to Produce Antibodies as Affected by the Inheritance and as Related to Familial Resistance to Tuberculosis, J. Exper. Med. 47: 437, 1928. Tjcwis, Paul A., and Loomis, D. : Ulcerative Types as Determined by In- heritance and as Related to Natural Resistance Against Tuberculosis : An Experimental Study on the Inbred Guinea Pigs, .1. Exper. Med. 47: 449, 1928. Macklin, Madge T.: The Role of Heredity in Disease, Medicine 14: 1, 1935. Mackie. T. T., Eddy, W. H., and Mills," M. A.: Vitamin Deficiencies in Gastrointestinal Disease, Ann. Int. Med. 14: 28, 1940. Madsen, T.: Vaccination Against Whooping Cough, ,T. A. M. A. 101: 187, 1933; The Abraham Flexner Lectures. Ser. 5, p. 216, Baltimore, 1937, Williams & Wilkins Co. Morgan, I. M. : Relation of Age to Immune Response of Mice to Formolized Equine Encephalomyelitic Virus, Proc. Soc. Exper. Biol. & Med. 42: 501, 1939. Morrison, A. P., Shaw, D. R., Kenney, A. S., and Stokes, J., Jr.: Complement Fixation Studies on the Sera of Individuals Vaccinated With Active Virus of Human Influenza, Am. J. M. Sc. 197: 253, 1939. Mudd, S., Pettit, H., Lackman, D. B., and Morgan, I. M. : The Antigenic Structure of Hemolytic Streptococci of Lancefield Group A, J. Immunol. 36: 381, 1939. Neff, F. C: Personal communication, 1941. Neufeld, F., and Etinger-Tulczynska, R.: Experimentelle Untersuchungen iiber die zeitlichen Schwankungen der natiirlichen Empfanglichkeit fiir Infektionen, Ztschr. f. Hyg. u. Infektionskr. 115: 573, 1933. Newsholme, A.: Elements of Vital Statistics and Their Bearing on Public Health, New York, 1923, D. Appleton-Century Co., Inc. Olitsky, P. K., Sabin, A. B., and Cox, H. R. : Acquired Resistance of Growing Animals to Certain Neurotropic Viruses in Absence of Humoral Antibodies or Previous Exposure to Infection, .7. Exper. Med. 64: 723, 1936. Park, W. H., Kereszturi, C, and Mishulow, L. : Effect of Vaccination With B. C. G. on Children From Tuberculous Families, J. A. M. A. 101: 1619. 19.33. NATURAL AND ACQUIRED IMMUNITY 125 Picado, C. : Proprietes antigeniques diflferentes des serums d 'Aniniaux jeunes et des serums d 'Animaux ages, Compt. rend. Soc. de biol. 102: (531, 1929. Pritchett, I. W.: Microbic Virulence and Host Susceptibility in Paratyphoid- Enteritidis Infection of White Mice. VI. The Relative Susceptibility of Different Strains of Mice to Per Os Infection With the Type II Bacillus of Mouse Typhoid (Bacillus pestis caviae), J. Exper. Med. 41: 195, 1925. Further studies, Ibid. 43: 161, 1926. Raistrick, H., and Topley, W. W. C. : Immunizing Fractions Isolated From Bact. Aertrycke, Brit. J. Exper. Path. 15: 113, 1934. Rivers, T. M.r Filterable Viruses, Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, Univ. of Chicago Press, p. 517. Rivers, T. M.: The Nature of Viruses, Physiol. Rev. 12: 423, 1932. Rivers, T. M., and Ward, S. M. : Further Observations on the Cultivation of Vaccine Virus in Lifeless Media, J. Exper. Med. 57: 741, 1933. Rivers, T. M., and Schwentker, F. F. : Louping 111 in Man, J. Exper. Med. 59: 669, 1934. Rivers, T. M., Ward, S. M., and Baird, R. D. : Amount and Duration of Immunity Induced by Intradermal Inoculation of Cultured Vaccine Virus, J* Exper. Med.' 69: 857, 1939. Robertson, E. C. : The Vitamins and Resistance to Infection, Medicine 13: 123, 1934. Robinson, E. S. : Methods of Preparation and Use of Smallpox Vaccine Viru.s. Virus and Rickettsial Diseases, Harvard Univ. Press, 1940, p. 201. Rogoff, J. M., and DeNecker, J.: The Influence of the Adrenals on the Toxicity of Morphine, J. Pharmacol. & Exper. Therap. 26: 243, 1926. Rogofl', J. M., and Ecker, E. E.: Suprarenalectomy and Susceptibility to Tetanus Toxin, Tr. Arch. Path. Lab. Med. Soc. 1: 309, 1926. Rosling, E.: Zur Kritic der Hirszfeldschen Hypothese iiber den genctischen Zusammenhang zwischen Blutgruppe und Schickschcr Reaktion, Ztschr. f. Immunitatsforsch. u. exper. Therap. 59: 521, 1928. Sauer, L. W. : Immunization With Bacillus Pertussis Vaccine, J. A, M. A. 101: 1449. 1933. Sauer, L. W.: Whooping Cough, Ibid. 100: 239, 1933. Sauer, L. W. : Preparation of Bacillus Pertussis Vaccine for Immunization, Ibid. 102: 1471, 1934. Sauer, L. W.: Whooping Cough, Ibid. 112: 305, 1939. Scammon, R. E.: New Phases of Work on Immunization and Prophylaxis, Morris' Human Anatomy, Philadelphia, 1925, The Blakiston Co., p. 30. Schott, R. G.: The Inheritance of Resistance to Salmonella Aertrycke in Various Strains of Mice, Genetics 17: 203, 1932. Scott, \V. .1. M. : The Influence of the Adrenal Glands on Resistance. II. The Toxic Efl'ect of Killed Bacteria on Adrenalectomized Rats, J. Exper. Med. 39: 457, 1924. Sherwood, N. P., Nigg, C, and Baumgartuer, L.: Studies on the Dick Test and Natural Immunity to Scarlet Fever Among the American Indians, J. Immunol. 11: 343, 1926. Simmons, J. S. : Immunization Against Infectious Disease in the United States Army, South. M. J. 34: 62, 1941. Smitli, W., Andrews, C. H., and Stuart-Harris: Great Britain Medical Re- search Council Special Report Series 228, 1938. Smith, Theobald : The Degree and Duration of Passive Immunity to Diphtheria Toxin Transmitted by Immunized Female Guinea Pigs to Tiicir Immediate Oif spring, J. M'. Research 16: 359, 1907. 126 IMMUNOLOGY Smith. W.. and Andrews, C. H. : Serological Eaces of Influenza Virus. Brit. J. Exper. Path. 19: 293, 1938. Snyder, L. H. : Studies in Human Inheritance, and the Linkage Eelation? of the Blood Groups, Ztschr. f. Immunitatsforsch. u. exper. Therap. 49: 464, 1926, Stewart, G. N., and Rogoff, J. M.: The Influence of Morphine on Normal Cats and on Cats Deprived of the Greater Part of the Adrenals, With Specific Reference to Body Temperature, Pulse, and Respiratory Fre- quency and Blood Sugar Content, J. Pharmacol. & Exper. Therap. 19: 97, 1922. Stokes, J., McGuinness, A. C, Langner, P. H., Jr., and Shaw, D. R. : Vac- cination Against Epidemic Influenza With Active Virus of Human Influenza, Am. J. M. Sc. 194: 757. 1937. Sutliff, W. D., and Finland, M.: Antipneumococci Immunity Reactions in Individuals of Different Ages. J. Exper. Med. 55: 837, 1932. Swingle, W. W., Pfifi'ner, J. J., Vars. H. M., and Parkins, W. M.: The EflFect of Hemorrhage on the Normal and Adrenalectomized Dog, Am. J. Physiol. 107: 259, 1934. Szent-Gyorg>-i, A. v.: On Oxidation. Fermentation, Vitamins, Health and Disease, Baltimore, 1939, Williams & Wilkins Co. Taylor, R. M., and Dreguss. M. : Experiment in Immunization Against In- fluenza With Formaldehvde-Inactivated Virus, Am. J. Hyg. 31: 31, 1940. Topley, W. W. C. and Wilson. G. S. : The Principles of Bacteriology and ' Immunity, New York, 1929. William Wood & Co., p. 767. Topley, W. W."^C., Raistrick, H., Wilson, J., Stacey, M., Challinor. S. W., and Clark, R. O. J.: Immunizing Potency of Antigenic Components Isolated From Different Strains of Bact. Typhosum, Lancet 1: 252, 1937. Toyoda, T., Moriwaki, J., and Futagi, Y. : Does the Dick Reaction With Streptococcus Toxin Indicate Susceptibility to Scarlet Fever? J. Infect. Dis. 46: 186, 1930. Veintemillas, F. : Vaccination Against Typhus Fever With tlic Zinsser- Castaneda Vaccine, J. Immunol. 36: 339, 1939. Vitamins, Chicago, 1932, American Medical Association. Von Dungern and Hirschfeld (1910). Cited by Ottenberg, R., and Bcres, O. : Newer Knowledge of Bacteriology and Immunology, Chicago, 1928. University of Chicago Press, p. 11. Warthin, A. S. : Old Age, New York, 1930, Paul B. Hoeber, Inc. Webster, L. T. : Microbic Virulence and Host Susceptibility in Mouse Typhoid Infection, J. Exper. Med. 37: 231, 1923. Webster, L. T.: Microbic Virulence and Host Susceptibility in Para- t^-phoid-Enteritidis Infection of White Mice. IV. The Effect of Selective Breeding on Host Resistance, J. Exper. Med. 39: 879, 1924. Webster, L. T., and Pritehett, I. W. : Microbic Virulence and Host Suscepti- bility in Paratyphoid-Enteritidis Infection of White Mice. A'. The Effect of Diet on Host Resistance, J. Exper. Med. 40: 397, 1924. Webster, L. T.: Microbic Virulence and Host Susceptibility in Paratyphoid - enteritidis Infection of White Mice. VIII. The Effect of Selective Breeding on Host Resistance. Further Studies, J. Exper. Med. 42: 1, 1925. Webster, L. T. : Inherited and Acquired Factors in Resistance to Infection ; Development of Resistant and Susceptible Lines of Mice Through Selective Breeding, J. Exper. Med. 57: 793, 1933. Inherited and Ac- quired Factors in Resistance to Infection; Comparison of Mice In- herently Resistant or Susceptible to Bacillus Enteritidis Infection With Respect to Fertility, Weight, and Susceptibility to Various Routes and Tj-pes of Infection, Ibid., p. 819. NATURAL AND ACQUIRKI) IMMUNITY 127 Webster, L. T., and Burn, C. G. : Biology of Bacterium Lepisepticum. III. Physical, Cultural, and Growth Characteristics of Diffuse and Mucoid Types, and Their Variants, J. Exper. Med. 44: 343, 1926. Webster, L. T., and Burn, C. G.: Biology of Bacterium Lepisepticum. IV. Virulence of Diffuse and Mucoid Types, and Their Variants, J. Exper. Med. 44: 359, 1926. Webster, L, T., and Hodes, H. L.: Role of Inborn Eesistance Factors in Mouse Populations Infected With Bacillus Enteritidis, J. Exper. Med. 70: 193, 1939. Wells, A. Q.: Tuberculosis in Wild Voles, Lancet 1: 1221, 1937. Wells, A. Q., and Brooke, W. S.: The Effect of Vaccination of Guinea Pigs With the Vole Acid-Fast Bacillus on Subsequent Tuberculosis Infection, Brit. J. Exper. Path. 21: 104, 1940. See also Editorial in J. A. M. A. 116: 509, 1941. Wells, J. R. : The Origin of the Immunity to Diphtheria in Central and Polar Eskimos. I. A Study of the Throat Flora, Am. J. Hyg. 18: 629, 1933. II. Epidemiological and Serological Studies, Ibid., p. 656. Zinsser, H. : Immunology of Infections by Filtrable Agents and Epidemiology and Immunity in Rickettsial Diseases. Virus and Rickettsial Diseases, Harvard Univ. Press, 1940, pp. 89 and 872. Zinsser, H., and Bayne- Jones, S.: Textbook of Bacteriology, ed. 8, New York, 1939, D.'Appleton-Century Co., p. 791. Zinsser, H., Enders, J. F., and Fothergill, L. D.: The Prophylaxis of Pneu- monia by Vaccines and Various Immunologic Constituents of Pneumo- coccal Cells, Immunity, ed. 5, Eesistance to Infectious Diseases, New- York, 1939, The Macmillan Co., p. 688. Supplementary References Almon, L., and Stovall, W. D.: A Study of ''Vi" Antigen of Felix and Pitt, J. Immunol. 31: 269, 1936. Campbell, D. H.: The Effect of Sex Hormones on the Normal Eesistance of Rats to Cysticercus Crassicollis, Science 89: 415, 1939. CHAPTER VII IMMUNITY MECHANISMS IN EXPERIMENTAL INFECTIONS The question of resistance to infection lias been studied experi- mentally with considerable profit since Ashoff and Maximow crystallized oiir present concept of the retieulo-endothelial system. This chapter will be devoted to summarizing results and conclu- sions of Gay, Cannon, Teale, Goodpasture and others who have investigated the mechanism of resistance employed by the animal body to localize infection or to free the blood stream of bacteria and to favor recovery from infection. Experimental Streptococcus Infections in Rabbits. — Gay (1923, 1926) and his colleagues studied the defensive mechanism of the pleural cavity of rabbits against experimentally produced infec- tion with a highly virulent hemolytic streptococcus. They first injected a sterile irritant (aleuronat) which caused an inflam- matory reaction in the pleura with the formation of granulation tissue. Polymorphonuclear leucocytes appeared in great numbers early in the reaction. Later there was an increase in mononuclear phagocytes both in the exudate and within the tissues. If virulent streptococci were introduced when the neutrophiles predominated, the animals died as rapidly as the controls. If they were injected after the mononuclear phagocytes were mo- bilized, the animals could withstand many lethal doses of strepto- cocci inoculated either into the pleural cavity on the side where granulation tissue had been produced or into the opposite side where none was present. In the latter case the protection was due to clasmatocytes coming over as reinforcements from the depot of mobilization in the opposite side. Gay and his associates speak of this as "transpleural mohilization" of clasmatocytes. Linton (1928), working in Gay's laboratory, transplanted irri- tated omentum (rich in clasmatocytes) into the peritoneal cavity of normal rabbits and found that the rabbit's resistance to intra- pleural infection with Streptococcus liemolyticus was greatly in- creased. 128 IMMUNITY IN EXPERIMENTAL INFECTIONS 129 Importance of Antibodies. — More recently Gay and Clark (1930)* have offered experimental evidence indicating that anti- Ijodies enhance the cellular defensive mechanism. In their opinion the reason that antibacterial, e.g., antistreptococcal immune serum, fails to combat a streptococcus infection is not a lack of antibodies but an absence of mobilization of clasmatocytes in the patient. They found that "in the case of experimental streptococcus empyema in the rabbit the course of the ordinary fatal infection is in no wise affected by the transfer of the pleural fluid contain- ing large numbers of mononuclear cells derived from an animal that is protected as a result of nonspecific irritation. The serum of a rabbit highly immunized against the streptococcus and con- taining antibodies for it, produces relatively slight effect in pre- vention or cure. ' ' Passive Immunization With Pleural Exudate. — "In contrast to this the pleural exudate, either acute (polymorphonuclear) or subacute (mononuclear) produced in an actively immunized animal does protect passively to a considerable degree. In a similar fashion normal exudate cells of either type in combination with the relatively ineffective antiserum give a high degree of protection." In other studies Gay and Oram (1931) report that clasmatocytes are much more resistant to Streptococcus leucocidin than the neu- trophils. They regard this as further evidence of the superiority of "tissue macrophages" over neutrophiles in combating strepto- coccus infections. Experimental Staphylococcus Infections in Guinea Pigs. — In regard to tissue resistance to staphylococcus infections Freedlander and Tooiney (1928) have studied nonspecific mechanisms and Can- non et al. specific factors in the skin of guinea pigs. The former compared the inflammatory response in the subcutaneous tissues of normal guinea pigs with the response in guinea pigs whose skins had been treated with nonspecific irritants. They observed that the latter were protected for short periods of time by tissue macrophages mobilized by nonspecific irritation. Cannon et al. (1930, 1932) have investigated tissue immunity to staphylococcus infection in normal and vaccinated guinea pigs. They find that when staphylococci are injected into the skin of the abdominal wall of normal guinea pigs there results an inflammation •Gay, F. P., and Clark, A. R. : J. Exper. Med. 52: 94, 1930. 130 IMMUNOLOGY characterized by an infiltration oL' neutroi)liilcs which actively phagocytize the bacteria. This process does not lead to a local- ization of the infection, but instead, the latter spreads throughout the subcutaneous tissue in the form of a cellulitis. On tlie other hand, wlien they inject staphylococci into the skin of previously immunized pigs the organisms are agglutinated into masses of various sizes, and there is an infiltration of cells from the subreticular layer of the cutis where vast numbers of "tissue macrophages" have accumulated as a result of the previous intra- cutaneous vaccination. ]\Iany of the clumps of bacteria are ]^hagocytized by the large mononuclear pliagocytes and the infec- tion remains localized ])ecause of phagocytosis and other factors. Local Fixation. — In discussing their results Cannon and Pacheco (1930) call attention to the significant work of Opie (1929) which enabled them to offer a rational explanation of the observed phenomena. Opie studied the Arthus phenomenon and concluded that when one injects an antigen into tissues containing antil)odies the resultant antigen-antibody complex acts as a tissue irritant, with a resulting inflammatory reaction characterized by an infiltration of neutrophiles, edema, and the deposition of fibrin. The small blood and lymph vessels are injured and become throm- bosed. He obtained similar results when he injected antigen and antibody simultaneously into the tissues of the rabbit. Of con- siderable significance is his observation that antigen injected into the skin of normal rabbits passes rapidly into the blood whereas antigen injected into immune animals is fixed at the i>oint of injec- tion. When along with these results one takes into consideration the work on opsonification discussed in Chapter V, it would appear that Cannon's explanation of tissue immunity to staphylococci observed in the immunized guinea pigs may be summarized as follows : Summary of Cannon's Work on Tissue Immunity to Staphy- lococci.— 1. As a result of intracutaneous vaccination there is a mobiliza- tion of mononuclear phagocytes within the subreticular layer of the cutis. 2. There is an accumulation of antibody (opsonins, agglutinins, etc.) within the tissues which is perhaps adsorbed by tissue cells. IMMUNITY IX EXPERIMENT^NX, INFECTIONS 131 Since in Cannon's opinion the antibodies are produced largely by the reticulo-endothelium, it is ])robable that antibody is also con- tained within the tissue macrophages. 3. When virulent staphylococci are injected after the guinea pigs have become sensitized, the bacteria adsorb antibody, there is an increase in the coliesive (sticky) propertj* of their surface and as a result of physical-chemical changes many of them agglutinate. This antigen-antibody complex should lead to anaphylactic inflam- mation. 4. The filming of bacteria by antibody with resulting surface changes constitutes the process of opsonification which favors phagocytosis. 5. The bacteria are pliagocytized and destroyed Avithin the food vacuoles of the clasmatocytes. The latter contain antibody wliich means that tliey may be affected by antigen-antibody reactions. Tliis may be a factor in the development of an intense secondary inflanuuation accompanied l)y an increase of neutrophiles and clasmatocytes. The organisms remain localized not only as a result of the edema and thrombosis of blood and lymph vessels but also because of the factors just mentioned. Immunity to Pneumococcus Infection. — There is more or less controversy in regard to the defensive mechanism against pneu- mococcus infection. Gay and Clark (1930) say that while mononu- clear mobilization by nonspecific irritation is effective against streptococci, it is of little value in pneumococcus infection unlass the pneumococci are first treated with a specific immune serum. Apparently when the pneumococci are opsonized the mobilized mononuclear defense is effective. Nakahara has also investigated the effectiveness of clasmatocytes mobilized in the walls of the ])eritoneum of mice by the intraperitoneal injection of olive oil. He found increased resistance to staphylococci, pneumococci and li. coli. Cannon and his associates, Walsh and Hartley (1936, 1938), have studied upper respiratory tract infections in normal, immune and allergic rabbits. They have also investigated the effect of various substances used in intranasal medication upon the de- 132 IMMUNOLOGY fensive mechanisms of the respiratory tract. The results of their work may be summarized as follows : 1. By intranasal vaccination with a formalin-killed culture of pneumococcus Type I or with an autolysate they were able to con- fer practically complete protection against living pneumococci in- troduced intranasally. 2. Such nonspecific irritants as formalin, tannic acid, alum or a paratyphoid vaccine used as intranasal stimulants did not con- fer protection against intranasal infection with pneumococci. 3. They found intranasal immunization superior to intragastric immunization with a pneumococcal vaccine. 4. They found no correlation between antibody titer in the blood and intranasal immunity since intranasally vaccinated rabl)!ts, having no demonstrable antibodies in their blood, resisted intra- nasal infection. 5. Successful intranasal vaccination did not prevent the aspira- tion of intranasally instilled pneumococci into the lungs. It did modify their passage from the lungs to the blood possibly because of a general immunity and perliaps because of a locally enhanced capacity of the lungs to inhibit growth and spread of the pneu- mococci reaching them. 6. They concluded that when rabbits are kept in a normal posi- tion the virulent pneumococci instilled intranasally do not ])ass through the epithelium of the upper respirator}^ tract but are aspirated into the lungs and then enter the blood. 7. They confirmed the work of Freund (1927) and Freund and Whitney (1928) that, following the development of general active or passive immunity, the antibodies are found rather uni- formly distributed throughout the tissues and the ratio of tissue antibod.y to serum antibody is usually between 1:10 and 1:15. This is called the T :S ratio. 8. When animals were vaccinated regionally as by intranasal histillation the regional tissue (mucous membrane) serum ratios of antibody was on an average of one to five. Cannon et al. state that this suggests that perhaps the antibodies are formed locally in regionally stimulated tissues and diffuse into the Ijlood. 9. Cannon and Walsh (1938) followed \\\^ the work mentioned above by studying the effect of many substances used in intra- nasal medication on the local defensive mechanisms. Their work IMMUNITY IN EXPERIMENTAL INFECTIONS 133 is of special interest in view of the increase in the number of cases of lipoid pneumonia reported in infants as resulting from intra- nasal medication. Cannon and Walsh found that mineral oil, which is a popular vehicle for menthol, eucalyptol, iodine, guaiacol, etc., readily reaches the lungs when instilled into the nose of rabbits. They ])oint out that it is quite liable to pass from the nose to the trachea and lungs of infants lying on their backs. This may account for mucli of the lipoid aspiration which is found at autopsy. The min- eral oil and many otlier fluids used for nasal medication such as argyrol, etc., can carry bacteria from an infected nose or naso- pliarynx to the lungs. Cannon and Walsh also cite other raseareh that indicates that many substances used in intranasal medication interfere with the ciliary movement or by otlier means interfere with the streaming of mucus over the mucous membrane and thus paralyze a pro- tective mechanism. Not only do mineral oil and some other sub- stances affect the cilia but when they reach the lungs they affect the permeability of the blood capillaries. They state, however, that some materials such as prontosil, 1 per cent thymol, 1 per cent menthol, 10 per cent glycerin or any of the solutions of the vaso- constrictors in isotonic saline are not likely to be injurious although some of them do affect the cilia. 10. Cannon and Hartley sensitized rabbits to egg albumin and then injected a mixture of pneumococci and egg albumin locally to see if the allergic inflammation would protect against the pneu- mococcus. As a result of this and a number of other similar ex- periments they conclude that allergic inflammation failed to pro- tect rabbits against infection with virulent pneumococci. These findings are in harmony with those of Rich (1933). He mixed a small dose of fowl cholera bacilli with pneumococcus polysaccharide and injected the mixture into a rabbit sensitive to the polysac- charide. The resulting allergic inflammation did not protect the ral)bit since a generalized infection ensued. Experimental Infection of the Chorio-allantoic Membrane of Chick Embryos was carried out by Goodpasture and Anderson (1937) with results that appear to throw light upon susceptibility as well as resistance. Goodpasture's timely observation, that while 134 IMMUNOLOGY resistance has been investigated extensively the problem of sus- ceptihility mechanisms has been neglected, shonld stimnlate re- search of great value to scientific medicine. The results of their experimental studies may be summarized as follows : 1. They suggest that the inoculation of the chorio-allantoic mem- brane of the chick embryos with pure cultures of bacteria is an excellent method for studying the early stages of invasion. 2. They produced experimental infections with Staph, aureus, Str. haemohjticus, Str. viridans, and A. aerogenes, E. typkosa, Br. abortus, C. diphtheriae, and 3Iyco. tuherculosis avium. 3. They found that either mesodermal cells (fixed or motile), or epithelial cells, or both, seem to act as host-cells for all of the organisms mentioned except Stajyh. aureus, Str. haemohjticus and C. diphtheriae. Str. virid/ms utilized the intracellular en- vironment of wandering and fixed mesodermal cells; A. aerogenes the wandering mesodermal and entodermal cells; E. typhosa utilized cells of the ectodermal epithelium ; Br. abortus utilized both ectodermal and mesodermal cells while Mj/co. tuherculosis avium preferred mesodermal elements. The staphylococcus and hemolytic streptococcus grew well in between the tissue cells and underwent phagocytosis but were ap- parently unable to utilize any living intracellular environment for growth. 4. These results caused Goodpasture (1937) to investigate care- fully the pathogenesis of typhoid fever. As a result of his study of excellent autopsy material he found young plasma cells in sec- tions of the lymphoid follicles of iliac and mesenteric lesions filled with small, gram-negative apparently unaltered bacilli which he judged to be E. typhosa. He found larger gram-negative bacilli in microphages of the lesions associated with remnants of lympho- cytes and macrophages. Goodpasture suggests that the young plasma cell is a host for E. typhosa in human cases and nourishes and protects the bacteria during the incubation period and through- out the. disease. Role of Clasmatocytes in Other Infections. — That the reticulo- endothelial system is an important factor in combating both bac- terial and protozoan infection is suggested by the results of a number of investigators. Buxton (1907) concluded that the re- sistance of rabbits immunized against typhoid is due largely to IMMUNITY IN EXPERIMENTAL INFECTIONS 135 the iiu'i-eased ability of the mononuclear phajiocytes to ensiili" and destroy typhoid bacteria. Cannon and Taliaferro (1931) carried out an extensive investigation of the cellular reactions in the tis- sues of canaries infected for the first time with Plasmodium cathcr- ncnum and also in superinfected birds. This last observation in- dicates that the i^rimary infection alters the reactivity of the luescnchyiuo so that ])lui.orted u])on the cellular reactions during primary infections and superinfections of Plas- modium Jirdsilianum in Panamanian monkeys. They say that the maci'ophages of the s]deen. liver and bone marrow are the ])rimary defense against malaria. They suggest that tlie reason for this is probably the slow circulation of the blood through these tissues which allows for direct contact of the blood and macro- phages wherea.s elsewhere in tlie body similar conditions do not exist. Tissue Resistance to Cysticekcus pisiformis. — In a study of experimental infections of C iisticerciis pisiformis, a larval ta]ie- worm, in passively inuuunized rabbits, Leonard (1940) showed that host resistance is ex])ressed parenterally as aii enhanced and accelerated tissue response; larvae in the liver were killed in these animals in aj^proximately one-half the time required for their death in normal animals. Leonard and Leonard , (1941) found that in passively imnuuiized rabbits, the intestinal wall, or some substance released by the host tissues within the intestine, plays a major part in the total resistance mechanism. This was shown by injecting artificially hatclied larvae into tlic nu\senteric veins, thus allowing them to I'cacli the liver witliout ]ienetrating the intestine. It was shown by this means that, in ])assively im- nuuiized animals, only 3 to 4 pei* cent of the larvae are capable of overcoming the resistance of the host intestine, while in normal animals, 85 per cent of the larvae are able to penetrate the in- testine and safely reach the liver. Removal of Bacteria from the Blood Stream. — Cannon, Sulli- van and Xeckermann* (1932) investigated the conditions in- *Cannon, P. R., SuUivan, F. L., ami Xeckennani), E. F. : J. Expor. Meil. 5.'»: 121, 1932. 136 IMMUNOLOGY fluencing the disappearance of living bacteria from the blood stream. They interpret the general mechanism as follows : "Staphylococci injected intravenously into normal rabbits circu- late throughout the blood stream in large numbers, probably mak- ing many passages through the organs and tissues of the body. In passing through the spleen and liver, especially, conditions more favorable for phagocytosis may obtain, particularly those de- pendent upon slow blood flow, availability of macrophages and leucocytes, mechanical conditions favoring filtration, etc. Chance contacts between phagocytic cells and the relatively unchanged staphylococci induce a certain degree of phagocytosis, as seen in the polymorphonuclear leucocytes in the lungs and in the mac- rophages and leucocytes of the liver and spleen. Eventually this mechanism removes the bacteria from the circulating blood. Their further fate doubtless depends upon the virulence of the micro- organisms and the digestive capacities of the macrophages or in other words the functional state of the phagocytes, both mac- rophages and polymorphonuclear-leucocytes. As Werigo showed in experimental anthrax infection, if these cells become inadequate, the bacteria again multiply and generalize. "In the immune animals, this normal mechanism disposed of the dead bacterial bodies during the preliminary period of im- munization, at which time many macrophages in the liver and spleen removed and digested the bacterial particles. "When at a later period large numbers of living staphylococci run the gaunt- let of those macrophages, there is an almost instantaneous swelling of the micro-organisms, an increasing obstruction to their free pas- sage through the immune liver and spleen and a tendency to clump- ing of the bacteria with a resulting retention of such affected micro- organisms in these organs." In regard to the relative importance of macrophages and neutro- pliiles Cannon, Sullivan and Neckermann, state that "in such a complex system, however, it is questionable whether too much emphasis should l)e placed upon the comparative significance of two groups of mesenchymal cells whose functions are so similar and apparently complemental. " They observed phagocytosis by both groups of cells. They do state, however, that the "primary re- action is mainly between the cocci, immune bodies and the cytoplas- mic surfaces of the macrophages, accompanied or quickly followed IMMUNITY IN EXPP:RIMENTAL INFECTIONS 137 by the aecuinulation of polymorphonuclear leucocytes attracted or retained there l)y clieniotactic or electrotrophic influences." Both types of cells engulfed and destroyed bacteria, tlie outcome of the battle depending upon the virulence and number of the latter. Teale (1935) reports the results of an extensive study of the relative importance of the retieulo-endothelial tissues and the cir- culating antibody in immunity. He reports that immunized an- imals whose blood did not contain agglutinin, germicidal power or protective antibody tested in passive transfer, were able to clear completely the peripheral circulation of highly virulent bacteria against which they had been immunized. He also gives protocols showing that normal animals, which he assumed had no circulating antibodies, were likewise able to free the peripheral circulation of virulent organisms although they could not prevent the occurrence of secondary waves of fatal bac- teremia as could the immune animals. These results are not out of line with the findings of others and do not, in the case of immune animals, exclude antibodies present in the tissues from playing a role in defense. Furthermore, in the immune animals the anamnestic reaction (hair-trigger mechanism) might operate and circulating antibodies appear folloAving the test injection of bacteria. In regard to his protocols on normal animals he states that ''the rabbit is generally stated to have no antibody against B. dys- cnteriae (Shiga) nor the organism of fowl cholera." He has ap- parently accepted this assumption in lieu of testing the rabbits he used. There seems to be good reason to question this assumption since Mackie and Finkelstein (1931, 1932) reported that normal rabbit blood is frequently germicidal for B. dysenteriae (Shiga), and Coriell, Miller, and Sherwood (1940) confirmed the conclusion of Mackie and Finkelstein and in addition found bactericidal anti- bodies for the organism of fowl cholera. Teale apparently as- sumes that such antibodies do not exist. In spite of these differences of opinion we are in agreement with his major thesis that resistance to infection does not necessarily parallel the content of circulating antibody. In fact we have cited other literature to that effect and also work done in this laboratory showing that no virucidal antibody could be demonstrated in cats exhibiting a solid immunity to a strain of vaccine virus that pro- 138 IMMUNOLOGY duced fatal infection in rabbits. We are, however, convinced that the presence of antibodies intimately associated Avith the tissue cells is of prime importance as an adjunct to cells of the reticulo- endothelial system in resistance. In immune animals the an- amnestic reaction may lead to an increase of tissue antibodies even though they are not in delectable amounts in tlie blood. Defensive Mechanisms in Peritonitis. — One of tlu' pathological states in the human thai frequently results in generalized infection and an overwhelming toxemia is peritonitis. Hertzlcr's (1919) extensive and very valuable study of the anatomy, physiology, pathology and defensive mechanism of the peritoneum deserves every medical student's attention. He found by experiment that isotonic solutions and particulate matter injected into the normal peritoneal cavity were rapidl}' absorbed directly into the blood, although he does not deny that absorption by way of the lymphatics occurs. Absorption is delayed when the intraabdominal pressure is sufficient to interfere with venous flow. Tympanitis may there- fore be of value in retarding absorption. He calls attention to the possibility of increasing absorption from the peritoneal cavity in such cases when the intraabdominal pressure is reduced too rapidly. If he produced an inflammatory exudate of the perito- neum before injecting fluid or particulate matter into the cavity, absorption was retarded or prevented. He discusses the method of formation and the importance of temporary adhesions in limiting the spread of infection within the abdomen. In regard to the nature of the cellular response in peritonitis he states that neutrophiles predominate for a period, and if the infection is being successfully cliecked, mononuclear cells make their appearance. On the other hand, "when patches of viable and degenerated leucocytes coexist, an advancing con- dition may be assumed." Altemeier and Jones* (1940) report an inteiesting comparison of postoperative results in fifty-one consecutive cases of resection for carcinoma of the rectum and sigmoid reported by Pratt and a series of experiments on experimental peritonitis in rabbits which they conducted. It was noted that the al)sence of postoperative peritonitis in the cases reported by Pratt apparently correlated with preoperative high voltage roentgen therapy one month to six weeks previous to operation. An investigation of the effect of such *Altemeier, W. A., anrl .Jone.s, H. C. : Kxporimental Peritnniti.=!, J. A. M. A. 114: 27, 1940. IMMUNITY IN EXPERIMENTAL INFECTIONS 139 treatment upon rabbits carried out by Altemeier and Jones showed that preoperative hif?h voltao:e roentgen irradiation was of value in developing immunity against experimental peritonitis. They offer no theory of the mechanism involved. Defense Against Viruses. — Tn regard to the defensive mech- anism against viruses, the prevailing opinion as expressed by Rivers (1928) and Ayeock (1931) seems to be that it is both cellular and virucidal or humoral in nature nnd that the inflam- matory response is secondaiy to cell injury. This is apparently borne out by the experimental work of Andrews (1930). Some of his conclusions are summarized as follows: (1) The virus of lierpes can be cultivated successfully in tissue cultures (rabbit testes) if dilute rabbit serum is employed. (2) Neither growth of the virus nor the formation of inclusion bodies is obtained in tissue cultures if immune serum is added to tlie culture before the virus or together with it. (3) The virus of herpes will grow and form inclusion bodies in tissue cultures Avhen immune rabbit testes and normal serum are used. (4) Normal tissue is infected by the virus witliin one-half ]iour at 17.5° C. or 37° C. if immune serum is not present ; the later addition of immune serum does not pre- vent growth of tlie virus nor the formation of inclusions. -Jamuni and Holden (1934) present evidence wliich indicates that l)oth leucocytes and immune serum play a role in 'immunity against herpes virus. The mononuclear cells are apparent!}" more efficacious than polymorphonuclear cells in disposing of herpes virus. They suggest that the virus is opsonized and then phago- cytized although they were unable to verify this point. Their j'esults indicate that phagocytic cells (immune or normal) bring about a greater virucidal effect than can be obtained with immune serum alone. Tolerance. — In addition to the various factors of defense dis- cussed in this chapter the phenomenon of ''tolerance" perhaps should be mentioned. Gunn (1923) regards this as very impor- tant especially as regards certain toxemic disea cs. He cites the specific congenital tolerance of the toad, rat and grass snake to toad poison which has a digitalis-like toxic action and is a glu- coside. He says that these same aninuils show a high degree of tolerance to members of the same group of glucosides. The re- sistance is due to an insusceptibility of tissue, especially that of 140 IMMUNOLOGY the heart, to these glucosides. This is of interest in view of the resistance of the rat to diphtheria toxin. This has been studied extensively by Coca and others. The toxin apparently circulates in the blood and does not combine with the tissues. Further work is apparently necessary before such a phenomenon can be desig- nated as a significant factor in active immunity in man. References Andrews, A. H. : Tissue Culture in the Study of Immunity to Herpes, J. Path. & Bact. 33: 301, 1930. Aycock, W. L.: Immunity in Virus Diseases, J. A. M. A. 97: 1199, 1931. Buxton, B. H.: Absorption From the Peritoneal Cavity, Part VIII, J, M. Eesearch 16: 251, 1907. Cannon, P. E. : Bacterial Localization and Growth in Normal and Immune Tissues, Am. J. Path. 11: 852, iy35. Cannon, P. K., and Hartley, G., Jr.: The Failure of Allergic Inflammation to Protect Babbits' Against Infection With Virulent Pneumococci, Am. J. Path. 14: 87, 1938. Cannon, P. E., and Pacheco, B. S. : Studies in Tissue Immunity, Am. J. Path. 6: 749, 1930. Cannon, P. E., Sullivan, F. L., and Neckermann, E. F.: Conditions In- fluencing the Disappearance of Living Bacteria From the Blood Stream, J. Exper. Med. 55: 121, 1932. Cannon, P. E., and Taliaferro, W. H. : Acquired Immunity in Avian Malari;i, J. Prev. Med. 5: 37, 1931. Cannon, P. E., and Walsh, T. E.: Studies on the Fate of Living Bacteria Introduced Into the Upper Eespiratory Tract of Normal and Intra- nasally Vaccinated Eabbits, J. Immunol. 32: 49, 1937. Cannon, P. E., and Walsh, T. E.: Lipoid Pneumonia and Some Potential Dangers of Intranasal Medication, Internat. Clin. 3: 109, 1938. Coriell, L., Miller, W. E., and Sherwood, N. P.: Studies on Natural Bac- teriolysins, 1940. (Unpublished work.) Freedlander, S. O., and Toomey, J. A.: The Eole of Clasmatocytes and Connective Tissue Cells in. Nonspecific Local Cutaneous Immunity to Staphylococcus, J. Exper. Med. 47: 663, 1928. Freund, J: Distribution of Immune Agglutinins in Serum and Organs of Eabbits, J. Immunol. 14: 101, 1927. Freund, J., and Whitney, C. E. : Distribution of Antibodies in Serum and Organs of Eabbits; Effect of Perfusion Upon Antibody Content of Serum and Organs, J. Immunol. 15: 369, 1928. Gallavan, M., and Goodpasture, E. W. : Infection of Chick Embryos With H. Pertussis Eeproducing Pulmonary Lesions of Whooping Cough, Am. J. Path. 13: 927, 1937. Gay, F. P., and Clark, A. E.: A Comparison of Indifferent Substances and Specific Antigen in Production of Local Streptococcus Immunity, Proc. Soc. Exper. Biol. & Med. 24: 20, 1926. Gay, F. P., and Clark, A. E. : Enhanced Passive Immunity to Streptococcus Infection in Eabbits, J. Exper. Med. 52: 95, 1930. Gay, F, P., and Morrison, L. F. : Clasmatocytes and Eesistance to Strepto- coccus Infection, J. Infect. Dis. 33: 338, 1923. Gay, F, P., and Oram, F. : Streptococcus Leucocidin and the Eesistance of Clasmatocytes, Proc. Soc. Exper. Biol. & Med. 28: 850, 1931. IMMUNITY IN EXPERIMENTAL INFECTIONS 141 (ioodpasture, E. W. : Coiicciiiiii}^' the Pathogenesif! of Typhoid Fever, Am. J. Path. 13: 175, 1937. Goodpasture, E. W., and Anderson, K. : The Problem of Infection as Presented by Bacterial Invasion of the Chorio-Allantoic Membrane of Chick Embryos, Am. J. Path. 13: 149, 1937. Gradwohl, E. B. H. : Clinical Laboratorv Methods and Diagnosis, St. Louis, 1938, C. V. Mosby Co. Gunn, J. A.: Cellular Immunity: Congenital and Acquired Tolerance to Nonprotein Substances, Physiol. Rev. 3: 41, 1923. Haden, R. L.: Discussion of Leucopenia, J. A. M. A. 116: 483, 1941. Hertzler, A. E.: The Peritoneum, Vol. I, St. Louis, 1919, C. V. Mosby Company. .Tatmini, A., and Holden, M. : The Role of Leucocytes in Immunity to Herpes, J. Immunol. 26: 395, 1934. Lawrence, J. S.: Leukopenia: A Discussion of Its Various Modes of Production, J. A. IVF. A. 116: 478, 1941. Leonard, A. B. : The Accelerated Tissue Response to Cysiicercus pisiformis iu Passively Immunized Rabbits, Am. J. Hyg. 32: 117, 1940. l^eonard, A. B., and I^eonard. A. E.: The Intestinal Phase of the Resistance of the Rabbit to Cystieercua pisiformis, J. Parasitol., 1941 (In press). Linton, R. W. : Mobilization and Transfer of Clasmatocvtes, Arch. Path. 5: ■ 787, 1928. Mackie, T. J., and Finkelstein, M. H. : The Bactericidins of Normal Serum: Their Characters, Occurrence in Various Animals and the Susceptibility of Different Bacteria to Their Action, J. Hyg. 32: 1, 1932. Mackie, T. J., and Finkelstein, M. H.: Natural Bactericidal Antibodies: Observations on the Bactericidal Mechanisms of Normal Serum, J. Hyg. 31: 35, 1931. Menkin, V.: Studies on Inflammation. XII. Mechanism of Increased Capillary Permeability. A Critique of the Histamine Hypothesis, J. Exper. Med. 64: 485, 1936. Menkin, V.: Studies on Inflammation. XIV. Isolation of the Factor Con- cerned With Increased Capillary Permeability in Injury, J. Exper. Med. 67: 129, 1938. Menkin, V.: A Note on the Differences Between Histamine and Leuko- taxine, Proc. Soc. Exper. Biol. & Med. 40: 103, 1939. Menkin, V.: Studies on Inflammation. XVIII. On the Mechanism of Leukocytosis With Inflammation, Am. J. Path. 16: 13, 1940. Mueller, E. F. : Evidence of Nervous Control of Leukocytic Activity by the Involuntary Nervous System, Arch. Int. Med. 3?": 268, 1926, Opie, E. L.: Inflammation, Arch. Int. Med. 5: 541, 1910. Opie, E. L.: Inflammation and Immunity, J. Immunol. 17: 329, 1929. Opie, E. L.: The Fate of Antigen (Protein) in an Animal Immunized Against It, J. Exper. Med. 39: 659, 1924. Rich, A. R.: The Mechanism Responsible for the Prevention of Spread of Bacteria in the Immune Body, Bull. Johns Hopkins Hosp. 52: 203, 1933. Rivers, T. M. : Filterable Viruses, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 881. Rivers, T. M.: Viruses in Relation to the Practice of Medicine, Pennsyl- vania M. J. 36: 489, 1933. Schilling, V.: The Blood Picture and Its Clinical Significance; Translated by R. B. H. Gradwohl, St. Louis, 1929, C. V. Mo.sby Co. Taliaferro, W. H., and Cannon, P. R.: Cellular Reactions During Primary Infections and Superinfections of Plasmodium Brasilianum in Panamanian Monkej's, J. Infect. Dis. 59: 72, 1936. 142 IMMUNOLOGY Teale, F. H.: Some Observations on the Kelative Importance of the Reticulo-Endothelial Tissues and the Circulating Antibody in Im- munity. I. Bacterial Immunity in Eelation to the Eole Played by the Circulating Antibody and the Tissues Following Intravenous Introduc- tion of the Bacteria, J. Immunol. 28: 133, 1935. Todd, J. C, and Sanford, A. H.: Clinical Diagnosis by Laboratory Methods, Philadelphia and London, 1931, W. B. Saunders Co., p. 293. Walsh, T. E., a,nd Cannon, P. E. : Immunization of the Eespiratory Tract. A Comparative Study of the Antibody Content of the Respiratory and Other Tissues Following Active, Passive and Regional Im- munization, J, Immunol. 35: 31, 1938. Walsh, T. E., and Cannon, P. E.r Studies on Acquired Immunity in Babbits to Intranasal Infection With Type I Pneumococcus, J. Immunol. 31: 331, 1936, Walsh, T. E., and Cannon, P. R.: The Problem of Intranasal Medication, Ann. Otol., Rhin. & Laryng. 47: 579, 1938. Walters, O. S.: Normal Ervthrocvte, Hemoglobin and Packed Cell Volume Standards in Young *Men. ' Studv of 100 Subjects, J. Lab. & Clin. Med. 19: 851, 1934. Supplementary References Angevine, D. M.: The Fate of Avirulent Hemolytic Streptococci Injected Into the Skin of Normal and Sensitized Rabbits, J. Exper. Med. 60: 269, 1934. Angevine, D. M. : The Fate of Virulent Hemolytic Streptococci Injected Into the Skin of Normal and Immunized Rabbits, J. Exper. Med. 64: 131, 1936. Menkin, V.: Inflammation and Bacterial Invasiveness, Am. J. M. Sc. 190: 583, 1935. Redmond, W. B.: The Cross-Immune Relationship of Various Strains of Plasmodium Cathemerium and P. Relictum, J. Infect. Dis. 64: 273, 1939. Sabin, F. R.: Cellular Studies in Tuberculosis, Am. Rev. Tuberc. 25: 153, 1932; also Tubercle 13: 206, 1932. Sabin, F. E., and Doan, C. A.: Biological Eeactions in Rabbits to Protein and Phosphatide Fractions From Chemical Analysis of the Human Tubercle Bacilli, J. Exper. Med. 46: 645, 1927. Sabin, F. E., and Doan, C. A.: The Eelation of Monocytes and Clasmato- cytes to Early Infection in Eabbits With Bovine Tubercle Bacilli. J. Exper. Med. 46: 627, 1927. Taliaferro, W. H.: The Mechanism of Acquired Immunity in Infections with Parasitic Worms, Physiol. Eev. 20: 469, 1940. Taliaferro, W. H., and Huff, C. G.: The Genetics of the Parasitic Protozoa, Am. A. Advance. Sc, Pub. No. 12, p. 57. Taliaferro, W. H., and Kliiver, C: The Hematology of Malaria (Plas- modium Brasilianum) in Panamanian Monkeys, J. Infect. Dis. 67: 121, 1940. Taliaferro, W. H., and Sarles, M. P.: The Cellular Eeactions in the Skin, Lungs and Intestine of Normal and Immune Rats After Infection With Nippostrong3'lus Muris, J. Infect. Dis. 64: 157, 1939. Taliaferro, W. H., and Taliaferro, L. G.: Active and Passive Immunity in Chickens Against Plasmodium Lophurae, J. Infect. Dis. 66: 153, 1940. CHAPTER VIII NATURAL AND IMMUNE ANTIBODIES Duriii.u' Iho decade l)elw(H'n 1880 and 1890 two Iheorios arose as to the natui-e of the l)ody's defense against infectious agont.s. The one, under the leadersliip of Fliigge and von Fodor in (iermany and Nnttall in Enoland, conceived of the body's defense as ])eino- due to chemical suljstances in the blood, while the other, under the leadership of IMetchnikoff, maintained tliat when infectious agents entered the l)ody, they were engulfed (phagocytized) by certain wandering and fixed tissue cells and ultinuitely destroyed by a process of digestion. The former is known as tlie humoral theory, while the latter is the cellular theory of immunity. The Humoral Theory of Immunity, according to Ledingham, had its origin in Lord Lister's (1880-81) experiments on the keeping quality of aseptically removed ox blood. Blood itself is a complex circulating body fluid which functions as a carrier of oxygen and of the nuti-itive, waste and other materials of the body's metabolism. About 30 to 40 per cent of it is made up of cellular material while the remainder is the fluid portion or plasma. This latter contains 90 to 92 per cent water and 8 to 10 per cent proteins, carbohydrates, fats, electrolytes and a wide variety of substances of unknown structure such as enzymes, antienzymes, antibodies, etc. When blood clots, there is scpieezed out a straw- colored fluid called scruui which differs from plasma in tliat tlie tibrinogen has been removed in the process of clotting. Blood from which fibrinogen has been removed is called defibrinated blood. Locke and Hirscii* state (1928) that chemical concepts of im- munity had become sufficiently crystallized by 1887 to lead Emmerich to make the following suggestion : "It should be an important task for investigators to seek out the substances which are associated with tlie inununity state and to ascertain in what chemical grouping they belong."' *"The Isolation of Substances with Immune Properties," by A. Locke and K. F. Hir.scb. in The Xetcer Knouledffe of Bacteriology and Immunology, edited by E. O. Jordan and I. S. Falk. Reprinted by permission of the University of Chicago Press. 143 144 IMMUNOLOGY Alpha Lysins. — Between the years of 1888 and 1890, Nuttall in England and von Fodor on the Continent published their investiga- tions as to the germicidal property of blood. By mixing defibrin- ated blood with bacteria and plating out after a period of incuba- tion, they were able to show that some defibrinated bloods pos- sessed mild germicidal properties. This seemed to be lessened or destroyed by heating to 60° C. for a short time or by standing for a longer time at room temperature. These observations were confirmed by Buchner (1889) who re- garded the germicidal property of blood as due to an unstable, thermolabilc substance which he called alexin. By animal in- oculation experiments, he showed that bacteria were destroyed by humoral elements in serous cavities of the body and in the subcutaneous tissues. Buchner conceived of resistance as due to alexin. This thermolabilc bactericidal substance is also called alplui lysin. Immune Lysins and Pfeiffer's Phenomena. — In 1894 Pfeiffer was studying cholera immunity and noted that when those guinea pigs which recovered from infection with Microspira comma were reinoculated intraperitoneally, the bacteria were rapidly destroyed and the animals survived, whereas in normal pigs very little de- struction of the organisms occurred and the animals succumbed. Pfeiffer made three other observations that are fundamental to our present concepts. He found first, that the immunity enjoyed by the recovered pigs was specific for cholera vibrios and not for other bacteria; second, he could convey this immunity to normal animals by injecting intraperitoneally a small amount of blood from immune animals together with the virulent vibrios; third, Pfeiffer found that heated immune serum when injected together witli the vibrios was effective in protecting tlic normal pigs. This WAS quite puzzling since he had previously confirmed Buchner s conclusions that in test tube and plating experiments, heated serum or defibrinated blood has apparently lost its germicidal property. Pfeiffer' decided that some substance from the endo- thelial cells lining the peritoneum was able to restore to heated serum its germicidal property. Pfeiffer's Immunity Unit. — It is interesting to note that Pfeiffer realized the importance of devi.sing some method for NATURAL. AND IMMUNE ANTIBODIES 145 measuring the strength of immune serum. This he did by select- ing a number of guinea pigs of uniform weight and inoculating a series with varying amounts of heated immune serum mixed with two milligrams of virulent bacteria, an amount sufficient to insure death in normal guinea pigs when inoculated alone. The least amount of immune serum that dissolved the bacteria and pro- tected the animal was called an ''immunity unit." Bordet's Explanation of Lytic Mechanism. — It remained, however, for Jules Bordet (1898) working in Pasteur's laboratory to investigate carefully and explain the phenomena described by Pfeiffer, His results may be briefly summarized as follows: 1. He confirmed Pfeiffer 's observation that normal guinea pigs could be protected by injecting them with immune serum. This is now called passive immunity as contrasted with active immunity enjoyed by animals that recover from an infection. 2. He further showed that fresh unheated serum from cholera immune guinea pigs would specifically kill and dissolve cholera vibrios in a hanging drop preparation under the microscope. He found normal guinea pig serum to be only slightly potent and not very specific. 3. In addition, he observed that the serum from immune guinea pigs, when heated to 55° C. for one-half hour and mixed with vibrios in hanging drop preparations, would frequently clump the organisms (agglutinate them), but would not kill or dissolve them. However, if he added a little fresh normal serum, which by itself had no germicidal properties, to the hanging drop of bacteria and immune serum, the vibrios were quickly killed and dissolved. Pfeiffer had been unable to render heated immune serum potent, l)ut Bordet definitely proved it possil)le. Thus Bordet showed that fresh serum from nonimmune as well as immune animals contains a thermolabilc or heat sensitive substance (called complement by Ehrlich) whicli will act upon and destroy bacteria suspended in heated immune serum. Bordet concluded that some specific thermostable (heat resistant) substance in the immune serum rendered the cholera vibrios sus- ceptible to the digestive action of complement. The chemical sub- stance in the immune serum that renders bacteria sensitive to complement has been called by various names such as protective 146 IMMUNOLOGY substance (body protector), immune body (Pfeiffer), sensitizer (Bordet), and amboceptor (Elirlich). Bordet showed that if he vaccinated guinea pigs with a killed vaccine, the protective sub- stance or sensitizer appeared in their blood after the lapse of several days and that it was the same as the sensitizer found in the blood of animals recovering from infection. It is now possible to develop these simple definitions: 1. Bacterial Amboceptor, Immune Body or Sensitizer. — This is a thermostable substance found frequently in small amouiits in the blood of normal animals but more frequently and in much greater concentration in the blood of vaccinated animals or animals which liave recovered from an infection. It has a specific affinity for the bacteria used in the vaccine or Avliich caused the infection and Ity combining with them renders them susceptible to union with a nor- mal enzjnne-like substance called complement which kills and in the case of cholera vibrios dissolves the bacteria. 2. Sensitized Cells. — When bacteria or any cellular antigen is united to its antibody or sensitizer it is then a sensitized cell. 3. Complement. — This is a thermolabilc enzyme-like substance found in the blood of all Avarm-blooded and some cold-])looded animals. It will combine with seiLsitized cells and bring about their death or lysis. Further Studies on Normal Bacteriolytic Substances. — In addi- tion to tiie alpha hjsins whicli require thermolabile complement for their activity other lytic agents such as p hjsins, leukins and lysozyme have been described. Eacli of tliese would seem to merit a brief discussion. Alpha Lysixs. — The question arose quite early as to whether the lytic mechanism described by Bordet for immune lysins would apply to normal lytic agents. i\Iuir and Browning (1908) working with guinea pig serum, concluded that the mechanism of normal bactericidal action was similar to the bactericidal action of immune serum. They further concluded that bactericidal complement (termed by them "bacteriophilic" complement) and hemolytic complement are separated varieties. Other research such as that of Irwin, Beach and Bell (1936), or Shrigley and Irwin (1937), and of Dingle, Fothergill and Chandler (1938) tends to substantiate this conclusion. Mackie and Finkelstein (1931, 1932) carried out NATURAL AND IMMUNE ANTIBODIES 147 an extensive investigation of thei-molabilc baeteriolysins. They concluded that the baeteriolysins are widely distributed in the animal kingdom, are specific for the bacteria they act upon and tliat they consist of thermostable antibody (amboceptor) and thermolabile complement. In their opinion the antibodies, de- veloped as a result of either vaccination or infection, have their precursors specifically differentiated in the serum of normal animals and that, in general, they are not substances formed de novo. Coriell. Miller, and Sherwood (1940), working in this labora- tory, confirmed many of the conclusions and extended the experi- mental findings of Mackie and Finkelstein. They found the serum of 20-hour-old kittens strongly bactericidal ; the serum of 3-day-old rabbits moderately bactericidal ; and the serum of very young chicks and chick embryos weakly bactericidal. Since these bactericidal substances were inactivated at 56° C. and could be specifically ab- sorbed they were apparently a lysins. /? Lysins, Leukins and Lysozyme. — These are bactericidal sub- stances having many properties in common with each other but dis- tinctly different from a lysins. While a lysins are quite active against typhoid-colon, Salmonella and dysentery groups as well as V. cholerae, B. pyocyancous, pneumococcus and streptococcus, the /3 lysin and leukins are active against anthrax-subtilus and proteus groups. Ledingham (1931) gives an excellent brief discussion of these substances. It appears that their titer is not increased by vac- cination and they are more thermostable than the a lysins. The (3 lysins are found in normal sera, the leukins are present in leucocytes, while lysozyme is present in various secretions and tissues of the body. It is especially present in tears and cartilage. Lysozyme is most active against a saprophytic micrococcus isolated by Fleming. While 13 lysins are more thermostable than a lysins they apparently are of complex nature. Pettersson (1928, 1929) says that the portion analogous to amboceptor is denoted the "activdble substance" and that corresponding to complement the "activating substance," which is destroyed at temperatures be- tween 63° and 70° C. Apparently the activable substance does not combine with the bacteria unless the activating substance is present. It is still unknown what role these substances play in the body's defense. 148 IMMUNOLOGY Natural and Immune Bacterial Agglutinins. — The capacity of normal or immune sernm to bring about the specific clumping (^■glutination) of bacteria depends upon its antibody content. The titer of normal agglutinins varies from zero to one in ten or one in twenty ; rarely is it found higher. On the other hand the titer of agglutinin following vaccination or recovery from in- fection is usually quite high in comparison. This subject will be discussed in a later chapter. Hemolysins, Hemagglutinins and Other Antibodies. — While l>ordet was busily engaged in explaining the mechanisms of l)ac- teriolysis by immune serum, an important observation of Buchnei- nttraeted his attention. The latter had observed that occasionally normal blood serum had the property of destroying and hemo- lyzing foreign red blood cells in vitro (outside the body). Bordet wondered whether this phenomenon was dependent upon a sensi- tizer or amboceptor and complement mechanism such as he had shown for bacteriolysis and whether he could make specific sensitizer or amboceptor appear in the blood stream of animals by vaccinating them with a suspension of foreign red blood cells. By a series of simple experiments he showed that in a normal rabbit whose blood serum had little if any effect upon sheep red cells, he could, by injecting it several times with washed sheep cells, cause the appearance in the blood stream of the rabbit, within about a week after the last injection, of an appreciable amount of sensitizer or hemolysin for sheep cells. He found that this new sensitizer or hemolytic amboceptor for red cells was thermolabile, that it did not possess the property of destroying the cells, but, like bacteriolytic sensitizer, it rendered the red cells susceptible or sensitive to the digestive or lytic action of complement. This re- sulted in the liberation of hemoglobin from the red cells, a phenomenon which is called hemolysis. The natural antibodies causing hemolysis and agglutination of foreign red cells are called heterohemolysins and heteroJiemagglu- tinins, respectively, while similar antibodies that result from vaccinating with foreign red cells are called immune liemolifsins and immune hemagglutinins, respectively. Landsteiner observed early in this century that human blood could be arranged into several groups, depending upon the prop- erty of human blood serum from one person clumping and fre- NATURAL AND IMMUNE ANTIBODIES 149 quently hemolyzing the red cells from another person. When antibodies are present in the blood of one member of a species for the red cells of another member of the same species the anti- bodies are called isohemolysins and isohernagglutinins, respectively. It is quite important in transfusing a patient to select as a donor some one whose blood cells are not clumped by the serum of the recipient. Downs, Jones, and Koerber (1929) have shown that these isohemolysins require complement for their action as do other hemolysins produced by injection of foreign red cells. Isohemo- lysins and isohemagglutinins have been studied in rabbits, cattle and other animals. These substances and the inheritance factors involved will be discussed in another chapter under Blood Group- ing. Isohemolysins in Paroxysmal Hemoglobinuria.— Another type of isohemolysin is described by Donath and Landsteiner (1904). This interesting type of hemolysin is apparently responsible for the clinical condition known as paroxysmal hemoglobinuria. They found in the serum or plasma of patients suffering from this dis- ease, antibodies (isolysins) which would combine with the patient's own red cells only at low temperatures. They reasoned that the red cells of patients liaving tliese antibodies would become sensi- tized in the skin capillaries when the surface of the body was exposed to cold and would combine with complement after sensi- tization. When the sensitized red cells with their complement were transported to the internal organs where a temperature of 37° C. would be encountered, hemolysis would occur. Mackenzie (1927) has reviewed this subject and gives an excellent discussion of the work of Donath and Landsteiner and others. Apparently the blood serum of almost every case of paroxysmal hemoglobinuria reacts with Wassermann antigens to give a posi- tive serological reaction. While 24 to 25 per cent give a history of syphilis, it appears that approximately 75 per cent are negative as far as history goes; the diagnosis being made upon the positive serological findings. This is interesting in view of the work on natural antibody-like substances (reagins) that are found quite extensively in the blood of lower animals and to some extent in the blood of normal human individuals. Kahn (1940, 1941) has shown that these antibodies which unite consistently with Kahn antigen and less extensively with Wassermann antigen do so more 150 IMMUNOLOGY effectively at low temperatures, while syphilitic reagiii or anti- bodies react more effectively at body temperature. According to Mackenzie, Burmeister found that the lysins, dissociated from sensitized erythrocytes, give a positive Was- sermann reaction. In his opinion there are some cases of paroxysmal hemoglobinuria which give a positive Wassermann test that are not syphilitic in nature while others show a relationship between the two diseases. For more recent work upon the subject of natural reagins or antibodies having an affinity for antigens used in the diagnosis of syphilis, sec the review of the literature by Kemp (1940) and experimental studies by Kahn (1940-1941), Sherwood, Bond and Clark (1941); and SherAvood, Bond, and Canuteson (1941). The interesting point about these normal biological reagins read- ing witli Wassermann antigen is that they are another example of antibodies that will react at low temperatures mucli more intensely than they do at body temperatures. There is no reason to assume that they are the same antibodies that are present in paroxysmal hemoglobinuria because these normal reagins are ])resent in tlie lower animals as well as in many cases of tuberculosis, leprosy, malaria, and in certain types of far advanced malignancies that do not show hemoglobinuria. It is possible that individuals with paroxysmal hemoglobinuria may have malaria, tuberculosis, and syphilis but the occurrence is probably a coincidence. Reagins. — Atopic reagins are antibody-like substances dis- covered l\y Prausnitz and Kiistner (1921) in the l)lood of patients suffering from hay fever or asthma. Kiistner was sensitive to fish. He found that when some of his serimi was injected intradermally into the forearm of an individual not sensitive to fish tlie area of skin so infiltrated became temporarily sensitive to an extract of fisli proteins. Reagins for the various pollens have been found in the blood of pollen hay fever and asthma cases. Coca suggested that these antibody-like substances be called "reagins" rather than antibodies. He took this position because, at that time, he did not believe that these substances developed as a result of antigenic stimulation in individuals possessing the necessary inheritance factor for allergy. This question will be discussed more fully in another chapter. NATURAL AND IMMUNE ANTIBODIES 151 LiPOiDOPHiLic Reagins. — Tlicsc are antibody-like siilistaiicos tliat will sensitize emulsions of certain tissne lipoids in tlie same way that bacteria are sensitized by their specific antibody. Just as sensitized bacteria may become agglutinated or may unite witli complement, so will sensitized lipoid particles undergo agglutina- tion if conditions are appropriate or they will combine with the complement if it is present. In man, i-eagins for acetone insoluble lipoids obtained from heart muscle or other animal tissue are found almost exclusively in the blood during the secondary and later stages of syphilis. A somewhat similar reagin, found occa- sionally in nonsyphilitics and ciuite extensively in the blood of the lower animals, can apparently be differentiated from syphilitic reagin by methods to be discussed in a later chapter. Precipitins. — These are antibodies produced against any un- altered soluble protein. They are usually found in tlie l)lood of any man or lower animal that has received injections of foreign protein as, e.g., antitoxin, normal horse serum, bacterial extracts, etc. The antibody sensitizes each colloidal particle of protein and this leads to a precipitation of these sensitized colloidal particles and to the union with complement if it is present. Anaphylactic Sensitizers. — Many persons have considered these interesting antibodies as identical with the ])recipitins but more recently some doubt has been cast upon this hypothesis. They are antibodies that have the rather unique property of attaching them.selves to smooth muscle and other tissue cells of the animal which produce them, several days after they make their appearance in the blood stream. This is rather a serious state of affairs for the animal, because on receiving another injection of antigen it would combine with these antibodies that are attached to the body cells and produce a serious physiological reaction called anapJwj- lactic shod'. Antitoxins.- — When a specific antigenic poison such as diph- theria toxin, tetaniLS toxin, l)otulinus toxin or streptococcus scarlet fever toxin is injected into an animal, a specific neutralizing anti- body called antitoxin is developed for the particular kind of toxin injected. It neutralizes but does not destroy the toxin. Small amounts of antitoxins may be found in some normal animals and in man. 152 IMMUNOLOGY Opsonins and Bacteriotropins represent normal and immune antibodies, respectively, that unite with specific bacteria and render them more easily engulfed by leucocytes. They are dis- cussed more fully in another chapter. Ablastin. — Tliis relatively new antibody was discovered by Taliaferro (1929, 1932). It develops in rats infected with Trypanosoma lewesi and prevents reproduction or cell division of the parasites without apparent injury to them. It differs from other antibodies in that it does not form a demonstrable union with the cells (trypanosomes) which it specifically affects. This type of antibody is not produced as a result of infection with T. hrucei, T. gamhiense, T. rJwdesiense, T. equiperdum, nor has Taliaferro observed it in his studies of the resistance of birds and mammals to certain malarial parasites. Antiaggressins. — Pathogenic bacteria vary in their ability to invade the tissues. Marked difference is exhibited by different strains of any one pathogen. Even some strains of C. diphtheriae seem to possess invasive powers. This is suggested by the work of Feierabend and Schubert (1929) and others to which attention has been called recently by Wells (1932). There are immunologists who interpret their experiments as showing that bacterial invasion is prevented by specific antibodies called by them antiaggressins. Their conclusions are based upon the acceptance of Bail's theory of aggressins. The veterinary immunologists have obtained star- tling results by immunizing domestic animals with antigenic ma- terial which they believe contains aggressins. Their work deserves serioiLs consideration by the medical student since their experi- mental studies are both extensive and intensive. Many important papers have appeared in the American Journal of Veterinary Medicine and elsewhere in veterinary literature. CoNGLUTiNiNS. — Ehrlich and Sachs (1902) discovered that in- activated bovine serum intensified hemolysis due to amboceptor and complement. Bordet and Gay (1906) and later Bordet and Streng (1909) investigated the phenomenon described by Ehrlich and Sachs and definitely showed that bovine serum contains a colloidal substance named by them conglutinin that unites only with sensitized cells that have adsorbed complement. It intensifies both the agglutination and hemolysis of such cells. They showed that this conglutinin will not unite with either unsensitized or NATURAL AND IMMUNE ANTIBODIES 153 sensitized cells alone but will unite with the latter only after they have combined with complement. These authors ako found that the substance resists heating to 56° C. and that it has biochemical properties somewhat similar to the albumins. Manwarinj? (1906) described a thermostable component of bovine serum whicli he named ''auxilysin." It is probably the same as conglutinin. Heterophile Antibodies and Heterophile Antigens. — In 1911 Forssman discovered that a great variety of apparently otherwise unrelated substances possessed in common an antigenic factor tliat would stimulate rabbits to produce hemagglutinins and hemolysins for sheep cells. These nonspecies specific antigenic substances have been called heterophile antigens and the hemagglutinins and hemolysins resulting from their injection are called heterophile antibodies. The heterophile antigen present in sheep cells is dis- tinct from the isophile or species specific antigen. This work was extended by Forssman and Hintze (1912), Forssman and Fox (1914), Pick (1913), Doerr and Pick (1913), and many others. More recent reviews and investigations are those of Tanigiechi (1921), Landsteiner and Simmons (1923), Powell (1926), Hooker (1926), Bull (1928), Jungeblut (1929), Landsteiner and Levine (1932) and Meyer and Morgan (1935). As a result of this work the following conclusions relative to heterophile antigens have been formulated and generally accepted. They have been reported as present in the tissues (especially the kidney) of the guinea pig, horse, cat, dog, camel, mouse, chicken, gills of carp and pike, in the plasms and urine of the horse, in the red blood cells of the sheep, goat and chicken and also in certain bacteria such as B. enteritidis of Gaertner, B. paratyphosus B and B. dysenteriae (Shiga). As a rule, with the exception of the chicken, when tlie heterophile antigen occurs in the tissues of an animal, it is absent from tlie red cells of that animal and conversely, when it is present in the red cells, it is absent from the tissues. It is interesting to note that while it is found in tlie chicken and in tlie mouse, it is not present in either the pigeon or the rat. While it is present in B. para- typhosus B, it is absent from E. typhosa. The Forssman antigen found in many animal tissues seems to be made up of a protein and perhaps a combination of lipoid and polysaccharide whereas the active substance in bacteria seems to be polysaccharides alone. Schiff and Adelsberger (1924) and others seem to have established 154 IMMUNOLOGY a relationship between tlie group A substance of human red cells and the polysaccharide of Forssman's antigen. Interest in hetero- phile antibodies has increased since Paul and Bunnell (1932) noted their presence in high titer in many cases of acute infections mononucleosis. The subject has been extensively investigated by Davidsohn, While the term heterophile antibodies has been used almost exclusively to indicate antibodies for nonspecies specific antigens of the Forssman type which will cause agglutination of old sheep red cells, it should be remembered that there are other examples of nonspecies specific antibodies. Among the well-known antibodies that cause cross reactions are those that are produced by im- munizing with Friedlander's bacillus. Such antibodies will ag- glutinate not only suspensions of B. friedVdnder but also suspen- sions of Pneumococcus Type II. Another example of heterophile antibodies are those found in the blood of typhus fever patients. These antibodies will react with both Proteus 0X19 and the Rickettsia of Mexican and European typhus fever. While these antibodies giving cross reactions are for nonspecies antigens and therefore are heterophile antibodies they do not cause agglutination or hemolysis of sheep cells. References Bordet, J.: Studies in Immunity, Translated by F. P. Gay, New York, 1909, John Wiley & Sons, Inc. Bordet, J., and Gay, F. P.: Sur les relations des sensibilisatrices avec I'alexine, Ann. Inst. Past. 20: 467, 1906. Bordet and Strang (1909): See Zinsser, H.: Kesistance to Infectious Dis- eases, New York, 1931, The Macmillan Co., p. 192. Buchner, H.: Centralbl. f. Bakteriol. Orig. 5: 817, 1889; 6: 1, 1889. Bull, C. G.: Heterophile Antigens and Antibodies. Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 733. Burmeister, J.: Cited from Mackenzie. Coriell, L., Miller, W. E., and Sherwood, N. P.: Studies on Natural Bac- teriolysins, 1940. (Unpublished work.) Davidsohn, I.: Heterophile Antigens and Antibodies, Arch. Path. 4: 776, 1927. Davidsohn, L: Heterophile Antibodies in Serum Sickness, J. Immunol. 16: 259, 1929. Davidsohn, I.: Further Studies on Heterophilic Antibodies in Serum Sick- ness, J. Immunol. 18: 31, 1930. Davidsohn, I.: Heterophilic Antibodies in Serum Disease, III, J. Infect. Dis. 53: 219, 1933. Davidsohn, I.: Infectious Mononucleosis, Am. J. Dis. Child. 49: 1222, 1935. NATURAL AND IMMUNE ANTIBODIES 155 Davidsolin, I.: Serological Diagnosis of Infectious Mononucleosis, J. A. M. A. 108: 289, 1937. Davidsohn, I., and Walker, Phoebe H.: Nature of Heterophile Antibodies in Infectious Mononucleosis, Am. J. Clin. Path. 5: 455, 1935. Dingle, J. H., Fothergill, L. D., and Chandler, C. A.: Studies on Haemophilus Influenzae. III. The Failure of Complement on Some Animal Species, Notably the Guinea Pig, to Activate the Bactericidal Function of Sera of Certain Other Species, J. Immunol. 34: 357, 1938. Doerr, R., and Pick, E.: Ueber den Mechanismus der primaren Toxizitat der Antisera und die Eig«nschaften ihrer Antigene, Biochem. Ztsclir. 50: 129, 1913. Die primare Toxizitat der Antisera, 2 Mitteilung, Ztschr. f. Imm. 19: 251, 1913. Donath, J., and Landsteiner, K. : Ueber paroxysmalen Hamoglobinurie, Miinchen. med. Wehnschr. 51: 1590, 1904. Downs, C. M., Jones, H. P., and Koerber, K. : Incidence and Properties of Isohemolysins, J. Infect. Dis. 44: 412, 1929. Ehrlich, P.: Studies in Immunity, Translated by C. Bolduan, New York, 1910, John Wiley & Sons, Inc. Ehrlich and Sachs (1902): See Zinsser, H.: Eesistance to Infectious Diseases, New York, 1931, The Macmillan Co., pp. 190-193. Feierabend, B., and Schubert, O. : Experimentelle Untersuchungen mit stammen von maligner Diplitherie, Ztschr. f. Immunitatsforsch. u. exper. Therap. 62: 283, 1929. Fliigge, C. : Die Microorganismen, ed. 2, Leipzig, 1886, F. E. W. Vogel. Forssman, .7.: Biochem. Ztschr. 37: 78, 1911. Cited by Topley & Wilson. 1935. Forssman, J., and Fex, J.: Ueber heterology Antisera. Biochem. Ztschr. 61: 6, 1914. Forssman, J., and Hintze, A.: Die heterologe Toxizitat der Antisera, Biochem. Ztschr. 44: 336, 1912. Hooker, S. B.: Heterophile Antigen-Antibody Reactions in Relation to the Serum Diagnosis of S\'philis bv Precipitation, J. Immunol. 11: 403, 1926. Irwin, M. R., Beach, B. A., and Bell, F. N.: Studies on the Bactericidal Action of Bovine Whole Blood and Serum Towards Brucella abortus and Brucella suis, J. Infect. Dis. 58: 15, 1936. Jungeblut, C. W., and Ross, A.: On the Presence of Heterophile (Forss- man's) Antigen in Bacteria of the Parat^-phoid-Dysentery Group, J. Immunol. 16: 369, 1929. Kahn, R. L., McDermott, E. B., and Marcus, M. S.: Effect of Temperature on the Kahn Reaction. Studies I, II, III, and IV, Am. J. SjTph. Gonor. & Yen. Dis. 25: 151, 157, 163, 173, 1941. Kemp, J. E., Fitzgerald, E. M., and Shepherd, M.: Occurrence of Positive Serological Tests for Sj-philis in Animals Other Than Man, With a Review of the Literature, Am. J. Svph., Gonor. & Yen. Dis. 24: 537, 1940. Landsteiner, K. : Ueber Unterschiede des folaten und miitterlichen Blut- serums und Uber eine agglutinations und fallungshemmende Wirkung des Normal Serums, Miinchen. med. Wehnschr. 49: 473, 1902. Landsteiner, K., and Levine, P. : On the Forssman Antigens in B. Para- Typhosus B and B. Dysenteriae Shiga, J. Immunol. 22: 75, 1932. Ledingham, J. C. G. : Natural Immunity, System of Bacteriology, Yol. 6, Medical Research Council, p. 34, 1931. Locke, A., and Hirsch, E. F.: The Isolation of Substances with Immune Properties, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 1049. Mackenzie, G. M.: Paroxysmal Hemoglobinuria, Medicine 8: 159, 1929. 156 IMMUNOLOGY Mackie, T. J., and Finkelstein, M. H.: Studies of Non-specific Complement Fixation With Particular Reference to the Interaction of Normal .Serum and Certain Non-antigenic Substances, J. Hyg. 28: 172, 1928. Metchnikoff, E.: Immunity in Infectious Diseases, Translated by F. G. Binnie, London, 1907, Cambridge University Press. Muir, R., and Browning, C. H.: On the Bactericidal Action of Normal Serum, J. Path. & Bact. 13: 76, 1908. Nuttall, G. F. H.: Ztschr. f. Hyg. u. Infektionskr. 4: 353, 1888, Paul, John R., and Bunnell, W. W.: Presence of Heterophile Antibodies in Infectious Mononucleosis, Am. J. M. Sc. 183: 90, 1932. Pettersson: Cited by Mackie and Finkelstein. Pfeiffer, R., and Issaefif: Ueber die specilische Bedeutung der Cholera- immunitat. (Bakteriologie), Ztschr. f. Hyg. 17: .".55, 1S94. Powell, H. M. : Heterophile Antigen in Bacteria, J. Immunol. 12: 1, 1926. Prausnitz, C, and Kiistner, H. : Studien iil)er die Ueberempfindlichkeit, Centralbl. f. Bakt. 86: 160, 1921, Schiff, F. von, and Adelsberger, L.; Ueber blutgruppenspezifische Anti- korper und Antigene. I. Mitteilung, Ztschr. f. Immunitatsforsch. u. exper. Therap. 40: 335, 1924. Sherwood, N. P., Bond, G. C, and Canuteson, R. I.: On the Possible Pres- ence of a Reagin-like Factor in Normal Human Serum, Am. J. .Syph., Conor. & Ven. Dis. 25: 179, 1941. Sherwood, N. P., Bond, G. C, and Clark, H. F.r Results Obtained With Kolmer, Kahn, Kline, and Eagle Tests on Animal Sera, Am. J. Syph., Conor. & Ven. Dis. 25: 93, 1941. .Shrigley, E. W., and Irwin, M. R.: Difference in Activity of Serum Complement From Various Animal Species, J. Immunol, 32: 281, 1937. Taliaferro, W. H.r Infection and Resistance in the Blood-Inhabiting Protozoa, Science 75: 619, 1932. Wells, H. G. : Chemical Aspects of Immunity, New York, 1929, Chemical Catalog Co. Wells, J. R. : Intradermal Method of Virulence Testing of Corynebacterium Diphtheriae, Am. J. Pub. Health 22: 308, 1932. Supplementary References Holtman, D. F. : Acquisition of Heterophile Antigen by Eberthella Typhosa and Salmonella Paratyphi During Culture on Artificial Media, J, Im- munol. 36: 405, 1939, Holtman, D, F,: The Acquisition of Heterophile Antigen From the Guinea Pig by Eberthella Typhosa and Salmonella Paratyphi, J, Immunol. 36: 413, 1939. Redfern, W, W,: A Study of the Primary Toxicity of Heterophile Immune Rabbit Serum for Guinea Pigs, and Its Apparent Relation to the Phenomenon of Anaphylaxis, Am, J. Hyg. 6: 276, 1926, Taniguchi, T.: Heterophile Antigen and Antibody: Relation to Lipoids; Heterophile and Isophile Cytolysins, J. Path. & Bact, 24: 217, 1921, Differences in the Character of Haemolytic Immune Sera of Different Origins for Sheep Corpuscles and on the Mechanism of Heterophile and Isophile Cytolysis: The .Separation of Combining and Antigenic Properties, Ibid., p. 356. II. Differences in the Charac- ter of Haemolytic Immune .Sera of Different Origins of Sheep Cor- puscles and on the Mechanism of Heterophile and Isophile Cytolysis, Ibid., p, 456. CHAPTER IX COMPLP^MENT Complement. — Definition. — Complement may be defined as a thermolabile enzyme-like colloid, present in the serum of animaLs, that has the ability to combine with sensitized eells and bring about their lysis under suitable conditions. The history of the discovery and naming of this su])stance is dis- cussed briefly in an earlier cliapter. It was regarded as an enzyme by Buchner who named it alexin and regarded it as of leucocytic origin. Ehrlicli regarded it as having a combining or haptophore group and a zjnnophore or enzyme-like group which was respon- sible for lysis. Some of its enzyme-like properties may be sum- marized as follows: (1) It is colloidal in nature. (2) The presence of a small amount brings about extensive chemical changes in the substrate (cells). (3) It is thermolabile. (4) The reaction is about as reversible as enzyme reactions. (5) The optimum temperature for its action is 37° C. (6) Antienzymes as well as anticomplcment have been reported. (7) There is considerable evi- dence that its activity is interfered with by the end products of its action. Nature of Complement. — Biochemically speaking, Ferrata (1907) reported that complement is composed of an albumin frac- tion, named by Brand (1907) "endpiece," and a globulin frac- tion which he calls "midpiece." Subsequent work by Whitehead, Gordon and Wornall (1925) indicates that the midpiece consists of a heat labile euglobulin fraction and a heat stable fraction named tlic third component. The latter can be removed from a serum by treatment with zymin (residue after acetone and ether extraction of yeast) and restored by adding guinea pig serum heated to 56° C. In 1926 the same authors reported that complement could be rendered inactive by treatment with appropriate concentrations of ammonia and that the activity could be restored b\- tlic addition of either heated or zyniin-treated serum. They named this ammonia sensitive fraction the ''fourth" component and de- termined that it is associated with the endpiece. Pillemer, Seifter 157 158 IMMUNOLOGY and Ecker (1941) have made an extensive study of the endpieee in- eluding the fourth component of complement. They say that their findings ''seem to indicate that the endpieee of complement is the calcium-carbohydrate-pseudoglobulin molecule" and that in the light of this "the fourth component would be the carbohydrate." They consider that the complex calcium-pseudoglobulin molecule is the carrier of the active carbohydrate factor. If their work is confirmed, the old conception of endpieee as an albumin will have to be abandoned. In another paper Ecker, Jones and Kuehn (1941) suggest that the euglobulin fraction of midpiece is closely associated with a phosphatide (cephalin). The importance of this observation is apparently unknown. Sensitized cells which have adsorbed the globulin fraction (midpiece) only are called ''per sensitized" cells. Preservation of Complement. — When serum rich in comple- ment is frozen, its potency as a lytic agent of sensitized cells is re- tained for one or more weeks. Kolmer suggests that this is prob- ably the oldest method of preserving complement. Morgenroth and others were able to preserve complement kept at -10 to -15° C. for several weeks. Kolmer found that either diluted or un- diluted complement kept frozen except when samples are removed retains its titer undiminished for 3 or 4 days, after which time it drops rather rapidly. The titer of undiluted complement kept at 4° to 9° C. is diminished in 24 hours, and shows progressive deterioration thereafter. Many laboratories preserve complement by freezing and desicca- tion in vacuo at a low temperature. This method has come into rather general use since the publication of Flosdorf and Mudd (1935, 1938) in which they describe the procedure and apparatus that operates successfully. The methods are applicable to the ])reservation of various kinds of immune sera and also bacteria. The first method depended largely upon the vacuum pump for removal of the water vapor given off during drying and the dried serum was called "lyophile" serum. In 1938 the same authors reported upon a simpler and better method of desiccation. In this method both chemical desiccants and physical evacuation were used. The method is called the Cryo-chem process. Complement preserved by this method is stored in the refrigerator and main- tains its titer for long periods of time. COMPLEMENT 159 A metliod wliicli lias l)eeii used with success in this laboratory is giv'en by Boerner and Lnkens (1939). This is a modification of the original procedure of Sonnenscheine. The serum is obtained and an equal volume of the following mixture is added : sodium acetate — 12.0 grams; boric acid — 4.0 grams; sterile distilled Avater q.s. — 100.0 e.c. The mixture is tlion stored in tlie ice ])ox and will keep for relatively long periods of time. Effect of Heat on Complp:ment. — The optimum temperature for complement action is 37° C, but it loses its activity within a few hours at this temperature. Manwaring (1906) studied the effect of higher temperatures on complement and found that notice- able diminution in titer occurs within 10 minutes at 49° C, al- though traces are present at the end of one hour. At 51° C, there is greater diminution in 10 minutes and complete inactivation in 35 minutes. When a temperature of 53° C. is employed for 14 minutes the lytic property of complement disappears completely. He found also that complete inactivation occurs in 12 minutes at 55° C, 8 minutes at 57° C 4 minutes at 59° C, and 2 minutes at 61° C. Reversibility of the Reaction. — Gramenitski (1912) suggests that the inactivation of complement at 56° C. is not an irreversible reaction. Brooks (1919, 1921) has confirmed these observations and concludes that under proper conditions a partial return of activity may occur. Just how inactivation is brought about by heat is not definitely known. It is thought that a change in colloidal dispersion is an important factor. Inactivation by Shaking. — This concept is supported by the observations of Jacoby and Schiitze (1910) that prolonged shaking will eventually lead to complement inactivation. This has been confirmed by a number of workers. Anticomplementary Effects of Inorganic and Organic Com- pounds.— Hektoen (1903), Manwaring (1904) and others have shown that definite concentrations of various inorganic salts in- hibit complement action. In 1913, Arkin reported that a number of inorganic substances, as Avell as lactic acid, interfere with phagocytosis and concluded that it is due to an effect upon com- plement. A few 3'ears later Sherwood (1917) found that many other inorganic and organic compounds will prevent the lytic action of complement when present in very small concentrations. 160 IMMUNOLOGY Mention has already been made of the effect of zymin and ammonia upon midpieee and endpiece, respectively. These results indicate the necessity of using chemically clean glassware in all im- munological work dealing with opsonins and complement fixation. Origin of Complement. — In regard to the origin of complement nothing is known definitely, nor is it absolutely settled that it exists as such in the circulating plasma. Buchner (1892), Hankins (1892) and Metchnikoff suggested, quite early, that it was of leuco- cyte origin. In connection with this, it is of interest to note that when sensitized red cells are phagocytized by leucocytes, they seem to be no more readily hemolyzed than unsensitized cells. A great many have reported that the complement titer of serum is higher, if it is allowed to stand on the clot in the refrigerator at 6 to 8° C, for 24 hours. It has been argued that this increase in titer is due to the leucocytes present. Regardless of theoretical considerations, the observation that an increase in titer frequently occurs under the conditions just mentioned is of practical importance in diag- nostic serology. A great many have offered experimental evidence that the liver is the source of complement. Sherwood, Smith and West (1916) investigated this question and concluded that it is neither the only, nor the principal source of complement. They point out important sources of error in the work of Dick and of Nolf and others who had concluded that complement is of hepatic origin. Desirable Qualities of a Complement. — ^For use in complement fixation, there are certain qualities that must be considered in choosing complement. It should be readily fixed by a sensitized antigen ; free from natural hemolysins, and agglutinins ; lysis should be reasonably rapid, and it should be sufficiently stable for use in overnight fixation at 6° to 8° C. Complement From Various Animals.^ — Guinea pig complement most nearly meets these requirements, although human complement is quite satisfactory. The complement titer varies from day to day in the same pig, and may be nil in apparently healthy guinea pigs. For these reasons it is recommended that pooled complement (from 3 or 4 guinea pigs) be used in complement-fixation tests. A satisfactory unit of guinea pig complement in the Kolmer- Wa.ssermann technique should be from 0.25 to 0.35 c.e. of a 1 :30 COMPLEMENT 161 dilution, and hemolysis should be practically complete after twenty to thirty minutes in a water-bath held at 37° C, although the period of incubation is one hour. Rabbit complement and dog com- plement are very unsatisfactory, since nonspecific fixation is quite commonly encountered. Bond and Sherwood (1939) found that the hemolytic property of snake serum is due to the action of a thermolabile complement and a naturally occurring hemolysin. The complement in snake serum possesses properties similar to guinea pig complement. The titer is usually high; it is thermolabile and deteriorates on standing. Furthermore it was bound by a specific bacterial antigen-antibody complex and also by the syphilitic antigen-reagin complex respec- tively. Specific Fixation of Complement. — In 1901 Bordet and Gengou observed that sensitized bacterial cells adsorbed or bound comple- ment and apparently proved that the complement that lysed sensi- tized bacteria was identical with the complement that laked sensitized red cells. This work, however, has not ended the con- troversy over whether there is one complement or many comple- ments, but it did give the scientific world the complement fixation technique. The subject of multiplicity or singleness of complement is discussed rather extensively by Zinsser, Enders and Fothergill (1939). Specific Complement Fixation by Precipitates. — The follow- ing year (1902) Cengou showed that precipitates formed in the I)ro<'ipitin reaction would bind complement. Gengou explained this as due to amboceptors or sensitizing antibodies present in the im- nume serum along with precipitins. According to the unitarian viewpoint supported by Zinsser and others, the sensitizer and precipitin are identical, and while flocculation does not require complement, the precipitate formed, which is composed of sensitizer and antigen, is able to adsorb complement. Experimental support for this has been offered by Gay (1905), Moreschi (1905), and others. It was Gay who noted the relationship to the precipitin reaction. Time of Occurrenck of Fixation. — Dean (1913) has shown that the rate and character of precipitates formed influence the fixation of complement. In his opinion the greatest fixation of 162 IMMUNOLOGY complement occurs early in the reaction before large aggregates are formed and when the optimum surface of sensitized colloid is available for absorption. Nonspecific Adsorption. — Cells whose surface contains lipoids seem to be able to adsorb complement in a nonspecific manner. Landsteiner and von Eisler suggest the importance of lipoids in certain nonspecific complement fixations. They conclude, how- ever, that the colloidal state is the important factor in this non- specific fixation. Wilde (1901) noted that emulsions of most bacteria adsorl) complement even when not sensitized. This is im- portant in view of the use of bacterial complement fixation in the diagnosis of certain diseases as well as in the identification of micro-organisms. Chemical and Physical Factors in Cell Sensitization. — In other chapters cell sensitization, or the union of antigen and anti- body, is discu.ssed extensively. The phenomenon of specificity which seems to depend upon the chemical constitution of the antigen forces one to recognize definite chemical factors in the process. The evidence seems to bear out Bordet's contention that adsorption plays a prominent part and that the antibody globulin appears to become insoluble in saline. The sensitized cells behave in cataphoretic experiments like particles of denatured globulin. The reaction occurs quite readily at 0° C. Purely chemical re- actions occur very slowly in aqueous systems at low temperatures, but the rate is greatly accelerated as the temperature is increased. Adsorption phenomena, on the other hand, are noticeably increased with the lowering of temperatures, and hence occur readily at 0° C. At the present time there are two concepts of adsorption that should be borne in mind. Adsorption may be illustrated by the removal of coloring matter from water by means of powdered charcoal. According to one theory, this is accomplished by the molecular capillary state of the surface, while according to an- other concept the valences of the molecules within the charcoal are all satisfied because they are united with each other, but those at the surface have unsatisfied valences or fields which unite with the coloring structure and physical state of the dye as well as the adsorbing surface. It would seem probable that either or both COMPLEMENT 163 mechanisms might be included under the term adsorption, since they would represent methods of surface binding of matter. Thiele and Embleton's Observations. — Another interesting observation concerning tlie union of antibody with antigen is re- ported in an excellent paper by Thiele and Embleton (1914). They showed that a given amount of washed red blood cells is sensitized bj^ definite amounts of antibody regardless of the concen- tration of antibody per cubic centimeter. In other words, if a sensitizing dose of amboceptor is added to one cubic centimeter of salt solution or 20 c.c, it is removed by adding the same unit of red cells to each tube. Bordet's and Ehrlich's Views on the Mechanism of Sensi- tization.— As previously stated, Bordet showed that this jn-ocess of sensitization of antigen occurs before complement could bring about lysis, since the latter acted only upon sensitized cells. Bordet concludes from his experiments that complement is adsorbed by the antigen-antibody complex with resulting lysis, while Ehrlich concludes that amboceptor chemically combines with antigen on the one hand and with a combining group of complement on the otlier. The complement is thus enabled to act on the antigen, producing lysis. Visible Phenomena op Cell Lysis. — At this point it might be well to visualize what can be seen when an adequate amount of complement is added to various sensitized antigens such as Microspira comma, E. typhosa, immune precipitates and sensitized red cells. In the case of a sensitized suspension of living Microspira comma there occurs at 37° C. at first a rounding up of the organisms, fol- lowed by their disappearance. They seem to be almost completely destroyed except for a residual granule. Living sensitized typhosus suspensions are killed, as can be demonstrated by plating methods, but the individual cells frequently appear to be unchanged. Sensi- tized streptococci, staphylococci and also sensitized precipitates seem to show no change. When sensitized red cells are mixed Avith an adequate amount of complement, there occurs after a few minutes a swelling of the cell and liberation of the hemoglobin; the stroma or cell framework remains. 164 IMMUNOLOGY Fixation of Complement by Products op Lysis. — Liefmann and Cohn (1910) showed that during hemolysis there are liberated products which render complement inactive. A number of workers have considered that complement functions as an enzyme and is bound by the products of lysis rather than by the antigen-antibody complex. Hill and Parker's Physicochemical Interpretation. — Hill, Parker and McKinstry (1925) give a physicochemical interpreta- tion to a simple equation which they believe explains all the phenomena of hemolysis by complement. In their paper they draw the following interesting conclusions: That complement is a catalyst. It enters into and combines with the cell under the in- fluence of amboceptor. That the latter (amboceptor) contains a ferment which can bring about the release of hemoglobin. Comple- ment acts as a catalyst for this reaction. They conclude that com- plement is fixed by some other fraction of the firmly bound amboceptor and that the quantity of this complement fixing sub- stance does not change during hemolysis. This hypothesis is in- teresting since it is based upon an equation which fits not only their own experimental results but also tliosc of Thiele and Emble- ton, and certain conce])ts advanced l)y Nolf in 1900. Undoubtedly there are many secondary factors introduced when the antigen undergoes visible physical and cliemical cliange such as occurs with Micro.spira comvut or with red cells, which ai'c not present in re- actions of complement with other sensitized bacteria or sensitized inert particles. Action of Complement Depends on Concentration. — While it is obviously difficult to be certain of the exact mechanisms involved, there is no difference of opinion over certain observed facts about complement. Thiele and Embleton find that whereas amboceptor unites with antigen regardless of the amboceptor concentration per cubic centimeter, complement action depends upon the exist- ence of a certain minimum concentration of complement per cubic centimeter of the solution. In other words, there might be enough complement in a test tube to hemolyze 50 or more units of red cells, l)Ut unless its concentration per cubic centimeter is adequate no liemolysis will result. Others have shown that while antigen and antibody unite readily at 0° C, complement and sensitized antigen do not, although they do unite at temperatures slightly above zero. COMPLEMENT 165 This union at low tem])eratures makes possible a primary incuba- tion of 6° to 8° C. in complement fixation work. Effect of Temperature on Complement Action. — In regard to the effect of temperature upon complement action, Zinsser says the si)eed and i'ompleteness of the union of complement and sensitized antigen increase as the temperature approaches 40° C. Discussion of Eagle's Work.— Eagle (1929) has reported a scries of experiments attempting to explain the mechanism of com- plement fixation. He finds, as others before him, that qualitatively speaking, tlie plienomenon of hemolysis appears to represent a inonomolccular reaction, and that shortly after hemolysis appears, there is an inci-ease in the rate of fixation of complement. He studied and compared fixation of complement by agglutinated bac- teria, immune precipitates and sensitized red cells, and noted a similarity in the curves, with the exception of this added fixation in the case of hemolysis. Liefmann and Cohn (1911) noted this, as did Thiele and Embleton (1914). While it is an interesting observation, it does not prove the monomolecular nature of the reaction, nor is it so regarded by Eagle. He concludes that ''the physical constants of fixation (temperature coefficient, velocity, quantitative relationships between the reactants) are those com- monly associated with adsorption processes and are the same in the three types of fixation studies. " While this is a good working explanation of complement fixa- tion, it should be remembered that his conclusions result largely ])ecause his data fit Freundlich's equation for adsorption processes and also because the figures he obtains for temperature coefficients are low as is the case in adsorption reactions. The Freundlich equation is usually used in the sense of circumstantial evidence rather than proof, and the problem is such that it is doubtful whether the temperature coefficients he reports are significant. Since this explanation of complement action is perhaps the most commonly accepted one, it can be used as a working hypothesis for the present. This admits that both chemical and physical fac- tors are involved, and assumes that complement is bound to the antigen-antibody complex preliminary to any lytic action that may occur, and also admits of the interference of lysis by secondary products liberated or formed in the process. 166. IMMUNOLOGY References Arkin, A.: The Influence of Strychnin, Caflfein, Chloral, Antipyrin, Cholesterol, and Lactic Acid on Phagocytosis, J. Infect, Dis. 13: 408, 1913. Boerner, F., and Lukens, M.: A Simplified Complement Fixation Technic for the Serologic Diagnosis of Syphilis, Am. Jour. Clin. Path. 9: 13, 1939. Bond, G. C, and Sherwood, N. P. : Serological Studies of the Eeptilia. II. The Hemolytic Property of Snake Serum, J. Immunol. 36: 11, 1939. Bordet, J., and Gengou, O.: Sur 1 'existence de substances sensibilisatrices dans la plupart des serums antimicrobiens, Ann. Inst. Pasteur 15: 289, 1901. Brand, E,: Ueber das Verhalten der Komplemente bei der Dialyse, Berl. klin. Wchnschr. 44: 1075, 1907. Brooks, S. C: The Eegeneration of Complement After Kadiation or Heating, J. M. Eesearch 41: 411, 1919. Buchner, E.: Cited in Metchnikoff, Resistance to Infectious Diseases, London, 1907, Cambridge University Press, p. 193. Dean, H. R.: The Relation Between the Fixation of Complement and the Formation of a Precipitate, Ztschr. f. Immunitats 13: 84, 1912. Eagle, H.: The Mechanism of Complement Fixation, J. Gen. Physiol. 12: 825, 1929. Ecker, E. E., Jones, C. B., and Kuehan, A. D.: Studies on the Adsorption of Complement, J. Immunol. 40: 81, 1941. Ecker, E. E., and Pillemer, L.: Anti-Coagulants and Complementary Activity. An Experimental Study, J. Immunol. 40: 73, 194L Ehrlich, P.: Studies in Immunity, New York, 1910, John Wiley & Sons, Inc. Ferrata, A.: Die Unwirksamheit der komplexen Hamolj-sine in salzfreien Losungen und ihre Ursache, Berl. klin. Wchnschr. 44: 366, 1907. Flosdorf, E. W., and Mudd, S.: Procedure and Apparatus for Preservation of "Lyophil" Form of Serum and Other Biological Substances, J. Immunol. 29: 389, 1935. Flosdorf, E. W., and Mudd, S.: Improved Procedure and Apparatus for Preservation of Sera, Microorganisms and Other Substanees^Cryo- chem-process, J. Immunol. 34: 469, 1938. Freundlich, H. : Kapillarchemie. Millard Physical Chemistry for Colleges, New York, 1931, McGraw-Hill Book Co., Inc., p. 133. Gay, F. P.: The Fixation of Alexines bv Specific Serum Precipitates, Centralbl. f. Bakt., O. 93: 603, 1905. Gengou, O. : Sur les sensibilisatrices des serums actifs centre les sub- stances albuminoides, Ann. Inst. Pasteur 16: 734, 1902. Gordon, J., Whitehead, H. R., and Wornall, A.: Action of Ammonia on Complement; Fourth Component, J. Biochem. 20: 1028, 1926. Gordon, J., Whitehead, H. R., and Wornall, A.: Calcium and Complement Action, J. Biochem. 20: 1036, 1926. Gramenitski, M.: Ueber die Eegeneration des Komplements, Biochem. Ztschr. 38: 501, 1912. Hankins, E. H.: Ueber den Ursprung und Vorkommen von Alexinen im Organismus, Centralbl. f. Bakt. 12: 777, 1892; Ibid. 12: 809, 1892. Hektoen, L.: The Action of Certain Ions Upon the Lysins in Human Serum, Tr. Chicago Path. Soc. 5: 303, 1901-03. Hill, A., Parker, G., and McKinstry, R. N.: Observations on the Deviation of Complement in the Wassermann Test, J. Path. & Bact. 28: 47, 1925. COMPLEMENT 167 Jacoby, M., and Schiitze, A.: Ueber die Inaktivierung der Komplemente durch Schutteln, Ztschr. f. Immunitats. 4: 730, 1910. Kolmer, J. A., Matsunami, T., and Trist, M. E.: Studies in the Standard- ization of the Wassermann Reaction. IV. A General Study of the Complements of Various Animals With Special Eeference to Human and Guinea Pig Complements and Methods of Collection, Am. J. Syphilis 3: 407, 1919. Liefmann, H., and Cohn, M.: Die Wirkuug des Komplementes auf die ambozeptorbeladenen Blutkorperchen, Ztschr. f. Immunitats. 7: 669, 1910; (II Teil) Ztschr. f. Immunitats. 8: 58, 1911. Manwaring, W. H.: On the Destruction of Complement by Heat, Tr. Chicago Path. Soc. 6: 425, 1906. Manwaring, W. H.: The Action of Certain Salts on the Complement in Immune Serum, J. Infect. Dis. 1: 112, 1904. Moreschi, C. (1905): Cited by Zinsser, Resistance to Infectious Diseases, New York, 1931, The Macmillan Co., p. 197. Nolf, P.: De I'origine du compliment hemolytique et de la nature de I'hemolyse par les serums. Bull. Acad, de Science de Belgique, 1908, Classe des Sc, p. 748. Pillemer, L., Seifter, J., and Ecker, E. E.: The Effect of Amino Compounds on the Fourth Component of Complement, J. Immunol. 40: 89, 1941. Pillemer, L., Seifter, J., and Ecker, E. E.: The Effect of Reducing Agents on the Fourth Component of Complement, J. Immunol. 40: 97, 1941. Pillemer, L., Seifter, J., and Ecker, E. E.: The Effects of Acids and Alkalies on the Fourth Component of Complement and the Role of Calcium, J. Immunol. 40: 101, 1941. Sherwood, N. P.: The Effect of Various Chemical Substances on the Hemolytic Reaction, .1. Infect. Dis. 20: 185, 1917. Sherwood, N. P., Smith, C, and West, R.: The Complement Content of Eck-Fistula Dogs, J. Infect. Dis. 19: 682, 1916. Thiele, F, H., and Embleton, D.: The Mechanism of Antibody Action, J. Path. & Bact. 19: 372, 1914-15. Whitehead, H. R., Gordon, J., and Wornall, A.: The "Third Component" or Heat-Stable Factor of Complement, J. Biochem. 19: 618, 1925. Wilde, M.: Ueber die Absorption der Alexine durch abgetodtete Bacterien, Berl. klin. Wchnschr. 38: 878, 1901. Zinsser, H.: Resistance to Infectious Diseases, ed. 4, New York, 1931, The Macmillan Co. Zinsser, H., Enders, J. F., and Fothergill, L. D.: Immunity, New York, 1939, The Macmillan Co., pp. 200-207. CHAPTER X ISOHEMAGGLUTININS— BLOOD GROUPS Isohemagglutinins. — Discovery. — The first recorded observation that the serum of a normal, healthy individual can bring about agglutination of the red cells of other normal individuals of the same species was made by Landsteiner in 1900. This preliminary report M^as followed by an intensive study of the sera and red cells of twenty-two individuals. From an analysis of the data ob- tained in this study, Landsteiner (1901) discovered that three definite blood groups were represented. The following year De- Castello and Sturli (1902) continued this work at the suggestion of Landsteiner. They not only confirmed the existence of the three groups, but found four exceptions which are the first re- corded examples of the fourth group. The}^ even discussed four possible groups which they designated hy numbers rather than by letters. Since the four exceptions tliat constituted a new blood group were bloods from young children, DeCastello and Sturli thought that they might be members of one of Landsteiner 's groups that had not yet acquired their full quota of necessary fac- tors. According to Ottenberg (1928) and also Zinsser and Coca (1931), Landsteiner immediately appreciated the existence of the four groups. He had previously postulated (1901) two agglutinins and two agglutinogens as was later done by Jansky (1907). Hektoen (1907) and Gay (1907) both confirmed the existence of the three human blood groups described by Landsteiner. Two years later Landsteiner (1909) published a more complete discus- sion of his work on isohemagglutinins. In 1910, Moss offered a classification of tlie 1)1 ood groups which has found wide acceptance. Apparently Hektoen (1907), Gay (1907) and Moss (1910) were not aware that previous workers (Landsteiner, et al.) had con- sidered more than three groups and had postulated two agglutinins and two agglutinogens since both Hektoen and Moss speak only of Landsteiner 's three groups and certain exceptions and both as- sume the existence of three agglutinins. Landsteiner (1928), Ottenberg (1928) and Snyder (1929) have reviewed the literature 168 ISOHEIM AGGLUTININS 169 on isohemagglutination and have discussed various classifications and theories relative to the inheritance factors involved. Kennedy (1931) published an extensive review of Jansky's work. This is of interest since the original publication is found in few libraries. Classification in Use. — At the present time there are three classifications of the blood groups in use. In the classifications of Jansky and Moss the groups are numbered. While groups I] and III are the same in both classifications, it should be remem- bered that group I of Moss corresponds to group IV of Jansky, and conversely, group IV of Moss is the same as group I of Jansky. Classification Based on Agglutinogen Content. — In the third and more recent classification, the groups are identified by the agglutinogen content of the red cells. The cells of the first or "0" group are not agglutinated by the sera of any group and hence may be regarded as not containing an agglutinable sub- stance or agglutinogen. One can imagine that the letter ''0" stands for the German word ohne meaning without. The red cells of the second or A group contain agglutinogen A; of the third, agglutinogen B, while cells of the fourth group contain both A and B agglutinogen and hence the group is called AB. The order of arrangement corresponds to that of the Jansky classification. A comparison of these three classifications with each otlier and with Landsteiner's original groups is summarized in Tal)]o I : Table I Comparison of Classification of Blood Grottps Jansky 1 11 III IV Moss IV II III I New Classification 0 A B AB Landsteiner's original c A B Type described by three groups DeCastello and Sturli Landsteiner's Three Groups. — In Table I it will be noted that Landsteiner used the letters A, B, and C to designate his three groups. He found that serum of group C agglutinated the red cells of groups A and B; the serum of group A agglutinated the cells of B but not those of C or A; and the serum of group B agglutinated the cells of A but not those of C or its own group B. On the other hand, the serum of the new type, or exception, 170 IMMUNOLOGY described by DeCastello and Stiirli (1902) failed to agglutinate the cells of any group while the corresponding red cells were agglutinated by the sera of each of the three Landsteiner groups. Mechanism Postulated by Landsteiner. — To explain these phe- nomena, Landsteiner (1901, 1928) says that he and later Jansky (1907) postulated the existence of two agglutinins, alpha and beta, and two corresponding agglutinogens, A and B. He fur- thermore assumed that there exists a reciprocal relationship be- tween them; i.e., when an agglutinogen is absent from the red cells of an individual the corresponding agglutinin will be pres- ent in the serum, and, conversely, when an agglutinogen is present in the red cells, the corresponding agglutinin will be absent from the serum. This explains why the red cells are not agglutinated by the homologoiLs serum. Thus, for group 0 where the red cells contain neither agglu- tinogen, the corresponding serum contains both agglutinins alpha and beta. In group A, the red cells contain agglutinogen A, and hence group A sera will contain only beta agglutinin. Likewise, group B sera will contain only alpha agglutinin and group AB neither agglutinin. This will be better appreciated from an inspection of Table II. Table II Agglutinogen content of cells Agglutinin content of corresponding group serum Jansky types Moss types 0 I IV A II II E a III III AB IV I Fig. 6 illustrates the phenomena observed from mixing the cells and sera of the four types. Moss's Classification in General Use. — Kennedy (1929) states that the classification suggested by Moss (1910) is used in 78 per cent of the hospitals of America. This is interesting in view of the fact that in 1921 a committee representing the American Association of Immunologists, the Association of Pathologists and Bacteriologists, and the Society of American Bacteriologists recom- mended the adoption of the Jansky classification. This was done ISOHEMAGGLUTININS 171 in recognition of the priority of Jansky's work. Since the majority of the hospitals insist upon using the classification of Moss it is necessary that this classification be taught to medical students. Sugg-ested Method for Learning the Classification. — Experi- ence in teaching has shown that if the student will associate the new classification "0,A,B,AB," which is based upon the agglu- tinogen content of the red cells, with some sentence such as "Oh,A,Be,A-be," keeping in mind the law of reciprocal relation- Cells (agglutiuogens) AgglU- HT Tir 1VI TT HI TTT MoSS I Moss Jansky tinin ^ ,'^°^\^\- a r^°''i ,T^ « .Y°'l T^T^ ^-B Content O (Jansky-I) A-(Jansky-II) B-(Jansky III) (Jansky IV) tn 12: ur TS. iz K/S Fig. 6. — Distribution of agglutinius and agglutinogens in human blood groups. ships between agglutinogen and agglutinin postulated by Land- steiner, he has no difficulty in remembering and understanding this classification. It is also our experience that he has no diffi- culty in remembering that the numerical groups of Jansky corre- spond to the "0,A,B, and AB" groups of the new classification and that in Moss's classification groups I and IV of Jansky's are reversed. Bj' learning the ncAV classification and the law of reciprocal relationships he has learned the agglutinogen and ag- 172 IMMUNOLOGY glutiiiin content of each group of both the new classification and that of Jansky and hence can readily determine the same for the groups of Moss. Time of Appearance of Agglutinogen and Agglutinins. — Since the discovery of the blood groups of Landsteiner, numerous in- vestigations have uncovered many interesting facts relative to the factors involved in isohemagglutination. It has been shown, for instance, that the agglutinogen content of the red cells is, as a rule, complete at birth. On the other hand, the agglutinins may or may not be present in the serum at birth, but may make their appearance at some time during the first four years of life. After the isohemagglutinating factors of a blood are complete, there is no qualitative change during the lifetime of the individ- ual. It has been shown that disease may increase or decrease the titer and that the titer may drop during the later years of life, but the group, when once determined, remains constant. A number of individuals have found agglutinins from the maternal blood present in the blood of the child at birth. These usually disappear from the blood stream by the tenth day. Various theories have been offered to explain their presence in the circulation of tlie newborn. It is thought that changes in permeability of ves- sels in the placenta may account for the phenomenon. Inheritance of Blood Group Factors. — The first evidence sug- gesting that the blood group factors might be inherited was pre- sented by Epstein and Ottenberg (1908). They typed a mother and seven sons of one family and four sons and both parents of another family. All members of the first family were found to be in group A, while all members of the second family were in group B. Two years later, von Dungern and Hirschfeld (1910) obtained data on seventy-two families covering tAvo generations and 248 individuals. From an analysis of this data, according to Ottenberg* (1928) and Snyder (1929), they concluded: "(1) A or B never occurs in the red cells of a child if not present in one of the parents. (2) When one of these substances is present in both parents it occurs in most of the children. (3) When only one parent has one of these particular substances, some of the •ottenberg and Bere;; : Newer Knowledge of Bacteriologrj- and Immunology, Jordan and Falk, Univ. of Chicago, 1928, p. 909. ISOHEM AGGLUTININS 173 children inherit it. (4) When a particular substance is absent from both parents, no child ever has it." Racial Distribution of Groups. — In 1919 L. and H. Hirschfeld published the results of their studies of the distribution of human blood groups among- sixteen nationalities including over eight thousand individuals. The data were obtained during the World War while they were serving as army physicians on the Balkan Front. They observed a significant difference in frequency of occurrence of the agglutinogens among different races. Since then, many additional studies have been made. Racial Types. — The data obtained by the Hirschfelds indicate that the nationalities may be grouped into three types (see Snyder, 1929, p. IIS) the European, Intermediate and Asio-AI'rican types. In the European type, which is made up of the English, French, Italians, Oermans, Austrians, Serbians, filreeks and Bul- garians, the frecpiency of the A factor is quite constant, varying from 41.8 in the Italians to 48 in the Germans. On the other hand, the B factor shows a progressive increase from 10.2 for the English to 20.4 for the Bulgarians. In the Intermediate group, composed of Arabs, Turks, Russians and Jews, the fre- quency of the A factor still exceeds that for B, but they are noticeably closer together. The Asio-African type is composed of the Madagascans, Senegalese, Annamese and natives of India. In this group the frequency of the B factor is equal to or defi- nitely greater than that for the A factor. In these determina- tions, the data for the A factor include A and AB, while those for the B factor include both B and AB. Ottenberg (1928) has also given an excellent summary jof the frequency of blood groups in different populations.* Frequency in Different Populations. — Apparently the fre- quency of the 0 group varies between wide limits. The Chinese show a frequency of 30 ; the Russians, 40.2 ; Germans, 40 ; French, 43.2; English, 46.4; Australian aborigines, 51; and the American Indians, 77.7 to 91.3 according to different investigators. High Incidence of 0 Group in the American Indian. — The American Indians have been studied by Coca and Deibert (1923), Snyder (1926) and Nigg (1926). It is interesting to note that all three studies have shown a high incidence for group 0 and a *It is interesting- to note reported variation in the incidence of the N factor. Vibeke Fabricius-Hansen finds it lower in the Greenland Eskimos and American Indians than in other peoples, J. Immunol. 38: 40.5. 1940. 174 tMMtJNOLOGtY very low incidence for B and AB. The frequency of B varied from 0.22 to 2.1 while AB varied from 0.0 to 0.22. Nigg (1926) has also published data from this laboratory covering her investiga- tions on the incidence of the various groups in 413 full-blooded Hawaiians. She found 36.5 per cent were of group 0, 60.8 per cent in group A, 2.2 per cent in group B and 0.5 per cent in group AB. Nigg (1929), as well as Coca and Deibert (1923), concludes that the highly civilized races are characterized by the presence of isoagglutinogens, whereas the primitive peoples in all parts of the world are characterized by the low incidence of iso- agglutinogens. This conclusion would seem to be borne out by the work of Goodner (1930) who found that of 223 pure Maya Indians studied, 97.7 per cent belonged to group 0. Rife (1932) likewise studied the incidence of blood groups among the Indians in certain Maya areas of Central America. He found that 122 out of 124 belonged to group 0. Recently Matson and Schrader (1933) reported that the Blackfeet and Blood tribes of American Indians show a high percentage of group A and a relatively low percentage of group 0. They found 76.5 per cent of 115 allegedly full-blooded Blackfeet and 20 out of 24 full bloods of the Blood tribe belonged to group A. Perhaps later work will explain these exceptions. Distribution of Ag-glutinogen in Lower Animals.— The distri- bution, among the lower animals, of substances either similar to or identical with the agglutinogens found in human red cells has been studied extensively by Landsteiner (1902), von Dungern and Table III Agglutination Tests on Erythrocytes of Other Animals S H M H . < < S OS cii oi coeuooMa^OQ Q xE Agglutinin solu- + +± +± +± + 000 + 000 + +±0 + tion from hu- man group II serum Agglutinin solu- 0 Tr? + Tr. 0 0 0 0 0 0 0 0 0 Tr? + 0 tion from hu- man group III serum 0 Negative. Tr. Trace of agglutination. Degree of agglutination indicated by ±, +, +±, etc. Prom Landsteiner and Miller: J. Exper. Med. 42: 869, 192.'5. ISOHEMAGGLUTININS 175 Hirschi'eld (1910-11), Hooker and Anderson (1921), Landsteiner and Miller (1925) and also Landsteiner and Levine (1929, A and B) . Table III taken from the paper by Landsteiner and Miller (1925) shows an interesting distribution of agglutinogens in the blood of the lower animals. While many of these are similar to certain agglutinogens in human blood they are not identical. For further information concerning the occurrence of receptors similar but not identical witli liuman substances A and B in the Fig. 7. — Graph showing distribution of agglutinogens in apes resembling those in man. Suggested by illustration in paper by Landsteiner, K., and Miller, C. P., Jr.. J. Exp. Med. 42: 871, 102.5. blood of the lower animals the student should consult the reviinvs of Wiener, Landsteiner and Snyder.* Bond! (1939) has shown that snake blood contains agglutinins for the A and B factors of human cells but that no A and B factors were present in the snake cells. Agglutinogen in Monkeys and Higher Apes. — In their studies on the bloods of the lower monkeys and also of the anthropoid apes, Landsteiner and Miller found an agglutinogen .similar but not identical to the human B factor, in the red cells of Lemurs. Platyrhinae, and Cercopithecidae. The red cells of the Gibbons ♦Ferguson identified nine antigens in tlie erythrocytes of cattle, J. Immunol. 40: 213, 1941. tBond has also found similar agglutinins and also a species specific one in turtles but not in alligator."?, J. Immunol. 39: 125, 133, 1940, 176 IMMUNOLOGY contained A, the Orangs, both A and B and the Chimpanzees, the A agglutinogen. The serum of the latter also contained agglu- tinins corresponding to those of the human 0 group. The biological relationships of the monkeys and apes, corre- lated with the various agglutinogens, are quite well illustrated in Fig. 7 suggested by the illustration used by Landsteiner and Miller (1925). Comparison of Agglutinogen in Human and Monkey Blood. — In regard to the agglutinogen found in the red cells of Lemurs and Platyrhinae that is similar to but not identical with the B factor of human cells, Landsteiner and Miller state that the dif- ference is detected when these monkey blood cells are used to absorb the beta agglutinins from type II human serum. They are unable to remove all of the agglutinin although unabsorbed type II serum agglutinates the cells and type III serum does not, or, at most, gives only slight agglutination. On the other hand, Landsteiner and Miller* (1925) have shown that the bloods of anthropoid apes "contain groups apparently identical with those of human blood." They were able to assign each blood to one of the four human groups. In regard to the bearing of these results upon the question of the origin of the blood groups in Man, they make the following statement: "If our findings in the anthropoids are taken into consideration, the simplest assumption seems to be Ihat tlie isoagglutinablc factors existed before Man and the anthropoids were differentiated from their common ancestor. If this assumption is not made, one is forced to the conclusion that identical mutations occurred in the evolutionary lines which developed into the Gibbon, Orang, Chimpanzee and Man at some later time." Human A and B Factors in Antklropoid Apes. — They also call attention to the interesting distribution of the agglutinogens in the anthropoid apes. Apparently, the Chimpanzee has only the A factor and its blood grouping A and 0 is comparable to that of the American Indian, while the Orangs contain both the A and B factors similar to those of human races other than the Indians. Medico-Legal Application of Blood Groups. — A great deal of attention has been given to the mechanism of inheritance of the agglutinogens A and B. Von Dungern and Hirschfeld regarded •Landsteiner and Miller; J. Exper. Med. 42: 841, 1925. ISOHEMAGGLUTININS 177 the two agglutinogens and their corresponding agglutinins as similar to independent factors described by Mendel in his experi- ments with peas. They considered that the agglutinogen factors A and B are dominant to the corresponding recessives a and b. They represented group 0 genetically, which is devoid of ag- glutinogens, by the double recessive aabb (see Ottenberg, 1928, 912). Table IV, taken from a paper by Ottenberg (1928), shows the genetic formulas of the four groups as suggested by von 13ungern and Hirschf eld : Table IV Genetic Formulas of the Four Groups* B aabb AAbb aaBB AABB Aabb aaBb AaBB AABb AaBb Table IV shows all of the possibilities according to the two- factor hypothesis of von Dungern and Hirschfeld. For a more comprehensive discussion of the genetic factors involved, the student is referred to an excellent book by Snyder (1929). Ottenberg (1928) has also included Table V showing the groups of the children resulting from all the possible crosses under this two-factor theory. Table V* PARENTS CHILDREN CHILDREN POSSIBLE NOT POSSIBLE O X 0 0 A, B, AB O X A 0,A B,AB 0 X B 0,B A,AB A X A 0, A B, AB A X B 0, A, B, AB A X AB 0, A, B, AB B X B O, B A, AB B X AB 0, A, B, AB AB X AB 0, A, B, AB 0 X AB (), A, B, AB Determination of Nonpaternity. — Ottenberg* calls attention to the possibility of "ruling out the reputed father as definitely not the father of the child being examined" by a reference to the •From "The Heredity of the Blood Groups" by Ottenberg, R., and Beres, D., in The Newer Knowledge of Bacterioloffy and Immunology, edited by E. O. Jordan and I. S. Falk. Reprinted by permission of the University of Chicago Press. 178 IMMUNOLOGY above table after the groups of the parents and child are deter- mined. It will also be noted that there are limitations to one's ability to determine paternity by means of isoagglutination tests. For a more comprehensive discussion of this subject the student is referred to the studies of Ottenberg (1928), Snyder (1929), Hooker and Boyd (1929), Wiener, Lederer and Polayes (1930), and Wiener (1933). Bernstein's Triple Allelomorph Theory. — For all practical pur- poses the assumption of Landsteiner that there are two agglu- tinogens and two agglutinins for which reciprocal relationships exist is quite satisfactory. However, the genetic formulas de- veloped from such an assumption do not give data that explain the racial distribution of blood groups; Bernstein (1925) de- veloped an hjT)othesis which enables him to write formulas that give data comparable to those observed. His theory is known as the three factor or triple allelomorph hypothesis. In this he assumes that the red cells of the 0 group contain a recessive ag- glutinogen designated by Snyder (1929) as 0 and that there exists a corresponding agglutinin o. He furthermore assumes that the 0 agglutinogen is present in some bloods of group A and also of group B but never in group AB, He postulates that all three agglutinins are present in all sera, but that there exists a recipro- cal binding between an agglutinin and its corresponding ag- glutinogen that prevents autoagglutination. Thus he assumes the existence of six genetic types. Snyder* has illustrated these assumptions in the following diagram. Group O A B AB Agglutinogen 66 Ao AA Bo BB AB Agglutinins ab(6) (a)b(6) (a)b6 a(b)(o) a(b)o (a)(b)6 In this diagram it will be observed that the agglutinins in- volved in a reciprocal binding with the corresponding agglutino- gens in the red cells are enclosed in parentheses. The reciprocal binding prevents autoagglutination. Conclusions concerning non- paternity are not in confiiet in the two theories except where AB parents are involved. Reference to Table V shows that accord- *Snyder: Blood Grouping- in Relation to Clinical and Legal Medicine, Wil- liams and Wilkins Co. Published by permission of Snyder and Williams and Wilkins Co, ISOHEMAGGLUTININS 179 ing to the von Dungern and Hirschfeld theory children of all types are possible in any matings which involve an AB parent. According to the Bernstein theory the following matings never result in 0 children : B x AB, AB x AB, A x AB. In 0 x AB matings A and B children may result but not 0 or AB children. While there is a general acceptance of Bernstein's hypothesis, Snyder says that it is not yet proved beyond all doubt. The student is referred to the studies of Ottenberg (1928) and "Wiener (1930) for a more comprehensive discussion of these two as well as other theories under consideration at the present time. Subgroups. — In 1911, von Dungern and Hirschfeld apparently demonstrated by means of agglutinin absorption experiments that individuals having the A factor fall into two subgroups, those that are purely A and those in which the A agglutinogen is linked with a second agglutinogen A'. Likewise, they apparently showed that there are two kinds of group B sera, those contain- ing only the a agglutinins and the other containing the a agglu- tinin always associated with an a' agglutinin. Others* (see Landsteiner, 1928) have shown that many group 0 sera contain a' as well as a and fB agglutinins. Importance of Quantitative Difference. — The existence of sub- groups for A has been confirmed by Schiitze (1921), Coca and Klein (1923), Gutherie and Huck (1923) and Landsteiner and Witt (see Landsteiner, 1928). Lattes and Cavazutti (1924) are of the opinion that these results are all due to quantitative dif- ferences in the agglutinability of corpuscles rather than the ex- istence of a third pair of agglutinins and agglutinogens. After carefully investigating the question raised by Lattes and Cava- zutti, Landsteiner (1928) concludes that the evidence at hand warrants the assumption of a'-A' factors. He states, however, that quantitative variations, as suggested by Lattes and Cava- zutti, may account for many blood differences reported in the literature. Table VI, showing the incidence of the third pair of agglutinating factors, is taken for the most part from Simson's (1926) detailed study with some rearrangement of data and an adoption of a uniform nomenclature. These subgroups are also designated as A^ corresponding to A' and Ao here called A, the corresponding agglutinins being a^ and a.- *See Supplementary References, p. 192, for papers by Wiener (1941). 180 IMMUNOLOGY « u < E-i 'A dS M P Tj M 1— 1 Lj t-i " isohemagglutinins 181 Presence of Subgroups Does Not Interfere with Routine Typing. — Since the a' agglutinin is almost always found associated with the a agglutinin in sera and the A' agglutinogen likcAvise associated with the A agglutinogen, it is evident that their exist- ence does not invalidate any conclusions as to blood grouping based upon the postulates of Landsteiner. In other words, their presence cannot be detected by the routine method of typing, but is detected only hy means of absorption experiments. Irregular Ag-g-lutination. — Landsteiner and Levine (1929) have made an extensive study of "isoagglutinin reactions of human blood other than those defining the blood groups." They cite the observation of Thomson (1928) that in five out of 3,500 bloods ex- amined he definitely proved an absence of isoagglutinins from sera of bloods belonging to groups 0, A, and B. In this series he observed 32 abnormal sera. Outherie and Huek (1924-25) found one serum of group B which contained only an agglutinin for one subgroup of A. In 1926, Landsteiner and Levine noted two sera of bloods belonging to group AB that contained an a agglutinin. Importance of Temperature. — In later studies (1929) they used very sensitive tests by employing diluted blood (2.5 per cent) and temperatures of 20°, 25°, 30° and 37° C. Most of the abnor- mal reactions occurred at temperatures below 37° C. At the latter temperature the abnormal agglutination either did not occur or it disappeared when cells agglutinated at 20° C. were warmed to 37° C. Landsteiner and Levine state that "for sev- eral sera the upper limit of activity was at 30° C, for others at 25° or even 20° C." In all they examined 500 sera. They found three sera of group AB that contained a' agglutinin, two sera of group AB and one of group A that agglutinated intensely group 0 cells and to a lesser degree cells belonging to subgroup A.. They further state that "in a total of about 500 sera examined there were at least 16 that gave reactions designated as +, i.e., about 3 per cent. Of 180 sera 12 or 6.6 per cent reacted weakly and 31 sera (about 17 per cent) showed traces of agglutination within the same group." They conclude that "one can speak of abnormally reacting sera but hardly of abnormal blood cells." M, N, and P Factors of Human Blood. — In 1928, Landsteiner and Levine demonstrated, by means of immune sera, three new 182 IMMUNOLOGY agglutinable factors distributed among the four blood groups. They found at that time no normal agglutinins in human sera corresponding to these three factors which they designated as M, N, and P agglutinogens. Subsequent studies have shown the M factor to be strictly defined whereas the property P is not. In regard to the latter, they make the following statement, "thus the property P is not strictly defined like A, B, or M, but desig- nates a group of related agglutinable factors." In regard to the N factor they say (July, 1929) that it, like P, shows fluctuation but not to the extent of the latter. Landsteiner and Levine (February, 1929) studied the distribution of these factors among the four blood groups of both white and colored individuals. They found the M agglutinogen present in 80.9 per cent of white and 71.9 per cent of colored individuals examined. The distribution among the four blood groups is quite uniform. The N factor is likewise uniformly distributed among the groups and shows the same incidence in botli colored and white individuals. Land- steiner and Levine found it present in 73.9 per cent of the white and 72.4 per cent of the colored individuals examined by them. In regard to the P factor they state, "The strongest reactions were almost four times as frequent in the colored as in the white individuals, whereas the weak reactions were much rarer in the former. Blood with negative reactions occurred only exception- ally among the colored." Landsteiner and Levine (1928) have shown that M and N fac- tors are inherited as Mendelian dominants, probably dependent upon a single pair of allelomorphic genes M and N. The resultant possible types are M, N, and MN. These factors occur as ag- glutinogens demonstrable only by immune serum. These immune sera are usually prepared by the injection of known J\I and N blood cells into rabbits. Recent work (Stuart, et al., 1936, 1937, 1939) has shown that rabbits, in order to produce potent antibodies, must not contain M factors in their tissues. Studies on the dis- tribution of these factors show that M and N occur regularly in human cells but are independent of the A and B factors. They may be used in medico-legal work in tlie same way as the A and B factors. However, by the determination of M and N as well as A and B the average chances of proving nonpaternity are doubled. ISOHEM AGGLUTININS 183 Extra Agglutinin 1. — hi July, 1929, Laiidsteiner and Levine called attention to an extra agglutinin which they found in two sera of group 0, three of group A, and one of group B. This new agglutinin "gave reactions with bloods of various grou])s. " In February, 1930, they report tlie results of further studies upon this new agglutinin and designate it as "extra agglutinin 1." They state that there is some apparent relationship between the agglutinin and the P factor. In July, 1930, Nigg reported her studies of two unusual type B bloods. Both contained an agglutinin demonstrable at 25° C. for many type 0 as well as type B cells. Agglutination did not occur, however, at temperatures above 30° C. She concluded that these agglutinins were identical with the "extra agglutinin 1" of Landsteiner and Levine. Since then, Landsteiner and Levine (1931) have carried out extensive studies on "The Dif- ferentiation of a Type of Human Blood by Means of Normal Animal Serum." They conclude that this "extra agglutinin 1" is specific for the P factor. Thus it is evident that occasionally^ the serum of groups 0, A, or B individuals may contain an extra agglutinin for an agglutinogen P that has been previously dis- cussed. When a serum of this kind is encountered and compati- bility tests are done at room temperature, some confusion may result unless the phenomenon is understood. Irregular Isoagglutination at Temperatures Below 37° C. — If one carries out agglutination tests using the sera and cells of different individuals and varies the temperature between 37° and 0° C, he will observe many individual variations not distinguish- able at body temperature. The number of these interesting vari- ations increases as the temperature is lowered. Many of the re- actions are due to the presence of a^ and a^ agglutinins and their corresponding agglutinogens but others are not tlioroughly un- derstood. AuTOAGGLUTiNiNS. — Landstcincr and Levine (1926) and Land- steiner (1928) call attention to the frequent occurrence of auto- agglutination at temperatures between 0° C. and 5° C. The autoagglutinins can l)e absorbed by the homologous red cells at 0° C. and after washing with saline at the same temi3erature, tlie agglutinins can be recovered by warming the suspension to 37° C. or even less. Marked autoagglutination has been observed in 184 IMMUNOLOGY a number of pathological conditions such as hypertrophic cirrho- sis of the liver, hemolytic jaundice, paroxysmal hemoglobinuria, Raynaud's disease, and trypanosomiasis in man and animals. Cold Agglutinins. — The term ' ' cold agglutinins ' ' is used by Li Chen-Pien (1926) and others to indicate autoagglutinins, but Landsteiner (1928) considers any hemagglutinins that bring about real agglutination only at low temperatures of either the individual's own corpuscles or those of others of the same species, "cold agglutinins." On the other hand, Snyder differentiates ])etween autoagglutinins and cold agglutinins for he says, "When the individual's red cells are agglutinated under such conditions (0°-5° C.) by his own serum, the reaction is spoken of as 'auto- agglutination.' When the agglutination occurs due to serum of another individual, the reaction is known as 'cold agglutination.' These two reactions are certainly related and may be identical." Landsteiner 's views seem to be in harmony with the term "cold agglutinins" and are hence recommended to the student as per- haps the most satisfactory. PsEUDOAGGLUTiNATiON. — The phenomenon of pseudoagglutina- tion occurs at low temperatures and may be intensified at 37° C. It is due to rouleaux formation in which the red cells adhere to- gether like piles of coins. The mechanism of rouleaux formation does not involve agglutinins and hence is an entirely different phenomenon from agglutination. Snyder (1929) says that the tendency for rouleaux formation is frequently increased in "rheu- matic fever, tuberculosis, pneumonia, cardiac diseases and in cer- tain physiologic conditions such as menstruation and pregnancy" or any condition showing an increase in the rate of sedimentation of the red cells. Inhibition of Rouleaux Formation, — Snyder cites the work of Lattes (1924) and Falgairolle (1926) who found that rouleaux formation is inhibited by adding lecithin and other substances to the serum. Snyder (1929) states that one part of kaolin to three parts of serum will also inhibit rouleaux fonnation. He also suggests the use of formalin and hypotonic salt solution. Shat- tock (1900) noted that diluting the serum with saline prevented pseudoagglutination. Rouleaux formation and true agglutina- tion of red cells are illustrated in Figs. 8 and 9 respectively. ISOHEMAGGLUTININS 185 Nature and Distribution of Red Cell Haptens. — In his study of cellular antigens Landsteinei- (1936) discusses the nature of the haptens which determine blood groups. They are alcohol-soluble substances, probably lipoidal in nature, one for the A and a sec- ond for the B factor. In his opinion they are somewhat analogous in nature to Forssman's antigen. Schiff and Adelsberger (1924) conclude that the A factor in human cells is heterophile in nature Fig. S. — Pseudoagglatination or rouleaux formation of red cells. (Microscopic.) Figr. 9. — True agglutination of red cells. (Microscopic.) since they found group reactions for sheep cells and those of group A (human). It is also of interest to note that the normal antisheep hemolysins frequently found in human blood are re- garded as heterophile antibodies. Landsteiner (1928) suggests that perhaps the antigens of the red cell may have a mosaic structure, since this concept apparently harmonizes with observed phenomena, such as the ease with which antibodies can be frac- tionated by absorption. Recent work by Landsteiner (1936) and 186 IMMUNOLOGY Schiff and Adelsberger (1924) has suggested that the group specific A hapten is a lipoid-carbohydrate complex owing its specific reactions to carbohydrate groups. Friedenreich (1939) has demonstrated that group specific substances identical or similar to the group A substance in human red cells are widely distributed in human tissues and secretions. In addition, similar group A substances are found in nature from such diverse sources as horse and bovine saliva and certain pneu- mococci. Summary. — It is hoped that the following partial summary of material in this chapter may be of help to the student. In 1901, Landsteiner discovered three of the four recognized blood groups within the human species and postulated two ag- glutinogens A and B and two agglutinins a and ^. He also sug- gested the law of reciprocal relationships according to which, when an agglutinogen is present in the red cells the correspond- ing agglutinin is absent from the serum, and conversely, when the agglutinin is present in the serum, the corresponding ag- glutinogen is absent from the red cells of that particular blood. The following year DeCastello and Sturli described four ex- amples of the fourth group. They considered a classification identical with that of Moss (1909-10), but did not feel warranted in recommending its adoption. Since the four examples of what they termed group 1 (AB) were all infants, they thought perhaps the latter had not developed a stable group. It is interesting to note that DeCastello and Sturli conceived that some type of what has been more recently called reciprocal binding may exist be- tween an agglutinogen and agglutinin which would prevent auto- agglutination. The classifications suggested by Jansky (1907) and Moss are given and compared with a new classification being used quite generally in the literature and based upon the ag- glutinogen content of the cells. The agglutinogens are present in the red cells at birth. The agglutinins may be present then, or make their appearance at some time during the first four years of life. When once established, the group remains stable except, perhaps, for quantitative variations in titer of agglutinins. The racial distribution of the blood groups is discussed briefly in connection with the two-factor and the triple allelomorph theory. ISOHEMAGGLUTININS 187 The latter, proposed by Bernstein, seems to fit the data at hand. For practical purposes in blood typing the assumption made by Landsteiner that there are two agglutinogens and two corre- sponding agglutinins is quite satisfactory. Evidence is presented indicating that the blood groups are inherited and that in some, but not all, cases of disputed paternity nonpaternity may be es- tablished for certain individuals. Attention is also called to tlie existence of subgroups and to the fact that their existence does not interfere with ordinary blood typing. The new agglutinable factors, M, N and P were discovered by Landsteiner et al. Normal agglutinins for M and perhaps for N have not been observed, but a few individuals, representing practically all the groups, have been found whose blood contains an agglutinin for P. This is the same as "extra agglutinin 1" of Landsteiner. Fortunately these reactions dis- appear, as a rule, at temperatures a])o\e 30° C. "Where typing is done at room temperature they may be very rarely encountered. The agglutinable factors present in the blood of lower animals and of the anthropoid apes are also mentioned in connection with their biological significance. Various irregular agglutinations are discussed and a definition of "cold agglutinins" is given. The antigenic factors of the red cells are discussed. Land- steiner's views concerning these are developed. Cellular antigens are complex. Two types of specificity exist, one for the species and a second indicating specific differences within a species. The species specific antigens of the red cell are represented by isopliile hemolysins, a nonspeeies specific antigen by the heter- ophile substances. Differences within a species are represented by the agglutinable factors A and B for human blood. The antigenic structure can be thought of as resembling a mosaic. Chemically speaking, the red cell antigens are made up of one or more protein structures and a number of peculiar lipoid-carbohy- drated complexes or haptens. The various haptens such as those for the A factor or the B factor confer definite specificity upon an antigen. The A substances seem to be widely distributed in the tissues of man and animals and even in bacteria. 188 IMMUNOLOGY References Bernstein, F. : Cited in Blood Grouping in Relation to Clinical and Legal Medicine, by Snyder, L. H., Baltimore, 1929, Williams & Wilkins Co. Bond, Glenn C: Serological Studies of the Reptilia. I. Hemagglutinins and Hemagglutinogens o:^ Snake Blood, J. of Immunol. 36: 1, 1939. Bull, C. G.: Heterophile Antigens and Antibodies. Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 733. Coca, A. F.: The Examination of Blood Preliminary to the Operation of Blood Transfusion, J. Immunol. 3: 93, 1918. Coca, A, F., and Klein, H.: A Hitherto Undescribed Pair of Isoagglutina- tion Elements in Human Beings, J. Immunol. 8: 477, 1923. Coca, A. F., and Deil)ert, O.: Studies of the Occurrence of the Blood Groups Among the American Indians, J. Immunol. 8: 487, 1923. DftCastello, A. V., and Sturli, A.: Ilelier die Isoagglutinine im Serum ge- sunder uiul kranker Menschen, Miinclien. med. Wchnschr. 49: 1090, 1902. Doerr, E., and Pick, R. : Ueber den Mechanismus der primaren Toxizitat der Antisera und die I]igenschaften ihrer Antigene, Biochem. Ztschr. 1: 129, 1913. Die primare Toxizitat der Antisera, 2 ^Vfitteilung, Ztschr. f. Immunitats. 19: 2.'51, 1913. von Dungern, E., and Hirschfeld, L.: Ueber nachwies und vererbung bio- chemischer Strukturen, Ztschr. f. Immunitats. 41: 531, 1909. Editorial: Transfusion of Preserved Blood, J. A. M. A. 113: 2061, ]939. Epstein and Ottenberg. See Ottenberg (1928). Falgairolle, P.: (1926.) Cited in Blood Grouping in Relation to Clinical and Legal Medicine, Baltimore, 1929, Williams & Wilkins Co. Forssman, J., and Fex, J.: Ueber heterologe Antisera, Biochem. Ztschr. 61: 6, 1914. Forssman, J., and Hintze, A.: Die heterologe Toxizitat der Antisera, Biochem. Ztschr. 44: 336, 1912, Friedenreich, V., Thyssen, G., and Hartmann, G.: On Serological Dif- ferences of the Group-Character A in Different Parts of the Human Organism, J. Immunol. 37: 435, 1939. Gay, F. P., and Southard, E. E.: On Serum Anaphylaxis in the Guinea Pig, J. Med. Res. 16: 143, 1907. Goodner, K. : Incidence of the Blood Groups Among the Maya Indians of Yucatan, J. Immunol. 18: 433, 1930. Gutherie, C. G., and Huck, J. G.: On the Existence of More than Four Isoagglutination Groups in Human Blood, Bull. Johns Hopkins Hosp. 34: 37, 80, 128, 1923. Hektoen, L. : Isoagglutination of Human Corpuscles, J. Infect. Dis. 4: 297, 1907. Hooker, S. B.: Heterophile Antigen- Antibody Reactions in Relation to the Serum Diagnosis of Syphilis bv Precipitation, J. Immunol. 11: 403, 1926. Hooker, S. B., and Anderson, L. M.: The Specific Antigenic Properties of the Four Groups of Human Erythrocj'tes, J. Immunol. 6: 419, 1921. Hooker, S. B., and Boyd, W. C: The Chances of Establishing Nonpaternity by Determination of Blood Groups, J. Immunol. 16: 451, 1929. Huck, J. G., and Gutherie, C. G.: Further Studies on Blood Grouping. I. The Antigenic Properties of Two Types of Group II Ervthrocytes, Bull. Johns Hopkins Hosp. 35: 23, 1924. Jansky, J.: Haematologische Studie u. pyschotikii. Arch. Bohemes de Med. "clin. 8: 85, 1907. (Translated by Kennedy, J. A.: J. Immunol. 20: 117, 1931.) ISOHEMAGGLUTININS 189 Kline, B. 8., and Young, A. M. : A Microscopic Slide Precipitation Test for Syphilis, J. Lab. & Clin. Med. 12: 477, 1926-27. Kline, B. S., Ecker, E. E., and Young, A. M.: The Incidence of Two Types of Group II Human Red Blood Cells, J. Immunol. 10: 595, 1925. Landsteiner, K.r (1936) The Specificity of Serological Reactions, Spring- field, 111., Charles C Thomas, p. 161. Landsteiner, K. : Zur kenntnis der antifermentativen, lytischen und agglu- tinierend«n Wirkungen des Blutserums und der Lymphs, Centralbl. f. Bakt. 27: 357, 1900. Landsteiner, K.r Ueber agglutinserscheinungen normalen menschlichen Blutes, Wien. klin. Wchnschr. 14: 1132, 1901. Landsteiner, K., and Levine, P.: The DiflFerentiation of a T^-pe of Human Blood by Means of Normal Animal Serum, J. Immunol. 20: 179, 1931. Landsteiner, K., and Levine, P.: On Isoagglutination Reactions of Human Blood Other Than Those Defining the Blood Groups, J. Immunol. 17: 1, 1929. Landsteiner, K., and Levine, P.: On Group Specific Substances in Human Spermatozoa, J. Immunol. 12: 415, 1926. Landsteiner, K., and Levine, P.: On the Cold Agglutinins in Human Serum, J. Immunol. 12: 441, 1926. Landsteiner, K., and Levine, P.: On the Forssman Antigens in B. Para- typhosus B and B. Dysenteriae Shiga, J. Immunol. 22: 75, 1932. Landsteiner, K., and Levine, P.: Observations on the Specific Part of the Heterogenetic Antigen, J. Immunol. 10: 731, 1925, Landsteiner, K., and Levine, P.: On the Inheritance of Agglutinogens of Human Blood Demonstrable by Immune Agglutinins, J. Exper. Med. 48: 731, 1928. Landsteiner, K., Levine, P., and Janes, M. L. : On the Developments of Isoagglutinations Following Transfusion, Proc. Soc. Exper. Biol. & Med. 25: 672, 1928. Landsteiner, K., and Miller, C. P., ,Tr.: Serological Studies on the Blood of the Primates. I. The Differentiation of Human and Anthropoid Blood, .L Exper. Med. 42: 841, 192.3. Landsteiner, K., and Miller, C. P., Jr.: Serological Studies on the Blood of the Primates. II. The Blood Groups in Anthropoid Apes, J. Exper, Med. 42: 8.53, 1925. Landsteiner, K., and Simms, S.: Production of the Heterogenetic Anti- bodies With Mixtures of the Binding Part of the Antigen and Pro- tein, J. Exper. Med. 38: 127, 1923. Landsteiner, K.: "The Human Blood Groups," Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, Univ. of Chicago Press, pp. 892-908, Lattes, L., and Cavazzuti, A.: Sur 1 'existence d'un troisieme element iso- agglutination, ,L Immunol. 9: 407, 1924. Lattes, L. : Cited in Blood Grouping in Relation to Clinical and Legal Medicine, Baltimore, 1929, Williams & Wilkins Co, Li Chen-Pien: Investigation in "Cold" or Autoagglutination, J, Immunol. 11: 297, 1926. Matson, G. A., and Schrader, H. F.: Blood Grouping Among the "Black- feet" and "Blood" Tribes of American Indians, J. Immunol. 25: 155, 1933. Moss, W. L.: Studies on Isoagglutination and Isohemolvsius, Bull, Johns Hopkins Hosp. 21: 63, 1910. Xigg, C: Studies on Agglutinogens of Human Bloods, .1. Immunol. 19: 1, 1930. Nigg, C: Study of the Blood Groups Among American Indians, Master's Thesis, Univ. of Kansas, 1926, 190 IMMUNOLOGY Nigg, C: A Study of the Blood Groups Among the American Indians, J. Immunol. 11: 319, 1926. Nigg, C: Studies of Isohemagglutination. Thesis for the Doctorate, Univ. of Kansas, 1929. Ottenberg, E.: Studies in Isoagglutination. I. Transfusion and the Ques- tion of Intravascular Agglutination, J. Exper. Med. 13: 425, 1911. Ottenberg, E., and Beres, D. : The Heredity of the Blood Groups. Newer Knowledge of Bacteriology and Immunology, 1928, Univ. of Chicago Press, p. 909. Pick, E., and Doerr, E.: Ueber ein« ueue Autigenfunktien der kristallinse des Auges, Centralbl. f. Bakt. I. (Orig.) 70: 435, 1913. Eife, D. W. : Blood Groups of Indians in Certain Maya Areas of Central America, J. Immunol. 22: 207, 1932. Schiff, F. von, and Adelsberger, L.r Ueber blutgruppenspezifische Antikorper und Antigene. I. Mitteilung, Ztschr. f. Immunitat. u. Exper. Ther. 40: 335, 1924. Schiitze, H.: Haemagglutination and Its Medico-Legal Bearing, With Ob- servations Upon the Theory of Iso-agglutinins, Brit. J. Exper. Path. 2: 26, 1921. Shattock, S. G. : Agglutination of Eed Cells in Pneumonia and Nature of Buffy Coat, J. Path. & Bact. 6: 303, 1900. Simson, F. W.: A Studv of the Third Agglutinating Sj'stem in Human Blood, J. Path. & Bact. 29: 279, 1926. Snyder, L. N.: (1926.) Eef erred to by Snyder, 1929. Snyder, L. N.: Blood Grouping in Eelation to Clinical and Legal Medicine, Baltimore, 1929, Williams & Wilkins Co. Stuart, C. A., Sawin, P. B., Griffin, A. M., and Wheeler, K. M.: Group- Specific Agglutinins in Babbit Serums for Human Cells, J. Im- munol. 31: 31, 1936; Ibid. 33: 393, 1937; Ibid. 37: 159, 1939. Wiener, A. S., Lederer, M., and Polayes, S. H.: Studies in Isohemagglutina- tion. IV. On the Chances of Proving Non-paternity, With Specific Eeference to Blood Groups, J. Immunol. 19: 259, 1930. Wiener, A. S. : On the Usefulness of Blood-Grouping in Medicolegal Cases Involving Blood Eelationships, J. Immunol. 24: 443, 1933. Zinsser, H., and Coca, A. F. : Eemarks Concerning Laudsteiner's Discovery of Isoagglutination and the Blood Groups, With Specific Eeference to a Paper by J. A. Kennedy, J. Immunol. 20: 259, 1931. Supplementary References Boyd, W. C, and Derew, M. A.: Proof of the Presence of Agglutinogen "A" in All the Erythrocvtes of Type "AB," J. Immunol. 24: 549, 1933. Buchanan, J. A.: A Consideration of the Various Laws of Heredity and Their Application to Conditions in Man, Am. J. M. Sc. 165: 675, 1923. Coca, A. F. : Note Concerning Differences Between the Clumping of Pseudo- agglutination and that of Isoagglutination, J. Immunol. 20: 263, 1931. Crile, George: Direct Transfusion of Blood in the Treatment of Hemor- rhage, J. A. M. A. 472: 1482, 1906. Gorter, E., and Grendel, F. : On Biomolecular Layers of Lipoids in the Chrenocytes of the Blood, J. Exper. Med. 41: 439, 1925. Halban, von. Dr. Joseph: Agglutinationsversuche mit mutterlichen kind- lichen Blut, Wien. klin. Wchnsehr. 13: 545, 1900. Ichida, K.: Mathematics-Statistical Considerations of the Inheritance of the Human Blood Groups, J. Immunol. 16: 81, 1929. Jenkins, E. L.:- Eandom Mating and Blood Groups, J. Immunol. 21: 279, 1931. ISOHEMAGGLUTININS 191 Jones, A. E., and Glynn Ernest: The Foui' Human Blood Groups, With Special Reference to Their Agglutination Titres and to Abnormal Donors, J. Path. & Bact. 29: 203, 1926. Jungeblut, C. W., and Smith, L. W.: Blood Grouping in Poliomyelitis, Its Eelation to Susceptibility and the Neutralizing Property of Con- valesc-ent Sera, J. Immunol. 23: 35, 1932. Kennedy, J. A.: Isohemagglutination: The Work of Jan Jansky With a Critical Analysis, J. Immunol. 20: 117, 1931. Kozelka, A. W.: Individuality of the Red Cells of Inbred Strains of Fowls, J. Immunol. 24: 519, 1933. Landsteiner, K. : Cell Antigens and Individual Specificity, J. Immunol. 15: 589, 1928. Landsteiner, K., and Levine, P.: On Individual Differences in Human Blood, J. Exper. Med. 47: 757, 1928. Landsteiner, K., and Levine, P.: On the Racial Distribution of Some Agglutinable Structures of Human Blood, J. Immunol. 16: 123, 1929. Landsteiner, K., and Levine, P.: On the Inheritance of Agglutinable Properties of Human Blood, J. Immunol. 18: 87, 1930. Landsteiner, K., and Levine, P.: Immunization of Chimpanzees With Hu- man Blood, J. Immunol. 22: 397, 1932. Landsteiner, K., and Miller, C. P.: Serological Studies on the Blood of Primates. II. The Blood Groups of the Anthropoid Apes, J. Exper. Med. 42: 853, 1925. Landsteiner, K., and Van der Scheer, J.: On the Antigens of Red Blood Corpuscles. The Question of Lipoid Antigens, J. Exper. Med. 41: 427, 1925. Landsteiner, K., and Van der Scheer, J.: On the Antigens of Red Blood Corpuscles. II. Flocculating Reactions With Alcoholic Extracts of Erythrocytes, J. Exper. Med. 42: 123, 1925. Landsteiner, K., and Witt, D. H.: Observations on the Human Blood Groups. Irregular Reactions. Isoagglutinins in Sera of Group IV. The Factor A, J. Immunol. 11: 221, 1926. Moss, W. L., and Kennedy, J. A.: Blood Groups in Peru, Santa Domingo, Yucatan and Among the Mexicans at the Blue Ridge Prison Farm in Texas, J. Immunol. 16: 159, 1929. Nigg, C: A Studv of the Blood Groups Distribution Among Polynesians, J. Immunol. 19: 93, 1930. Parr, L. W.: Studies in Isohemagglutination, J. Immunol. 16: 99, 1929. Ottenberg, R., and Johnson, A.: A Hitherto Undescribed Anomaly in Blood Groups, J. Immunol. 12: 34, 1926. Strandskov, H. H.: A Statistical Study of the Relative Goodness of Fit of the Two Proposed Theories of Human Blood Group Inheritance, J. Immunol. 21: 261, 1931. Thomsen, O.: Ztschr. f. Immunitat. u. exper. Therap. 57: 30, 1928. Cited by Landsteiner and Levine (1929). Todd, C, and White, R. G.: On the Hemolytic Immune Isolysins of the Ox and Their Relatix)n to the Question of Individuality and Blood Rela- tionship, J. Hyg. 10: 185, 1910. Unger, L. J.: The Therapeutic Aspect of Blood Transfusion, J. A. M. A. 73: 813, 1919. Precautions Necessary in the Selection of a Donor for Blood Transfusion, Ibid. 76: 9, 1921. Wheeler, K. M., Sawin, P. B., and Stuart, C. A.: Group- Specific Agglutinins in Rabbit Serums for Human Cells. V. Inlieritance of the A Character, J. Immunol. 36: 349, 1939. Wiener, A. S., Rothberg, S., and Fex, X. A.: Heredity of the Agglutinogen.s, M and N of Landsteiner and Levine. III. Medico-legal Application for the Determination of Non-paternity, J. Immunol. 23: 63, 1932. 192 IMMUNOLOGY Wiener, A.: Heredity of the Agglutinogens M and N of Landsteiner and Levine. II. Theoretics. Statistical Considerations, J. Immunol. 21: 157, 1931. Wiener, A. S., Lederer, M., and Polayes, S. H.: Studies in Isohemagglutina- tion. III. On the Heredity of the Landsteiner Blood Groups, J. Im- munol. 18: 201, 1930. Wiener, A. S., Lederer, Max, and Polayes, S. H.: Studies in Isohemag- glutination. I. Theoretical Consideration, J. Immunol. 16: 469, 1929. Wiener, A. S. : Subdivisions of Group A and Group AB. II. Isoimmuniza- tion of A, Individuals Against A^ Blood; With Special Reference to the Role of the Subgroups in Transfusion Reactions, J. Immunol. 41: 181, 1941. Wiener, A. S., and Kosofsky, I.: Quantitative Studies on the Group-Specific Substances in Human Blood and Saliva. I. Group-Specific Substance B, J. Immunol. 41: 413, 1941. Yamakami, K.r The Individuality of Semen With Reference to Its Property of Inhibiting Specifically Isohemagglutination, J. Immunol. 12: 185, 1926. Zinsser, H. : Isoantibodies, Resistance to Infectious Diseases, New York, 1931, The Macmillan Co., p. 276. Zinsser, H., Enders, J. F., and Fothergill, L. D. : Isoantibodies and the Blood Groups. Immunity: Principles and Application in Medicine and Public Health, New York, 1939, The Macmillan Co., p. 258. CHAPTER XI NATURE, FORMATION, ACTION AND MEASUREMENT OF ANTIBODIES Introduction. — When one reviews the basic facts that have been established beyond reasonable doubt about the plasma pro- teins, he discovers that an important one is missing, since no one knows with certainty the source of these plasma proteins. The immunologist usually defibrinates the blood and works with the serum proteins wliich remain. These represent a virtual spectrum of colloids ranging from the water-insoluble and soluble euglobu- lins through the pseudoglobulins to the water-soluble albumins. In the circulating plasma they probably represent a very complex arrangement of colloidal particles varying in size, amount of bound water, chemical content and structure, electrical charges, etc., and play many roles in the body's economy. By adding sodium sulphate to the extent of 13, 17 and 21 per cent to different portions of serum, Howe precipitated three frac- tions of proteins which are called euglobulins, pseudoglobulin 1 and pseudoglobulin 2, respectively. The albumins remained in solution but are precipitated by higher concentrations of sodium sulphate. Others have separated the serum proteins into these and additional fractions by other methods. The most recent and perhaps the most successful one is that of Ti.selius, who separates proteins into different fractions according to their rate of move- ment between two electrodes. Antibodies. — In previous chapters attention has been called to research indicating that the blood plasma is virtually a reservoir of traces of reacting substances or groups having more or less specific affinities for many red cells of the same and different species, for various kinds of bacteria, lipoids or lipoid-carbo- hydrate complexes and various bacterial poly.saccharides. These specific reacting factors are called natural antibodies and the sub- stances for which they have specific affinities are called either antigens or partial antigens (haptens as Landsteiner calls them). They are real antigens if they can stimulate some animal body to produce specific antibodies and partial antigens or haptens if 193 194 IMMUNOLOGY they react well with antibodies but either do not stimulate their production or are very weak stimulators. Just why these antibodies are present in normal blood is not definitely known. Some of them, like the isohemag£?lutinins, heterohemagglutinins and lysins, are apparently normal physi- ological products and it is possible that part of the antibodies foi' infectious agents of variou.s kinds likewise are normal physiological products. Sutliff and Davies (1937) studied nine infants over a five-month period in an attempt to see whether there was any relationship between the strain of pneumococcus in the naso- pharynx and the type of antibody in the blood. They report that no relationship could be noted although the pneumocoecidal action of whole blood against Type II pneumococcus increased in three infants and for T}T)e III pneumococcus in one infant. They suggest the advisability of investigating the possible physiological origin in future work. It is also possible that an undetermined amount of the so-called normal antibodies for bacteria may owe their presence to unrecog- nized infection Avilh tlie organism corresponding to the antibody. The presence of the bacteria in the body due to unrecognized or recognized infection or as a result of vaccination would represent antigenic stimulation. Presumably this would result in antibody production. Antigens. — As a ])relude to any further discussion of antibodies it would seem desirable to recall some of the pertinent facts about antigens that have either been mentioned or that appear in later chapters. Both the colloidal state and solubility in the body fluids seem to be properties of all true antigens. For a long time it was thought that only complete proteins could stimulate specific antibodies and tlierefore l)e regarded as true antigens. It is now quite generally agreed that the nitrogen con- taining polysaccharide si)ecific for Type I pneumococcus and a fetv other bacterial polysaccharides and perhaps a few lipoid- carbohydrate complexes from cells and tissues can stimulate specific antibodies when injected into certain animals. It was likewise thought for a long time that antibodies would react only with complete proteins, but Zinsser and Parker (1923) discovered non- protein substances (haptens) in bacteria that reacted with bac- NATURE OF ANTIBODIES 195 terial antibodies. Heidelberger and Avery found that such sub- stances in pneumococci are polysaccharides. It is now known that high molecular carbohydrates are important as determinants of specificity of many bacterial agglutinin and precipitin reactions (Landsteiner, 1936, p. 44) and Schiff and Adelsberger* seem to have shown that human red cell haptens are either complex poly- saccharides or lipoid-carbohydrate combinations. Landsteiner and his associates have shown that a wide variety of substances may function as haptens (see Chapters XVII and XVIII). In a later chapter attention is caUed to the conception of Ehrlich supported by experimental proof of WeUs and Osborne that antigenic specificity depends upon the chemical constitution of the antigen. Landsteiner (1936, p. 73-74) calls attention to the possibility of a single antigen having several reacting groups and that, therefore, a number of antibodies may be formed for one antigen. He and van der Scheer (1938) have also shown that separate antibodies may be formed for portions of an antigenic molecule. It is apparently established that cells contain many antigenic factors, and it is thought that the cell antigens present a mosaic structure. It is apparent that the surface antigenic factors will play a more important role in serological reactions than antigenic factors buried beneath the surface. Their poten- tialities may be masked. For a more extensive discussion of the complex nature of antibodies the student is referred to a paper by Marrack and Carpenter (1938). Nature and Origin of Antibodies.— The immunologists have found that the antibodies are precipitated with the euglobulin or pseudoglobulin fractions of serum. According to Landsteiner (1936, p. 94) it is not yet decided whether antibodies are proteins or substances intimately bound to tlie proteins. There is strong evidence indicating that they are protein in nature and therefore are frequently called antihody-glohulins or immune-glohtdins. They unite readily with their antigen at 0° C, are not dialyzable, and resist drying for long periods of time. Fahey and Green (1938) obtained from normal horse serum three water-insoluble proteins (euglobulins) by isoelectric precipi- tation in a very low salt concentration. They named these Pj, Pn, and Pi 1 1 fractions. In a second communication Green, McKhann, Kapnick and Fahey applied the same methods to a study of frac- •See discussion by Landsteiner (supplementary references). 196 IMMUNOLOGY tionation of the globulins of Types 1 and 2 antipneumoeoc- cal horse serum. They describe a new fraction of water-in- soluble globulin which contains most of the antibody. They named the fraction Piy. It seems to be made at the expense of Pii since the latter had almost entirely disappeared when Piv appeared. The authors suggest that the new antibody globu- lin may be a modified form of the normal globulin fraction Pn- Ando, Takeda and Hamano (1937, 1938) report isolating two fractions (A and B) of immune rabbit serum that contain anti- bodies. Fraction A is a water-soluble globulin and contains anti- bodies sucli as the antitoxins and the sliiga-dysentery and per- haps Paratyphosus B. antipolysaccharide antibodies. Fraction B is water insoluble and contains antibodies for pneumococci, E. tifphosa, P. pestis and many other bacteria. They call attention to the fact that these antibodies characterizing one fraction may be found to some extent in the other. We have noted, in unpub- lished work, that Avhile antibodies were present in either the euglobulin or pseudoglol)ulin I fraction they were found pre- dominating first in one and then in the other. Perhaps imperfect methods of separation are partly responsible for these results. Horsfall and Goodner (1935, 1936) liave reported interesting differences in antipneumococcal sera ol)tained from different an- imal species. The.y were able to divide the ten Type I antipneu- mococcal sera from the ten animal species studied by them into two groups. In group one, of which the horse-antiserum is a proto- type, the antibody contains the lipoid lecithin, the presence of which seems to be necessary for agglutination or precipitation while the antibody of the rabbit, the prototype of group two, con- tains the lipoid cephalin which is essential for agglutination. They found the antibodies from which the essential lipid was removed Avould nevertheless combine with its antigen but neither agglutina- tion nor precipitation would follow. Furthermore they found tluit while the antibodies of group one combined with the correspond- ing pneumococcal polysaccharide, the resulting complex fails to fix complement. On the other hand, the complex resulting from the union of antibodies of the second grou]i with pneumococeus polysaccharide fixes complement. Another interesting difference is in the immunological response the two groups of animals give to the injection of Type I pneumo- NATURE OF ANTIBODIES 197 eoccal polysaccharide. The animals in group one respond with antibody production while those of group two give no antibody- response to its presence in a free state in the body. The sera com- prising group one are horse, man, mouse, cat, dog and goat, while those of group two are rabbit, guinea pig, rat, and sheep. The same authors (Horsfall and Goodner) have also shown that the antibody molecule in horse antipneumococcus serum is larger than the corresponding one in rabbit pneumococcus immune serum. This difference in size of the antibody molecule probably explains, in part at least, why the antiserum obtained from rab- bits is more effective in the treatment of pneumonia than antiserum fi'om the horse. Presumably the rabbit serum antibody molecule can diffuse more readily into the tissues. Another difference be- tween antibody from horse and that from the rabbit is observed in the Quellung reaction where the antibody from the horse fails to give the reaction in contrast with the positive results obtained with antibody from the rabbit. According to Chase and Landsteiner (1939) the molecular weights of Types I and III pneumococcal antibodies have been determined in Svedberg's laboratory to be 930,000 for horse, cow and pig and 157,000 for the rabbit and monkey. Goodner and Horsfall (1937) have also shown that antipneumo- coccus horse serum contains at least three antibodies which precipitate the specific polysaccharide but differ in their ability to protect mice. They employed Heidelberger 's method of sepa- rating antibodies from immune precipitates in hypertonic salt solution. By employing 10 per cent sodium chloride they sepa- rated two fractions of antibody. One of these antibodies was a water-soluble pseudoglobulin-like substance, P, which had low protective capacity and a second water-insoluble (euglobulin- like) antibody, E, which exhibited more protective power but formed precipitates less rapidly than P. Chase and Landsteiner suggest that these antibodies may both be lecithoproteins. Origin of Antibodies. — Buchner originally conceived of anti- bodies as being formed from the antigens injected but his theory soon gave way to the ''side chain theory" of Ehrlich. Recently Manwaring and others have attempted to revive Buchner 's theory although modifying it to some extent. Heidelberger (1932) in discussing Manwaring 's theory cites a statement by Doerr "that 19S IMMUNOLOGY arsenic in an atoxyl-azo antigen leads to an arsenic-free antibody, although the arsenic acid radical determines the specificity. ' ' This is further borne out by the work of Heidelberger and Kendall, who found that R-salt-azobenzidineazo-egg albumin givas rise to colorless antibodies although the colored part of the antigen is the portion determining specificity, Heidelberger further calls attention to tlie failure of quantita- tive studies to support Manwaring's views since he says, "As much as eight mg. of circulating antibody may be produced per milli- gram of antigen injected, and since this is only a portion of the to- tal antibody produced, the amount seems inconsistent with the idea that specific antigen fragments are present, though the evi- dence is not conclusive." He then cites the work of Hooker and Boj^d and also of Topley, who disagree with Manwaring. In a later paper Heidell)erger, Kendall, and Soo Hoo (1933) state Hint they obtained a total response for tlie rabbit of over 100 mg. of circulating antibody for every milligram of antigen injected. Buclmer's original tlieory was soon supplanted by Elirlich's side-chain theory. He conceived of antibodies as chemical food receptors produced by the tissue cells in excess as a result of stimu- lation resulting from injury or demand. Since his theory is only of historical interest it will not be discussed. Heidelberger (1932) reviews the work of Breinl and Haurowitz who conclude that the presence of antigen in the tissues "dis- turbs the mechanism of globulin synthesis, proba1)ly in the amino- acid-peptid stage, modifying tlie method of union or the spatial relations of the globulin components so that a new globulin, an antibody, is formed which reacts specifically with the antigen by virtue of the distortion caused by the presence of the antigen at the moment of synthesis, for if the antigen can only affect amino acids having affinity for it, these should retain that affinity after their synthesis into globulin." Sabin (1939) suggests that antibodies represent proteins cast off by the clasmatocytes. While no one seems to know positively how they are formed, there is a great deal of evidence indicating they are formed by cells of the reticulo-endothelial system. Unitarian Theory of Antibodies. — Zinsser (1939) says that this theory does not imply that only one antibody will be pro- duced against a complex cellular antigen. He says, however, that NATURE OF ANTIBODIES 199 according to the unitarian theory a single antigen, in a pure state, would stimulate the formation of one variety of antibody capable of uniting with the antigen. This union of antigen and antibody could result in agglutination, precipitation, complement fixation, bactericidal phenomena, opsonization or anaphylactic sensitization depending upon the physical state of the antigen itself, "tlie na- ture of the cooperative substances (alexin, leucocytes, tissue cells) , and by the environmental conditions under wliich tlie observations are made." Delves (1937) as well as Zinsser, Enders and Fothergill (1939, pp. 175-178) cite considerable experimental evi- dence in support of this theory. While it has been apparently established that an antigen may give rise to more than one antibody, yet it is conceivable that any one of the antibodies may serve some or all of tlio various capacities mentioned by Zinsser. It is possible, as suggested by the work of Horsfall and Goodner mentioned earlier in this cliap- ter, that antibodies from some species of animals may not promote complement fixation although they lead to precipitation. Witli these points in mind we are accepting the general tenets of his Unitarian theory until more evidence against it is available. Mechanism of Antigen-Antibody Union. — There are at least two theories explaining antigen-antibody union that are being dis- cussed at present. According to the film hypothesis supported by Eagle, Mudd and others, the antibody is specifically adsorbed to the antigen forming a partial or com])le1e film on the surface. The antibody globulin becomes insoluble in saline (denatured) as a result of this union with antigen. Eagle suggests that the specificity of the antibody globulin is due to its hydrophilic (water-loving) groups which become attached to the antigen, thus orienting the hydrophobic ends of the molecules outward toward the water which accounts for the denaturing of the antibody pro- tein. Marrack, on the other hand, cites the quantitative studies of Heidelberger and Kendall as offering evidence against the film hypothesis. He thinks the antibody molecule combines as a closely packed group of molecules and not as a film. As a working theory we are tentatively accepting the film hypothesis as out- lined by Eagle and also Mudd. There are also two lines of thought relative to the mechanism of agglutination and precipitation which may result from antigen- 200 IMMUNOLOGY antibody union. The one theory first suggested by Bordet and sup- ported by experimental data outlined in Chapter XII assumes that the agglutination or precipitation is due to the reduction in re- pelling force between the cells or colloidal particles and the ex- istence of cohesive properties of the sensitized antigen. Accord- ing to this theory these physical changes in state of aggregation are secondary phenomena in which chemical forces do not play a role. The second theory, supported by experimental work of Heidel- berger and Kabat (1937), statas that chemical forces play a role in the second stage wliich is agglutination of cells or precipitation of colloidal particles. Opposition to this latter concept is offered by Hooker and Boyd (1938) and Eagle (1938). The explanation we offer in Chapter XII is largely in harmony with the explana- tion offered by Bordet, Eagle, Hooker and Boyd and others. We realize there is considerable evidence in support of the op- posing view but until there is a general acceptance of it we will hold to the older theory. Method of Measuring- Antibodies. — Antitoxins. — Ehrlich suggested a method of standardizing antitoxin, which, while used to some extent, is being superseded by the Ramon flocculation technique. Ehrlich was working with diplitheria toxin and determined the smallest amount tliat would kill a 250 gi-am guinea pig in four or five days. This he called a minimum lethal dose or an M.L.D. He then measured out 100 M.L.D.'s and called that amount an Lq dose of toxin. Tlie amount of antitoxin that just neutralized tliis he called a luiit of anti- toxin. This amount corresponds to what is now called an " old unit." He then determined the least amount of toxin tluit, when mixed with an ''old" unit of antitoxin, would kill a 250 gram guinea pig in four to five days. This amount of toxin he called the L, dose. The least amount of antitoxin which protects the guinea pig against the L^ dase of toxin is the "new unit." In Chapter XIV the method of titrating antitoxin by the Ramon flocculation technique is given. Eaton (1936) discusses the flocculation re- action with purified diphtheria toxin. This method is also ap- plied to standardizing tetanus and other antitoxins. Unit of Tetanus Antitoxin.— The American immunity unit of tetanus antitoxin has been defined as ''ten times the least NATURE OF ANTIBODIES 201 quantity of antitetanus soruiii necessary to save tlie life of a 350 gm. guinea pig for ninety-six hours against the official dose of standard toxin furnished by the Hygienic Laboratory of the Public Health and Marine Hospital Service." Compared to a unit of diphtheria antitoxin it has slightly more than ten times the protective power. The method of standardizing tetanus anti- toxin was suggested by Rosenau and Anderson (1907). As men- tioned above, the Ramon technique is now used extensively. Scarlet fever antitoxin is measured in terms of its protective value for skin test doses of toxin in accordance with the work of Dick and Dick (1924). Agglutinins. — In determining the titer of a clumping or ag- glutinating serum, as, e.g., one that agglutinates E. tiiphom, the antigen is not diluted, as in the precipitin ring test. On the contrary, a series of serial dilutions of the immune serum is made. A number of small test tubes are placed in a rack and to each is added a uniform and measured amount of the various dilutions and an equal amount of a standard turbid suspension of the antigen, e.g., E. typhosa. After shaking and incubating, nota- tion is made of the highest final dilution of the immune serum con- taining the antibody that gives perceptible clumping, and this is the titer of the immune serum. Titers as high as 1 :5,000 or 1 :10,000 are readily produced in rabbits by vaccination. The suspension of bacteria used in agglutination work is usually diluted to match a known turbidity standard. Precipitins. — The strength or titer of a precipitating immune serum is commonly described in terms of the highest dilution of the antigen that gives a perceptible ring precipitate when strati- fied over the undiluted immune serum in a small test tube. Titers as high as 1 : 1,000,000 have been produced. Cannon and Marshall (1940) have called attention to an old but more rational metJiod of determining the titer of precipitating antibodies. They prepare a standard suspension of collodion particles, film them with antigen and mix an equal volume of the filmed particles with a corresponding volume of serial dilutions of the antibody as in an agglutination test. After incubation for 30 minutes at 37° C. the tubes are centrifuged at low speed to bring together antigen-antibody coated particles. The contents of the tubes are then resuspended and examined for agglutination. 202 IMMUNOLOGY The highest dilution of antibody giving perceptible agglutination of the particles filmed with antigen is the titer of the precipitin serum. Bacteriotropins. — The content of bacteriotropins in a patient's serum is estimated by mixing equal parts of the serum, bac- terial suspensions and a suspension of leucocytes, incubating and determining, by microscopic examination of stained smears of the incubated mixture, the average number of bacteria ingested per leucocyte. A normal serum is treated in like manner and the aver- age number of bacteria engulfed per leucocyte is determined. If the mixture containing patient's serum showed, e.g., four bacteria phagocytized per white cell, and two bacteria per white cell in the mixture containing normal serum, the opsonic index would be 4 —or 2. In this example the patient's serum would cause twice as much phagocytosis of the organism used as normal serum. Antiaggressins. — There is considerable doubt in the minds of many, of the existence of such an antibody as an antiaggressin. According to Weil and others it is present in the albumin fraction of the serum. The antiaggressin content is estimated from the amount of serum that protects guinea pigs against a predetermined number of minimum fatal doses of the organisms. Hemolysins. — In order to measure the strength, i.e., determine the titer of an hemolytic immune serum, one must adopt definitions of what constitutes a unit of each of the three constituents used, i.e., red cell suspension, sensitizer and complement. Kolmer's definition of each of these may be expressed as follows : Kolmer has defined a unit of red cell suspension as one-half cubic centimeter of a 2 per cent suspension of washed sheep red cells in normal saline. Washed packed cells, obtained by eentrifuging defibrinated sheep blood and repeatedly resuspending in saline with subsequent centrifugalization, constitutes a 100 per cent sus- pension. From the final packed cell sediment a 2 per cent suspen- sion is prepared. Other authorities have arbitrarily adopted different kinds of red cells as well as different volumes and weights of suspensions in their definitions of what each regarded as a unit. These will be mentioned in the chapter on Complement Fixation. A hemolytic amboceptor or sensitizer unit, according to Kolmer, is 0.5 c.e. of the highest dilution of immune serum that will NATURE OF ANTIBODIES 203 sensitize one unit of red cells so that they will be completely hemolyzed by 0.30 c.c. of a 1 :30 dilution of guinea pis comple- ment after incubation for 1 hr. in a 37° C. water bath. A unit of complement is the least amount of a 1:30 dilution of complement that will completely hemolyze one unit of red cells sensitized by two units of hemolysin. Kohner always titrates complement in the presence of a test dose of antigen. Method of Titrating Hemolysin. — In order to determine ex- l)crimentally the amount of immuiic sei-um that contains a unit of hemolysin or to ascertain tlic amount of normal guinea pig serum that contains one unit of complement, certain standards have to be arbitrarily adoj^ted. Kolmcr, who has carried out an extensive investigation of various methods used to titrate hemolysin and complement, recommends that after all reagents arc added their final volume shall be 3.0 c.c. in each tube. He has adopted 0.5 c.c. of a 2 per cent suspension of washed slieep red cells as a iniit for the red cell suspension and noriual saline containing 0.1 gm. of magnesium sulphate per liter as a diluent. He makes up a series of dilutions of the immune serum using a separate test tube for each dilution and carefully labels each tube indicating the dilution it contains. These dilutions are usually as follows: 1:1,000. 1:2,000. 1:3,000, 1:4,000, 1:5,000, 1:6,000, 1:8.000, 1:10,000, 1:12,000, 1:16,000. He then dilutes fresh normal guinea pig serum (complement) 1:30 by adding one part of complement to twenty-nine parts of normal saline. After this he places ten Wassermann test tubes in a rack and adds the reagents accord- ing to the following protocol : Hkmotasix Titration COMPLEMENT ^^^ TUBE HEMOLYSIX, 1:30 SOLUTION SUSPENSION NO. 0.5 c.c. DILUTION C.C. ^•^- ^•^- 1 1: 1,000 0.:: 1.7 0.5 2 1: 2,000 0.3 1.7 0.5 3 1: 3,000 0.3 1.7 0.5 4 1: 4,000 0.3 ].7 0,5 5 1: 5,000 0.3 1.7 0.5 6 1: 6,000 0.3 1.7 0.5 7 1: 8,000 0.3 1.7 0.5 8 1:10,000 0.3 1.7 0.5 9 1:12,000 0.3 1.7 0.5 10 1:16,000 0.3 1.7 0.5 204 IMMUNOLOGY The contents of each tube should be mixed and incubated in a water bath at 37° C. for 1 hour. The unit is the highest diki- tion of hemolysin that gives complete hemolysis. Plate IV shows such a titration in w^hich tube 5 (1:5,000) contains 0.5 c.c. of the highest dilution giving complete hemolysis. The unit or titer in this case is 0.5 c.c. of a 1 :5,000 dilution. Complement Titration. — For complement titration Kolmer uses a 1 :30 dilution of complement and always titrates this in the presence of a test dose of antigen contained in 0.5 c.c. of saline. He makes a point of using only cold saline in the titration. The titration requires 9 tubes for the varying amounts of complement shown in Plate IV, 2 and a tenth tube containing only saline and red cells (not shown in the plate). He sets up the complement titration according to the following protocol : CoMPLEjrENT Titration COMPLEMENT ANTIGEN DOSE C.C. SALINE ^HEMOLYSIN 2 PER CENT ^ TUBE C.C. (1 :.•?()) SOLUTION C.C. ?r. c.c. T'NITS) RED CELL SUS- PENSION C.C. 0.5 0.5 cr - » 1 0.1 0.5 1.4 2 0.15 0.5 1.4 w 0.5 0.5 CO 3 0.20 n.5 1.3 ~i 0.5 0.5 ~l 4 0.25 0.5 1.3 O 0.5 0.5 O 5 0.30 0.5 1.2 0.5 0.5 6 0.35 0.5 1.2 o 0.5 0.5 o 7 0.40 0.5 1.1 (-• 0.5 0.5 h-1 8 0.45 0.5 1.1 c- 0.5 0.5 tr 9 0.50 0.5 1.0 o 0.5 0.5 o c 10 o.n 0.0 2.5 "I 0.0 0.5 Unit of Complement. — The tube containing the least amount of complement that shows complete liemolysis is said to contain one exact unit of complement. In the illustration (Plate IV, 2) tube four contains the least amount of complement in which complete hemolysis results. In this example the exact unit is 0.25 c.c. of a 1 :30 dilution. Kolmer defines a full unit as 0.05 c.c. more than an exact unit. This would be contained in tube five to which 0.30 c.c. .of the 1 :30 dilution of complement had been added. The method of antigenic titration used by Kolmer and tlie various standards he has set up will be discussed in the chapter oil complement fixation. Fig. 1. — Tube 5 contains 1 unit of anil)oceptor. /< iL^ L^ i J) 4 i I 3i I — I — [ — n ^ W W W Fig. 2. — Tube -i contains 1 unit of complement. Tube .5 contains 1 full unit of complement. I'LA'lE IV. TiTUATION" OF 1 1 KMOLVTIl ' AMIJOCEPTUU AM) CUMIM.KMKNT. NATURE OF ANTIBODIES 205 The reason for describing Kolmer's technique and omitting oth- ers is that the committee on Adherence to Conventional Technic in the Performance of Reliable Serologic Tests for Syphilis, ap- pointed, I believe, by the Surgeon General, has approved the Kolmer complement fixation technique. For this reason it is be- ing used, or is coming into use, in control and local laboratories throughout the TTnited States. It is lioped that the student will realize that many of the requirements laid down by Kolmer are empirical ones whih^ the principles underlying the tests are basic. It is true that empirical requirements such as the order of mixing, time of incubation, dilutions of amboceptor and complement used in titration, volume of fluid containing a unit or test dose, tlie unit of red cells, the temperature and time of inactivation, etc., all are based U])on good reasons mostly for economy of time, re- agents or for convenience. A good standard technique could be set up in which either slight or marked variation from Kolmer's would be used and results obtained that would be equally as ac- curate. Unfortunately there are bad modifications as well as good ones and it seems that in the interest of good serology it is best to recommend one good uniform technique as has been done by the committee. The basic things in all of these tests are the nature of the antigens, the laws governing the union of antigen and anti- body, the binding of complement, the lysis of cells and the physical and chemical constitution of the reagents employed. These are not empirical things. References Ando, K., Manako, K., and Takeda, S.: Studies on Serum-fractions. V. The Fraction of Antidiphtheric Horse-serum Precipitable by Anti- serum Prepared With the Floccules of Diphtheric Toxoid-Antitoxin, J. Immunol. 34: 295, 1938. Ando, K., Takeda, S., and Hamano, M.: Studies on Serum-fractions. VI. The Close Serologic Relationship of Different Antibacterial Anti- body-globulins, J. Immunol. 34: 303, 1938. Bordet, J.: See Chapter XII. Boyd, W. C, and Hooker, S. B.: Note on the Mechanism of Specific Ag- glutination, Proc. Soc. Exper. Biol. & Med. 39: 491, 1938. Cannon, P. E., Baer, R. B., Sullivan, F. L., and Webster, J. R.: The In- fluence of Blockade of the Reticuloendothelial System on the Forma- tion of Antibodies, J. Immunol. 17: 441, 1929. Cannon, P. R., and Marshall, C. E.: An Improved Serologic Method for the Determination of the Precipitative Titers of Antisera, J. Immunol. 38: 365, 1940. Chase, M. W., and Landsteiner, K.: Immunochemistry, Ann. Rev. Biochem. 8: 579, 1939. 206 IMMUNOLOGY Delves, E.: Immunological Studies With Purified Serum Proteins Bearing on the Unitarian Theory of Antibodies, J. Infect. Dis. 60: 55, 1937. Eagle, H.: Some Effects of Formaldehyde on Horse Antipneumococcus Serum and Diphtheria Antitoxin, and Their Significance for Theory of Antigen- Antibody Aggregation, J. Exper. Med. 67: 495, 1938. Eaton, M. D.: The Flocculation Eeaction with Purified Diphtheria Toxin, J. Immunol. 30: 361, 1936. Ehrlich, P.: Studies in Immunity, Translated by Bolduan, New York, 1910, John Wiley & Sons, Inc. Goodner, K., and Horsfall, F. L., Jr.: Properties of Type Specific Proteins of Antipneumococcus Sera. The Mouse Protective Value of Type I Sera with Reference to the Precipitin Content, J. Exper. Med. 66: 413, 1937. Goodner, K., and Horsfall, F. L., Jr.: Properties of Type Specific Proteins of Antipneumococcus Sera. Immunological Fractionation of Type I Antipneumococcus Horse and Eabbit Sera, J. Exper. Med. 66: 425, 1937. Goodner, K., and Horsfall, F. L., Jr.: Properties of Type Specific Proteins of Antipneumococcus Sera. Immunochemical Fractionation of Type I Antipneumococcus Horse and Rabbit Sera, J. Exper. Med. 66: 437, 1937. Green, A. A., McKliann, C. F., Kapnick, I., and Fahey, K. E. : A Fraction- ation of the Globulins of Types 1 and 2 Antipneumococcal Horse Serum, J. Immunol. 36: 245, 1939. Heidelberger, M.: Ann. Rev. Biochem. 1: 655, 1932. Heidelberger, M., Kendall, F. E., and Soo Hoo, C. M.: Quantitative Studies on the Precipitin Reaction. Antibodv Production in Rabbits Injected With an Azoprotein, J. Exper. Med. 58: 137, 1933. Heidelberger, M., and Kabat, E. A.: Chemical Studies on Bacterial Agglutination. III. A Reaction Mechanism and a Quantitative Theory, J. Exper. Med. 65: 885, 1937. Heidelberger, M., and Kabat, E. A.: Quantitative Studies on Antibody Purification; Dissociation of Antibody from Pneumococcus Specific Precipitates and Specifically Agglutinated Pneumococci, J. Exper. Med. 67: 181, 1938. Horsfall, F., and Goodner, K.: Lipids and Immunological Reactions. II. Further Experiments on the Relation of Lipids to the Type-Specific Reactions of Antipneumococcus Sera, J. Immunol. 31: 135, 1936. Howell, K. M.: Origin of Antibodies, Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press. Landsteiner, K., and van der Scheer. On Cross Reactions of Immune Sera to Azoproteins; Antigens with Azocomponents Containing 2 Determinant Groups, J. Exper. Med. 67: 709, 1938. Manwaring, W. H.: A Critique of the Ehrlich Theory, with an Outline of the Enzyme Theory of Antibody Formation. Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 1078. Alarrack, J., and Carpenter, B. R. : The Cross Reactions of Vegetable Gums with Type II Antipneumococcal Serum, Brit. J. Exper. Path. 19: 53, 1938. Mudd, S.: See Chapter XII. Sabin, F. R.: Cellular Reactions to a Dye-protein With a Concept of the Mechanism of Antibody Formation, J. Exper. Med. 70: 67, 1939. Sutliff, W. D., and Davies, J. A.: A Study of the Origin of Naturally Occurring Immune Bodies for Pneumococci in Infants, J. Immunol. 32: 43, 1937. NATURE OF ANTIBODIES • 207 Tiselius, A.: Biochem. J. 31: 1464, 1937. (Cited by Chase and Land- steiner, 1939.) Weil, E.: See Eeview of Literature on Aggressins by Scott, J. P., J. Bait. 22: 323, 1931. Wells, H. G., and Osborne, T, B.: (Cited by Wells in Chemical Aspects of Immunity, New York, 1929, Chemical Catalogue Company, p. 68.) Zinsser, H., Enders, J. F., and Fothergill, L. D.: Immunity, New York, 1939, The Macmillan Co., p. 175. tiupplcmcntarii References Boyd, W. C: The Time Factor in Solubility of Precipitates in Excess of Antigen, J. Immunol. 38: 143, 1940. Hooker, S. B., and Bovd, W. C: Antibodies to Strychnine, J. Immunol. 38: 479, 1940. Howe, A, E.: A Note on the Calculation of Antibody- Antigen Eatio, J. Immunol. 37: 77, 1939. Landsteiner, K.: The Specificity of Serological Eeactious, Baltimore, 1930, Charles C. Thomas. Libby, K. L.: A Simplified Photronreflectometric Technic for the Titration of the Antibody-Potency of Antipneumococcal Horse and Rabbit Serum, J. Immunol. 35: 289, 1938. Little, P. A.: On a Rapid Method for the Standardization of Antimeningo- coccal Horse Serum Type I, J. Immunol. 35: 117, 1938. CHAPTER XII MECHANISM OF ANTIGEN-ANTIBODY REACTIONS CELLULAR AGGLUTINATION Period of Discovery and Early Investigations. — The ]ieriod of discovery of the phenomenon of bacterial agglutination by im- mune serum began with Charrin and Roger (1889) and ended with Bordet (1895). The next year the value of the reaction in diagnosis and its relative specificity^ was described by Gruber, Gruber and Durham, Widal, Griinbaum and others. Gruber 's Theory of Agglutination (1896). — Gruber suggested the name ''agglutination" for the phenomenon and offered a theory explaining it. He believed that the clumping was caused by the action of agglutinin (antibody) upon the bacterial cell membrane whereby the latter became more viscous. In his opinion this change in the membrane caused the bacteria to stick together. This was really the first suggestion that "cohesive forces" might be a factor as shown recently by Northrop and De Kruif (1922). Bordet 's Early Work. — It is quite probable that all of this work was inspired by Bordet 's observation (1895) of the phe- nomenon of agglutination while lie was engaged upon tlie study of the phenomenon of Pfeiffer. The next year (1896) lie turned his attention to the mechanism of agglutination and published the results of his experimental work two years later (1898). Discovery of Precipitins. — In the meantime, Kraus (1897) made an important discovery that has directly or indirectly in- fluenced our conceptions of antigen-antibody I'eactions in general. He observed that a specific pi'ecipitate was formed when cholera- immune serum was mixed Avith filtrates from an old broth culture of the vibrio of Asiatic cholera. This was the first work done on precipitins. Two years later Tchistovitch noted that the blood serum of rabbits vaccinated against horse serum yielded a specific precipitate when mixed with horse serum. Similar results were ob- tained when eel serum was used as an antigen. Dineur's Hypothesis of Agglutination. — Following Kraus 's discovery and prior to Bordet 's publication, Dineur (1898) ad- 208 MECHANISM OF ANTIGEN-ANTIBODY REACTIONS 209 vanced an liy])otliesis which was based upon Kraiis's work but was really the beginning of our knowledge of flagellar agglutinins although Gruber had suggested that flagella might be important. Dineur conceived of the agglutination reaction as due to the forma- tion of an "adhesive" substance on the flagella with the subse- quent interlacing of the latter. Bordet's Objections to Current Theories. — Bordet carefully considered Gruber 's hypothesis that bacteria were agglutinated because the agglutinin caused them to become sticky. He could understand how changes in the coll membrane might make them ''adhere" Avhon tliey came in contact wnth each other, but this tlieory did not explain wliy they came together. In fact, there was no experimental evidence to show that a change in the membrane occurred. He knew from experience that hemagglutinins were developed in animals when red cells were injected and he watched the agglutination of red cells by immune serum under the micro- scope and could see no change in the membrane. Since red cells were agglutinated by their immune serum and since they did not possess flagella, he felt that flagella were not necessary factors in the phenomenon of agglutination, as suggested by others. Be- sides, Widal had shown that formalized cultures were agglutinated as readily as living suspensions and this he regarded as additional evidence. He then compared hemagglutination, bacterial agglu- tination, serum precipitation, and bacterial protein precipitation by their respective immune sera and saw that while they differed in that each antigen had in the beginning combined with its specific antibody, yet the end-result was quite similar; i.e., clumping or precipitation. Bordet's Two-Phase Theory. — Thus Bordet considered two steps or phases in immunological reactions of this kind: First, one which involves specificity, the union of antigen and antibody, and second, a common mechanism of agglutination or precipitation. It was this second phase, involving a mechanism of agglutination common to all, that he set about investigating. At the beginning of his work he was very much impressed with Duclaux's theories as to the mechanism of coagulation and his conclusion that ag- glutination is a phenomenon of coagulation. 210 IMMUNOLOGY Bordet's Definition. — This led Bordet* to adopt the followmg definition for agglutination, "It is the union into masses of or- ganized scattered particles by some peculiar influence that changes the properties of molecular adhesion." "The agglutination of bacteria is due to a change in molecular adhesion between the l)odies of the bacilli and the surrounding fluid.." Bordet's Experbients on Effect of Electrolytes on Chol- era Vibrios. — At that time it was apparently well known tliat clay forms a fine, homogeneous emulsion in distilled water, but that agglutination and rapid precipitation of the particles occurs when a small amount of sodium chloride is added. This suggested an experimental approach to the study of bacterial agglutination. Bordet made a suspension in salt solution using 24-liour-old cultures of Microspira comma and 10 c.c. of saline to each culture. To this homogeneous suspension he added sufficient anticholera- immune serum 1o produce complete agglutination of the bacteria. He then centrifuged and decanted the supernatant fluid and resuspended the sediment in enough distilled water to make a rather thick emulsion. This lie added to two test tubes in equal amounts. To tube No. 1, he added distilled water and to tube No. 2, physiological saline. These tubes were then shaken and centrifuged and resuspended as before, i.e., the sediment of tube 1 in distilled water and of tube 2 in saline. The tubes were then shaken and allowed to stand. Bordet says, "It is found that clumps form rapidly in llio tube containing salt solution, ])ut that the bacteria renuiin indefinitely in suspension in the tube contain- ing distilled Avater. " He also removed some of Ihc distilled water emulsion and added a small amount of sodium chloride and ob- tained rapid agglutination. Bordet thiLs concluded lliat the first phase was a specific ad- sorption of antibody (agglutinin) by the antigen (bacteria) and that the second j^hase was one of agglutination in which the sen- sitized cells were clumped upon the addition of electrolytes. It should be noted that the anti])ody was not removed by washing in either distilled water or salt solution. For a brief discussion of the physical chemical concepts involved in flocculation, the student is referred to the disciLssion of colloids included in the appendix. ♦Reprinted by permission from Bordet and Gay'.s Stivdies in Imimmity, published by John Wiley & Sons, Inc. MECHANISM OF ANTIGEN-ANTIBODY REACTIONS 211 Ehrlich's View. — This two-phase hypothesis of Bordet was vigorously opposed by Ehrlieh* who conceived of agglutinin as having chemical groups that caused clumping. Referring to Bor- det's work he says, "The attempt has been made to interpret the immunity reactions from the standpoint of colloid chemistry. . . . I see absolutely no advantage in such a method, and I have grave fears that it will result in checking further progress along this line. Structural chemistry, on the otlier hand, has not only served to explain all the phenomena in imuuniity studies but has also proved a valuable guide in indicating the lines along which further progress might be made. ' ' This difference of opinion between those holding to the physico- chemical theories and those equally ardent advocates of theories based upon structural chemistry led to an interusive study of anti- gens and the factors governing specificity and to the mechanism of the immunological reactions. The phenomenon of specificity has been discussed at lengtli in Chapters XVIF, XVIII, and XIX. Subsequent Lines of Research. — In the studies of the meciiani.sm of clumping or agglutination of l)actoria by immune sennn, three interesting lines of investigation have contributed to our present concepts. These may be enumerated as follows : 1. Studies to determine whether the law of multiple propor- tions holds when bacteria combine with agglutinin. 2. Studies of the effect of varying salt concentrations on mem- brane potential and cohesive forces of unsensitized and sensitized particulate matter that has previously been made to adsorb a specific protein and bacteria before and after the adsorption of ag- glutinin. 3. Studies relative to the nature of cellular surfaces and molecu- lar orientation before and after the adsorption of specific agglu- tinins. This work has been done largely by I\Iudd and IMudd (1924, 1926, 1927) using their ' ' interfacial tension technic." Studies on Effect of Dilution on Adsorption. — In 1902, Eisenberg and Volk found that if a constant amount of bacteria were mixed with equal amounts of varying dilutioiLs of immune serum, the bacteria removed a greater percentage of the agglutinin content from the higher dilutions. In the lower dilutions where •Reprinted by permis.sion from Studiffi in Immunitij by Ehrlich-Bolduan, published by John Wiley & Sons, Inc. 212 IMMTJNOLOGY the immune serum was more concentrated, they found that while the same amount of bacteria actually adsorbed more agglutinin than from the higher dilutions, yet the amount adsorbed was a smaller percentage of the total amount present than in the latter. Effect of Fractional Addition of Antigen. — In 1905, Craw showed that the way bacteria were added to immune serum in- fluenced the amount of agglutinin adsorbed He found that if tlie bacteria w^ere added all at once to the immune serum they adsorbed more agglutinin than if added in fractional amounts with appre- ciable time intervals between. This is similar to tlie Danysz phe- nomenon where more toxin is neutralized by a given amount of antitoxin added all at once than when added in fractional amounts with some time intervening. Bordet's experiment of dye adsorp- tion with filter paper illiLstrates this. Bordet's Experiment Explaining Danysz Phenomenon. — If a dilute solution of a dye is prepared and a definite sized square of filter paper is added, in small pieces at considerable time intervals, the first pieces added will be intensely dyed while the last added may not be colored at all. These results suggest that the adsorp- tion of agglutinin by bacteria is in accordance with the law of adsorption. It appears that the union does not follow any law of simple proportions. Heidelberger and Kendall's Theory of Reaction Mecha- nism.— Since it is thought that the mechanism involved in the formation of immune precipitates is identical with the one involved in the agglutination of bacteria by immune serum, the recent theory of Heidelberger and Kendall* (1935, 1937) relative to the mechanism involved in a bacterial precipitin reaction is of interest. In the summary of their paper (1935) they say that ''the pre- cipitin reaction between the specific polysaccharide of Type III pneumococcus and homologous antibody found in the horse can be accounted for quantitatively by assuming the chemical combination of the components in a bimolecular reaction, followed by a series of competing bimolecular reactions which depend upon the relative proportions of the components. These reactions would lead to the formation of larger and larger aggregates until precipitation ulti- mately occurred. The mathematical formulation of this theory on ♦Heidelberger and Kendall: J. Exper. Med. 61: 563, 1935. MECHANISM OF ANTIGEN-ANTIBODY REACTIONS 213 the basis of the mass law is described. The derived expressions are showTi to be in accord with the experimental findings and the constants used in these expressions are shown to have definite significance." In subsequent studies Heidelberger and Kendall (1937) have shown that the same mathematical expressions hold for rabbit as for horse precipitins in their reactions with pneumo- coccus polysaccharides. An excellent summary of their work is given by Chase and Landsteiner (1939). Reference to later work of Heidelberger and also of Hooker, Eagle and others, liearing upon the controversies over the mecha- nisms involved in cell sensitization and agglutination respectively, is given in Chapter XI. Early Cataphoresis Experiment. — The second line of investiga- tion whicli lias contributed to our present views involved experi- ments to determine the action of bacteria suspended in a liquid medium through Avhich a known electric current was passed. Such studies on the cataphoresis of bacteria have been very fruitful. The pioneer work in this field was probably inaugurated by Bech- old (1904) and also by Neisser and Friedemann (1904). They noted that bacteria that had adsorbed agglutinin clumped more readily between the electrodes than bacteria that had not adsorbed agglutinin. It suggested to them that perhaps adsorption of ag- glutin was associated with a loss of electrical charge by the bac- terial cells. In 1905, Pauli explained the agglutination of col- loidal particles by electrolytes as due to the neutralization of charges present on the dispersed particles. Powis ' Work and the Critical Potential. — The next important contribution was made by Powis (1914) who studied membrane po- tential changes in oil emulsions and found that coagulation oc- curred when a critical P.D. of about 30 millivolts was reached. Similarity to Denatured Proteins. — In the same year Tullocli suggested that sensitized bacteria, i.e., bacteria that had adsorbed agglutinin, were comparable to denatured proteins. Buchanan's Suggestion (1919). — ^Buchanan reviewed the whole subject of agglutination in 1919 and suggested that in a bacterial suspension, the repelling forces resided in the similarity of charges on the bacterial cells and these forces are opposed by those of sur- face tension operating to cavLse agglutination. 214 IMMUNOLOGY In 1921 Coulter showed that the most favorable pH for the clumping of unsensitized red cells is 4.75 and that this shifts to 5.3 when agglutinin is adsorbed by them. Since this latter is the isoelectric point for serum globulin and since agglutinins like all antibodies are precipitated out with the serum globulins, it sug- gests that the red cells are filmed or coated with antibody globulin when they adsorb agglutinin as illustrated in Figs. 10 and 11. LoEB (1923). — As further evidence lending support to the con- ception that globulin is adsorbed by the red cells is the result of Loeb's (1923) studies on the ability of collodion particles to ad- sorb protein. He shows after adsorption of a film or partial film of protein the collodion particles acquire the properties of the adsorbed substance. According to Northrop (1928) this phenome- non had been observed first by INIeyers and Lottermoser in 1901. Northrop and DeKruif (1922).- — Almost simultaneously with the appearance of Coulter '.s work, Northrop and DeKruif (1922, 1923) published the results of their careful investigation of the mechanism of agglutination. They studied tlie effect of pH on the specific adsorption of antibody globulin Avhich corresponds to Bordet's phase one and found that it does not seem to affect it ma- terially since the adsorption of antibody globulin occurs through- out a fairly wide range of pH values and even when both elements are similarly charged. Shiblfa's Summary of Northrop and DeKruif 's Work. — Their final conclusions including the results of their studies on phase two of Bordet are concisely summarized by Shibley* (1926) as follows: ''(1) Agglutination is to be considered in terms of two antago- nistic forces ; a repelling force, due to like electrical charges, which tends to keep the bacteria apart, and 'cohesive force,' wliicli makes for adhesion. In any bacterial sus]>ension, all factors that make the repelling force relatively greater than the cohesive force make for stability; and conversely, all factors tliat reduce the re-, pelling force or otherwise make the cohesive force relatively greater, lead to flocculation. (2) In the case of unsensitized bacteria, electrolytes in lower concentrations, < 0.01 N, affect primarilj^ the potential, and in higher concentrations, > 0.01 - 0.1 N, affect primarily the cohesive force. (3) As long as the •Shibley: J. Exper. Med. 44: 667, 1926. Fio-. 10. Fig. n. R. Blodd Fig. 10. — Red cell before adding ininmne serum containing hemagglutinin. Fig. 11. — Red cell after adding hemagglutinin partly or completely tllmed witli antibody globulin ab.sorbed from immune serum. MECHANISM OF ANTIGEN- ANTIBODY REACTIONS 215 cohesive force is unaffected, ag'glutination occurs wlicnever the charge is reduced by electrolytes to a point below a critical level of about 15 millivolts; that is, the unaffected cohesive force now becomes relatively greater than the force of repulsion. (4) Salt in high concentration depresses the cohesive force of unsensitized bacteria so that no agglutination occurs even thougli tliere may be no measurable charge ; i.e., the cohesive force is now so small that it is always less than the repelling force. (5) When bacteria are treated with immune sera, their cohesive force is in some manner protected from this depressing effect of strong salt (e.g., physiologic salt solution, etc.) and agglutination is determined solely by the charge; that is, whenever the potential of the sensi- tized bacteria is I'educed by electrolyte to a point below 15 milli- volts, the suspension agglutinates." Shibley continues by saying that ''this explains the observation of Bordet, confirmed by Northrop and DeKruif in the course of the work being cited, that electrolytes are essential for specific ag- glutination. That is to say, the salt, routinely used in the ordinary reactions, reduces the charge on the bacteria so that this charge comes to lie in the 15 millivolt agglutination zone, and tlie cohesive force of the sensitized bacteria, being insusceptible to the depress- ing effect of the electrol;^i;e, is now relatively greater than the repelling force and flocculation occurs. (6) Their results refute the idea that combination of antibody and organism is caused by difference of sign of the charges carried by the two substances ; but are in agreement witli the assumption that the agglutinin forms a film on the surface of the organism." Effect of Sex.sitiz.\tion on Charge.— Shibley then proceeds to offer more experimental evidence in support of the hypothesis that the union of antigen and antibody consists in the coating of the former by the latter. He reports that A\'hile both normal and im- mune sera depress the charge in the pneumococcus, the effect of normal serum is slight when compared with the marked reducing effect of specific immune serum. He studied the effect of specific immune serum for Type I pneumococcus, a strain of a hemolytic streptococcus, Esch. coli, E. tiiphosa, S. paratijphosi A, Myco. tu- berculosis and for the Flexner, Shiga and J\It. Desert strains of dysen- tery bacillus. All of tlie specific imnume sera possessed the charge-re- ducing effect except the three for dysentery bacilli. The three 216 IMMUNOLOGY immune sera for tlie latter possessed instead a charge-elevating ef- fect. Since the dysentery bacilli possessed a low charge (3 to 5 millivolts) while the other bacteria mentioned possessed high charges (23 to 40 millivolts), Shibley concluded that the effect of specific immune serum on its respective antigen is to bring the charge to a common potential level (8-14 millivolts). This is due to the fact that while the antigens differ as to charge and chemical constitution, when they unite with antibody they are alike in that they are coated or filmed with antibody globulin. Since the latter becomes denatured when specifically adsorbed, all of the particles of filmed antigen behave as particles of denatured protein. c.cc ±zS, --CJ Fig. 12. — Measurement of cohesive force of bacteria. T, Torsion balance to measure pull necessary to disrupt films (description by Northrop and De Kruif). C. G., Cover glass. F, Film of bacteria. S, Slide. G-, Glass container. Techniques. — Measuring Cohesive Force. — In order to obtain a rough measure of the cohesive force (stickiness) of bacteria, North- rop and DeKruif introduced an interesting technique. This is de- scribed by Northrop as follows : "It occurred to the writer that it might be possible to measure this sticking or cohesive force by de- termining the force required to separate two films of the suspen- sion. This turned out to be the case. The measurement was made by coating two pieces of glass with a thick smear of the suspension. The glass was warmed slightly in order to cause tlie particles to adhere to it, and the two films were allowed to rest together in the MECHANLSftl OF ANTIGICN-ANTIBODY REACTIONS 217 solution to be .studied. The force required to tear the films apart was then determined by a torsion balance." This is illustrated in Fig. 12. Extent of Surface Coating. — According to Northrop and DeKruif, it is not necessary for the entire surface of the cells to be coated with agglutinin for clumping to occur. They believe that if an eighth of the area is coated, it will suffice. Shibley found that a])parently tliis is more than is necessary provided tlie other factors arc satisfactory. Studies of Bactcrkil Surfaces. — Mudd (1924-1927) and Freund (1925) have made extensive studies attempting to determine the nature of the surface of various types of cells before and after ad- sorption of antibody. The former used the "interfacial tension method" in his investigation and obtained rather significant re- sults. While Mudd and Mudd were primarily interested in the Fig. 13. — Interfacial tension technique of Mudd. (After Mudd and Mudd, J. Exper. Med. 43: 148, 1926.) phenomenon of opsonification and in phagocytosis, their results definitely support the work of Shibley, Northrop and DeKruif and others in regard to Bordet's phase one Avhere the cells become coated with antibody globulin. Interfacial Tension Technique. — This "interfacial tension" technique is described by Mudd and Mudd as follows : "A drop of oil is drawn across a carefully cleaned slide. A small drop of dilute blood cell suspeiLsion is drawn along the slide a short distance from and at right angles to the streak of oil. One end of a clean oblong cover slip is touclied to the slide and to the oil so that the oil wets the under surface of the cover slip along one end. The other end of the cover slip is now lowered onto the slide, thus spreading the oil into a film under one side of the slip and the blood into a film adjoining it. In the best preparations the blood film does not cover quite all of the area under its end of the slip." This they also illustrate (Fig. 13). 218 IMMUNOLOGY Behavior of Cells, — By means of this technique they investi- gated first the behavior of red cells before and after they had adsorbed hemagglutinin. Before adsorption they readily passed from the aqueous phase into the oil phase indicating that their sur- faces Avere richly endowed with material readily miscible with oil. Referring to Harkins' work (see Fig. 26) (Appendix, p. 000) this would be interpreted as indicating that the nonpolar groups of the molecules were out. Change in Surface Molecular Orientation. — After adsorp- tion of agglutinin the surface appeared to contain predominantly polar groups, since the cells were no longer miscible with oil but were readily miscible wnth the aqueous phase (Ringer's solution). Using the same technique they also studied acid-fast bacteria and observed the same phenomenon. Both Freund and ]\Iudd and Mudd regard the surface of the unsensitized acid-fast bacteria as con- taining both protein and lipoid. From their work they conclude that the acquisition of polar groups results from adsorbed antibody globulin. This specific adsor])tion of antibody globulin constitutes the first phase of Bordet's hypothesis. Antigenic Components and Antibodies. — In Chapters XI and XIX the various possible antigenic components of the bacterial cell were discussed. The student should bear in mind that accord- ing to present concepts as many different antibodies may be pro- duced as a result of infection or immunization as there are true antigenic components or hapten-protein combinations. In the case of motile bacteria there are flagellar (H) as well as somatic (0) antigens. The rough colony type of bacteria con- lain 0 antigens (protein) which stimulate the production of cor- responding antibodies and the smooth colony types contain carbo- liydrate-protein complexes that stimulate antibodies that react citlier M'ith the combination or with the hapten portion in vitro. The latter, however, is frequently not a true antigen by itself. Nonprotein Carriers of Haptens. — Recently Zozaya (1932) has Referring to Harkins' work (see Fig. 26) (Appendix) this would be interpreted as indicating that the nonpolar groups of tlie He considers that the physical attachment of the hapten to a col- loid makes it an antigen. This revolutionary concept is in line with Zinsser's suggestion that antibody formation is largely a cell surface phenomenon and depends among other things upon MECHANISM OF ANTIGEN-ANTIBODY REACTIONS 219 the antigen's being in the colloidal state. In the lihenomenon of adsorption of antibodies by the cell which occurs in Bordet's phase one, the student should remember that the immune serum will con- tain antibodies for eacli Iriio antigenic constituent of the cell. Summary Some of the important points about the mechanism of cellular agglutination by immune serum may be summarized as follows: 1. There are two phases to the reaction as suggested by Bordet. 2. The antibodies are ]:)resent in the immune serum intimately associated with the globulin fraction. 3. During the first phase the cells specifically adsorb the anti- ])odies corresponding to the antigens present as a surface film com- ]iosed of antibody globulin. This readily occurs at 0° 0. and also at 37° C. to 56° C, and also thi'ough a fairly wide range of pH values. It is probably of molecular lliickness since i1 is not de- tectable by microscopic examination. 4. When the anti])ody glo])ulin is adsorbed, it seems to ])ccome denatured and causes tlie cells that have adsorbed it to behave as particles of denatured serum globulin with an increase of the co- hesive forces under proper conditions. 5. The presence of a proper concentration of sodium chloride leads to a change in membrane potential to within a critical value of 13 to 15 millivolts Avithout a reduction of the cohesive forces. 6. This alteration of membrane potential to within the critical value in combination with the increased cohesive force is respon- sible for the clumping and sticking together of the cells. 7. Reference is made to the newer concepts of cell sensitization and agglutination offered by Heidelberger and Kendall. References Bechold, H.: Die Ausflockung von Suspensionen bzw. Kolloiden und die Bakterienagglutination, Ztschr. f. Physick. Chem. 48: 385, 1904. Bordet, J.: 1895. Cited by Bordet, Studies iu Immunity, New York, 1909, John Wiley & Sons, Inc. Bordet, J.: Le Mecanisme de 1 'agglutination, Ann. Inst. Pasteur 10: 193, 1896. Ibid. 13: 225, 1899. Also Studies in Immunity, translated by Gay, New York, 1909, John Wiley & Sons, Inc., pp." 142-164. Buchanan, E. E.: Agglutination, J, Bact. 4: 73, 1919. Coulter, C. B.: The Isoelectric Point of Eed Blood Cells and Its Relation to Agglutination, J. C4en. Physiol. 3: 309, 1921, 220 IMMUNOLOGY Craw, J. A.: On the Mechanism of Agglutination, J. Hyg. 5: 113, 1905. Dineur: (1898) See Bordet's Studies in Immunity, New York, 1909, John Wiley & Sons, Inc., p. 147. Duclaux, E. : Traite de Microbiologic 2: 263, 706, 1899, Masson et Cie Editors, Libraires de 1 'Academic de Medicine, Paris. Eagle, H.: Some Effects of Formaldehyde on Horse Antipneumococcus Serum and Diphtheria Antitoxin, and Their Significance for the Theory of Antigen- Antibody Aggregation, J. Exper. Med. 67: 495, 1938. " Ehrlich, P.: Studies in Immunity, Translated by Bolduan, New York, 1910, John Wiley & Sons, Inc., p. 573. Eisenberg, B., and Volk, E.: Unt«rsuchungen iiber die Agglutination, Ztschr. f. Hyg. u. Infect. 40: 155, 1902. Preund, J.: The Agglutination of Tubercle Bacilli, Am. Eev. Tuberc. 12: 124, 1925. CJ ruber, IVF., and Durham, H. E.r Elne neue Methode zur raschen Erkennung des Oholeravibrio und des T_\7)husbacillus, ^fiinchen. med. Wchnschr. 43: 285, 1896. Gruber, M.r Cited in Bordet's Studies in Immunity, New York, 1909, John Wiley & Son, Inc., p. 14.'5. Griinbaum, A. S.: Preliminary Note on the U.se of the Agglutinative Action of Human Serum for the Diagnosis of Enteric Fever, Lancet 2: 806, 1896. Heidelberger, M., and Kendall, F. E.r The Precipitin Reaction Between Type III Pneumococcus Poly.saccharide and Homologous Antibody. II. Conditions for Quantitative Precipitation of Antibody in Horse Sera, J. Exper. Med. 61: 539, 1935. Heidelberger, M., and Kendall, F. E.: The Precipitin Reaction Between Type III Pneumococcus Polysaccharide and Homologous Antibody. III. A Quantitative Study and a Theory of the Reaction Mechanism, J. Exper. Med. 61: 563, 1935. Ibid. 65: 487, 647, 1937. Hooker, S. B., and Boyd, W. C: The Nonspecificity of the Flocculation Phase of Serologic Aggregation, J. Immunol. 33: 337, 1937. Kraus, E.: (1897) Ueber specifische Reactionen in Keimfreien Filtraten aus Cholera, Typhus, and Pestbouillononculturen, erzengt durch homologues serum, Wien. klin. Wchnschr. 10: 736, 1897. Landsteiner, K.. and Chase, M. W.: Immunochemistry, Am. Rev. Biochem. 8: 579, 1939. Loeb, J.: The Influence of Electrolytes on the Cataphoretic Charge of Colloidal Particles and the Stability of Their Suspensions. I. Ex- periments with Collodion Particles, J. Gen. Physiol. 5: 109, 1923. The Influence of the Chemical Nature of Solid Particles on Their Cataphoretic, P. D. in Aqueous Solutions, Ibid. 6: 215, 1924. Mudd, S., and Mudd, E. B. H.: The Penetration of Bacteria Through Capillary Spaces. IV. A Kinetic M«chani.sm in Interfaces, J. Exper, Med, 40: 633, 1924. Certain Interfacial Tension Relations and the Behavior of Bacteria in Films, Ibid., p. 647. The Surface Composition of the Tubercle Bacillus and Other Acid-Fast Bacteria, Ibid. 46: 167, 1927. On the Mechanism of the Serum Sensitization of Acid- Fast Bacteria, Ibid. 46: 173, 1927. Neisser, M., and Friedemann, U. : Studien iiber Ausflockungsercheinungen, Miinchen. med. Wchnschr. 51: 465, 1904. Northrop, J. H.: The Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, pp. 782-801. MECHANISM OF ANTIGEN-ANTIBODY REACTIONS 221 Nortliroj), .(. II., and DcKruif, P. H.: Stability of Bacterial Suspension; Agglutination of Bacillus of Rabbit Septicemia and of Bacillus Ty- phosus by Electrolytes, J. Gen. Physiol. 4: 639, 1922. Stability of Bacterial Suspension; Agglutination in Presence of Proteins, Normal Serum, and Immune Serum, Ibid. 4: 655, 1922. The Stability of Bac- terial Suspensions. IV. The Combination of Antigen and Antibody at Different Hydrogen Ion Concentrations, Ibid. 5: 127, 1923. The Stability of Bacterial Suspensions. V. The Eemoval of Antibody from Sensitized Organisms, Ibid. 5: 139, 1923. The Stability of Bacterial Suspensions. VI. The Influence of the Concentration of the Suspension on the Concentration of Salt Required to Cause Complete Agglutination, Ibid. 5: 605, 1923. Pauli, Wolfgang: Physical Chemistry in Medicine, New York, 1907, John Wiley & Sons, Inc. Authorized translation by Martin H. Fischer. Powis, F. : Die Beziehung zwischen der Bestandigkeit einer Oelemulsion und der Potentialdifferenz an der ol-wassergrenzflache und die Ko- agulation kolloider Suspensionen, Ztschr. f. Physik. Chem, 89: 186, 1915. Shibley, G. S.: Studies in Agglutination. I. The Agglutination of Strepto- cocci, J. Exper. Med. 39: 245, 1924. Studies in Agglutination. IT. The Relationship of Reduction of Electrical Charge to Specific Bac- terial Agglutination, Ibid. 40: 453. Studies in Agglutination. III. On the Mechanism of the Agglutination of Bacteria bv Specific Ag- glutinating Serum, Ibid. 44: 667, 1926. Tchistovitch, T. : Ictudes sur 1 'immunisation contre le serum d'anguilles, Ann. Inst. Pasteur 13: 406-425, 1899. Topley, W. W. C, and Wilson, G. S.: The Principles of Bacteriology and "immunity, vol. 1, New York, 1929, Wm. Wood. & Co., pp. 133-187. Tulloch. W. J.: The Mechanism of Agglutination of Bacteria bv Specific Sera, Biochem. J. 8: 293. 1914. Wells, H. G.: The Chemical Aspects of Immunity, New York, 1929, Chem- ical Catalog Co. Wida], F.: Serodiagnostic de la ficvre typhoide. Bull, et mem. Soc. mcd. d. hop. de Paris 6: 26, 1896. Zinsser, H.: Infection and Resistance, ed. 4, New York, 1931, The Mac- millan Co., pp. 214-228. Zozava, J.: Carbohvdrates Adsorbed on Colloids as Antigens, J. Exper. Med. 55: 325, 1932. CHAPTER XIII PRECIPITINS Introduction. — Shortly after Gruber and Durham (1896) and Widal (1896) had demonstrated the relative specificity of bac- terial agglutinins, Kraus (1897) discovered bacterial precipitins and found them correspondingly specific. He mixed antieholera inunune serum with the clear sterile filtrate from a broth culture of the cholera vibrio and incubated the mixture at 37° C. After a short time there appeared a cloudiness followed by flocculation in the tubes. The precipitate settled out, after standing over- night, leaving a clear supernatant fluid. He likewise found that when he mixed a sterile filtrate from a broth culture of Eherthella typJiosa with antityphoid immune scrum a precipitate developed. When antityphoid immune serum w^as mixed with the filtrate from vibrio cholera or when antieholera serum was mixed with the filtrate from E. typhosa no precipitate developed. Thus Kraus showed that the reaction is specific. Kraus' work was confirmed by Nicolle (1898) and extended by Tchistovitch (1899) and Bordet (1899). The former immu- nized animals with horse and eel serum and noted the develop- ment of specific precipitins in their sera while the latter produced precipitins for cow's milk. The following year Myers (1900) pro- duced precipitins for crystalline egg albumen. Kraus named the antigen and antibody involved in this type of reaction precipitiTwgen and precipitin respectively. This ter- minology is similar to that used for tlie antigen and antibody responsible for cellular agglutination where the former is called the agglutinogen and the latter agglutinin (antibody). According to Uhlenhuth (1909), von Fish became interested in Bordet 's observation that precipitins could be produced for cow's milk and extended this by comparing the precipitins for cow's milk and human milk. He concluded that these proteins could be differentiated by the precipitin test. This work was confirmed by Morgenroth (1900) and Wassermann ajid Schiitze. According to Hektoen and Welker (1924), Leblanc (1901), working with 222 PRECIPITINS 223 beef serum, found that the eui>iobulin, pseudoglobulin and albumin fractions could not only be differentiated from each other by the precipitin test but could also be shown to differ from beef hemo- globin. Schur (1904) says that Wa.ssermann (1900) deserves the credit for first applying the precipitin test to tlio differentiation of liuman and animal proteins. Uldenliuth (1901) is usually creditod with the introduction of the tcsl as a medicolegal ]U'ocedui'e. Early Investig-ations. — Group Reactions.— While Kraus, Was- sermann and even Uhlenhutli at first regarded the test as strictly specific Nuttall (1901) early recognized group reactions. Nuttall's observation was immediately confirmed by Uhlenhuth and others and due allowance made for this in the technical procedures worked out by them. Uhlenhuth 's conclusion that the test is specific, pro- vided the antigen dilution used is at least 1:1,000, has been re- peatedly confirmed and is at tlie present time an integral part of precipitin technique used in medicolegal cases. Closely related species are differentiated by absorption and precipitin tests. Value in Tracing Biological Relationship. — Shortly after Wassermann's (1900) observation that the blood of different species could be differentiated by the precipitin test, Nuttall (1901, 1902, 1904) investigated the biological relationship within the animal kingdom by means of the precipitin reaction and also recom- mended that it be used in medicolegal cases. In this work he employed not only the routine qualitative procedure but also a new quantitative technique which he had devised. It is obvious from a perusal of the early monographs of Nuttall (1904), Kraus (1904), Uhlenhuth (1909) that extensive investiga- tions were conducted relative to precipitins, their formation, nature, specificity, effect of physical and chemical agents upon them, the simultaneous existence of antigen and antibody in sera, the mechanism of the reaction, the importance of precipitins in elucidating various chemical, biological and medical problems, and the source and nature of the precipitates formed in the reac- tion. This is very evident fi'om the following summary of conclu- sions which Nuttall (1904) draws from a survey of the literature and from his ovm experiments. 224 IMMUNOLOGY Nuttall's Conclusions. — He concludes that the rabbit is per- haps the most satisfactory animal for use in obtaining potent immune serum. He used the intraperitoneal method for antigen inoculation and, beginning with a small dose, administered a series of 5 or 6 graded doses, gradually increasing the amounts at three to six day intervals and bled for antibody titration seven to twelve days after the la.st injection of antigen. He noted a fluctuation in precipitin content during immunization and observed that precipitin persisted in the blood stream in apparently undiminished titer for about one month. (The titer frequently drops much eariier.) He confirmed the observation of Tchisto- vltcli (1899) that precipitins disappear from the blood stream if injections are administered over too long a period. In his opinion antigen and antibody may both be present in the blood without precipitation occurring. (ThLs has since been confirmed by Weil and others and a fairly logical explanation has been offered by Dean.) In regard to measuring the strength of an immune serum, he suggests that the titer may be expressed either in terms of the volume of precipitate formed or "by giving the highest dilu- tion of blood (antigen) with which it reacts, the quantities of interacting substances being stated." In his opinion quantitative determinations are best recorded in terms of the volume of pre- cipitate formed under standard conditions and procedure. He thinks there is evidence indicating the exi.stence of iso-precipi- tins, auto-precipitins and anti-precipitins being artificially formed in the bodies of treated animals. In regard to normal precipitins he says certain sera may contain them but they are not specific. He considers that precipitins are antibodies intimately asso- ciated with serum globulin. They are more resistant to heat than complement and do not require the latter for their activity. Bac- terio-precipitins are inactivated at 58-60° C, while precipitins for animal proteins are inactivated at 68-70° C. As the source of the precipitate he reasons that since the amount formed is fre- quently out of all proportion to the amount of antigen present, the precipitate very likely comes from the immune serum. Subse- quent work has definitely established the truth of Nuttall's as- sumption. In his conclusions attention is also called to the observation that the presence of heated precipitin serum (pre- cipitoid) interferes with the reaction between fresh, unheated PRECIPITINS 225 precipitin and antioen (Mueller, 1902, Eisenberg, 1902, Niittall 1904). lie slates tliat both antigen and antibody resist desic- cation but tliat antibody is, in general, more unstable than antigen. In Nuttall's opinion both antigen and antibody are probably destroyed by tryptie digestion, although he noted that putrefaction of antigen or immune serum does not prevent the formation of a specific precipitate when they are mixed. He found that traces of acids or alkalies reduce the amount of precipitate formed but that, within limits, the concentration of sodium chloride is without effect. He also confirmed Kraus's observation tliat the ]irccipitate is soluble in an excess of precipitable substance. In regard to the effect of temperature of incubation upon the rate of the reaction he states that low temperatures retard and high temperatures accelerate it. Temperatures between 5° and 37° C. do not seem to influence the quantity of precipitate formed. He used room temperatui'e incubation in his biological investiga- tion. Most of these conclusions have been repeatedly confirmed by others and fairly plausible explanations offered for many of the phenomena. Extent of Early Use of Precipitin Test. — It is interesting to note that the precipitin test was used quite early for the identi- fication of bacteria, the diagnosis of disease, the study of biologi- cal relationships, the identification of human blood and of semen in cases of murder and attempted rape, respectively, and in the detection of food adulteration. It was also used in many im- munological studies such as those of Obermayer and Pick (1906), Landsteiner (1903, 1924, 1928) and others on antigenic specificity, the persistence of antigens within the circulation, the mechanism of antigen-antibody reactions as well as many other problems. Subsequent work has dealt largelj^ with the problems raised by these early investigators. Much of it is discussed in the chapters on antigens and specificity. The remainder of this chapter will be devoted largely to a discussion of technique, to the medicolegal aspects of the reaction and some of the factors influencing the formation of precipitates. Preparation and Prerequisites of a Satisfactory Immune Serum, — l-'or medicolegal work or for use in research it is quite 226 IMMUNOLOGY necessary to have a precipitating serum that is clear, of high potency, and that is specific. Satisfactory sera are best prepared from rabbits or roosters. Inoculation of Anim.\i.s. — It seems that any of several methods of immunization yields satisfactory results. Since different indi- viduals of the same species vary in their capacities to produce antibody, one should inoculate several rabbits in order to be certain of obtaining a satisfactory antiserum regardless oC the particular technique adopted. Inoculation of Animals According to Kolmer. — Kolmer recommends that 0.5 c.c. of the antigen be injected intravenously eveiy day for three weeks and a trial titration made ten days after the last injection. If the titer is low he suggests that 5.0 c.c. be given intraperitoneally and twenty-four hours later another series of intravenous injections be started. Inoculation of Animals According to Dean. — Dean (1931) recommends that two series of injections be given. Each series con- sists of six to eight injections, each of 2 c.c. of the antigen at five day intervals. The first few injections are given intravenously, and the remainder intraperitoneally. After a re.st of three to six months the second series is given. Methods Used in This Laboratory. — Various methods are used in this laboratory. One is essentially that recommended by Kol- mer, another is quite similar to the procedure followed by Nuttall. One or two c.c. of the antigen (serum undiluted, egg white 50 per cent or crystalline egg albumen 1 to 5 per cent) is injected into the marginal ear vein of a rabbit. This is followed three to six days later by either another intravenous injection of the same amount or by an intraperitoneal injection of 4 or 5 c.c. of the antigen. A third, fourth and frequently a fifth injection is given intraperitoneally at 3 to 6 day intervals and the animals bled for antibody titration 7 to 10 days after the last injection. Normal (0.85 per cent) saline is used as a diluent. Where a rooster is used for precipitin production, one injection of 20 c.c. of whole blood or other antigenic material is given intra- peritoneally. After ten or twelve days blood is removed from the heart and titrated for precipitin content. Hektoen recommends PRECIPITINS 227 that 1.8 per cent saline be used in preparing dilutions of rooster serum to avoid nonspecific reactions. When to Bleed Anbials. — In collecting blood from an im- munized animal to be used in precipitin work, it is better to bleed the animal just before feeding or after moderate fasting to avoid cloudy or opalescent sera. Undoubtedly much of the difficulty reported in the early literature was due to failure to observe this precaution. Obtaining Immune Serum. — After it is determined that the precipitin content is sufficiently high to react by the ring technique to be described later, wdth dilutions of antigen of 1 :1,000 or higher, the animals are bled to death u]ider aseptic conditions. It is desirable to obtain the serum in a sterile condition, but if con- tamination occurs the antisenun may be sterilized by filtration, placed in sterile containers, sealed and kept in the refrigerator. Preservatives should not he added, the serum should not be heated or frozen. All cloudy or opalescent sera should be discarded. Titration After Uhlexhutii. — The next step would be the titra- tion of the antiserum. According to Uhlenhuth (1909, p. 815) he prepared three dilutions of antigen 1 :1,000, 1 :10,000 and 1:20,000 using physiological (0.85 per cent) saline as a diluent. He then placed 4 small, clean test tubes in a rack and with a sterile pipette added one cubic centimeter of sterile saline to tube IV and one cubic <^entimeter of the 1 :20,000 dilution of antigen to tube III, a corresponding amount of the 1 :10,000 dilution to tube II and of the 1 :1,000 to tube I. He next took a sterile one cubic centimeter pipette graduated to 1/100 of a cubic centimeter and added 1/10 of a cubic centimeter of the clear immune serum to each tube. The tubes were not shaken and were incubated at room temperature and observed for five minutes. Tube IV re- mained clear, but he says that a good serum should give a positive reaction in tube I within two minutes and that within 3 to 5 minutes tubes II and III may become cloudy and a precipitate form and settle out. The titer is recorded as the highest dilution giving a definite reaction. Technique of Nuttall. — Nuttall (1904) in his routine qualita- tive work employed a similar technique. As a rule he used one cubic centimeter of a 1 :100 to 1 :200 dilution of antigen and to each 228 IMMUNOLOGY tube added 0.05 c.c. of immune serum. The tubes were incubated at room temperature, apparently witliout shaking, and observed closely during the first tive minutes and at intervals for 24 hours. Adequate controls were also included. Quantitative Technique of Nuttall.— In his quantitative technique he employed as a standard, representing 100 per cent precipitate, the amount formed when 0.1 c.c. of antiserum is added to 0.5 c.c. of a 1 :100 or 1 :200 dilution of homologous antigen. He considered it necessary that the ratio of antiserum to antigen be 20 :1 to 200 :1 or more. After the antiserum is added the tubes are shaken. Time of Incubation. — The tubes are then incubated for twenty- four hours, the supernatant fluid decanted, and the precipitate drawn up into capillary tubes 12 cm. long and having a lumen of 1 mm. The dry end of each capillary tube is sealed and the tubes are allowed to stand vertically for 24, 48 and 72 hours and the volumes (height) are measured. The Ring Test.— He says that Ascoli (1903) suggested stratify- ing immune serum under varying dilutions of antigen and deter- mining the highest dilution of antigen that produces a ring of precipitate at its junction with immune serum. This is essentially the ring technique of Fornet and IMiiller (1910) and the one Avhich Hektoen (1928) seems to prefer. In this test small clean vials or test tubes having a diameter of approximately 0.5 cm. are placed in a special rack. A series of dilutions of antigen varying from 1:1,000 to 1:10,000 or 1:20,000 are prepared. By means of a capillary pipette a measured amount of saline (0.85 per cent) is put into the last vial, the same amount of the highest dilution of antigen is put into the next vial and so on until vial I receives the same amount of the 1 :1,000 dilution of antigen. With an- other capillary pipette a measured amount of clear immune serum, to be titrated, is stratified in the bottom of the saline control and a corresponding amount stratified under each antigen dilu- tion, beginning with the highest dilution and proceeding to the lowest. Instead of stratifying the immune serum under the anti- gen dilutions as recommended by Hektoen (1928) some prefer to layer the antigen dilutions over 0.1 c.c. of immune serum that has previously been placed in each vial. The tubes are incubated at room temperature and the titer is recorded as the highest PRECIPITINS 229 dilution of antigen that yields a definite ring at the junction with immune serum. Small round-bottomed fermentation vials may be used in this work. A satisfactory serum should react within two to twenty minutes at room temperature. Range of Specificity. — As soon as the titer of an immune serum, e.g., antihuman, has been determined, it is necessary to ascertain the range of its specificity. In blood work this is accom- plished by setting up a series of titrations, using dilutions of various other bloods, as, for example, Hektoen mentions fish, chicken, rabbit, guinea ]>ig, ]"at, cat, dog, swine, sheep, beef, lioi'se, goat, monkey and luunan. In the case of liigh titcrcd antihuman precipitin serum, one will find tliat except for the blood of anthropoid apes, cross reactions either will not occur or will be evi- dent in very low dilutions such as 1 :10. Ape blood may react in dilutions of 1 :100 or 1 :200, but not in high dilutions sucli as 1 :1,000, 1 :r),000 or 1 :10,000 which the homologous human blood gives. When one is working with an antibeef serum one finds that sheep l)lood (closely related species) gives reactions in fairly high dilutions ; absorption experiments, however, will differentiate clearly l)etween the two. Otlier closely related species are tlie horse and mule ; dog, wolf and fox ; domestic fowl, turkey, goose, duck and pigeon ; hare and rabbit. Practical Use of the Test. — According to Fornet and Midler (1910) and Hektoen (1928) the ring test is satisfactory for all medicolegal work. Dean (1931), however, feels that it is satis- factory for preliminary work but that one should consider the influence of optimal proportions of antigen and antibody upon the reaction in performing the final test. Adsorption and Agglutination Technique. — In Chapter XI attention is called to a paper by Cannon (1940) in which he de- scribes a more rational method of titrating precipitins. This method is delicate and sharply specific. It promises to be of value for the quantitative study of the relationship of precipitins to various types of hypersensitive reactions. Optimal Proportion of Immune Serum and Antigen. — In order that the student may ))etter appreciate some of the conditions necessary for the formation of immune precipitates preparatory to a discussion of the Ramon floeeulation technique for the titration 230 IMMUNOLOGY of toxins and antitoxins to be discussed in the next chapter, it might be well to consider briefly the question of optimal propor- tion of reagents and a few other factors. Proportion of Immune Serum and Antigen Used by Nuttall. — Nuttall (1901, 1904) realized that certain ratios of immune serum to antigen favor the formation of immune precipitate. In his work he used ratios varying from 20 :1 to 200 :1 and higher. Danysz (1902) observed the formation of a precipitate when ricin was completely neutralized by antiricin. He found that the ratio of ricin (toxin) and antiricin (antitoxin) that gave the most voluminous precipitate also was the ratio necessary for complete neutralization of toxin by the antitoxin. In 1909 Cal- mette and JNIassol obtained similar results with venom and anti- venom. They concluded that this offered a reliable method for measuring tlie antitoxin content of immune serum. While a great many have offered experimental evidence to show that the toxin- antitoxin precipitation is due to bacterial protein and its bac- terial precipitin, Bayne-Jones (1928) in an excellent review of the literature as well as his own work concludes that the reaction is between the toxin and antitoxin and not between bacterial protein and bacterial precipitin. Importance of Optbial Proportions of Antigen and Antibody. — In view of the importance which zoning plays in both the toxin- antitoxin and precipitin reactions it would seem desirable to re- view Dean's (1931) extensive investigations and also those of others on the influence of optimal proportions of antigen and antibody in the latter reaction. Dean found that when he mixed e.g. a 1:5 dilution of mitihorse serum with varying dilutions of antigen (horse serum), the largest amount of precipitate occurred with a 1:8 dilution of antigen. When he used a 1:10 dilution of antiserum, the largest precipitate occurred in the tube to which he had added a 1 :16 dilution of antigen, etc. He obtained similar results with antit>T)hoid serum and typhoid filtrates. He also noted that when either antiserum or antigen was present in relative excess the reaction was delayed. He says that the importance of optimal proportions has been confirmed by Opie (1923), Parker (1923) and Morgan (1923). The latter worked with the soluble specific substance of the pneumococcus and the homologous type of pneumococcus immune serum. More recently PRECIPITINS 231 Dean and Webb (1926) have developed a method for the quan- titative determination of either antigen or antibody depending upon the optimal ratio of one to the other within the mixture. This is called the Optivwl Proportion Method. Coarse Test of Dean and Webb. — In this test they first de- termine the titer i-oughly by preparing four dilutions of anti- serum, i.e., 1 :5, 1 :10, 1 :20, and 1 :40 and ten sets of dilutions of antigen ranging from 1:10 to 1:10,000. Dean and Webb (1926) state that the volume of horse serum dilution in each set is 1 c.c, and the quantity of horse serum is halved progressively in each tube of the series. To each tube of set A they add 1 c.c. of un- diluted antiserum, to each tu])e of set B, 1 c.c. of 1 to 5 dilution of antiserum. They include as controls one set of antigen and also tubes containing only antiserum and saline, the two latter to serve as antiserum controls. Optbial Proportions Fine Test. — The object of the experiment is to determine which tube in each set containing varying mix- tures of antigen with antibody shows the most rapid formation of a precipitate In this way they determine the mixture in which the speed of the reaction is greatest. For the experiment cited they found that the optimal ratio of antigen to antiserum was between 1 :16 and 1 :32. With this information they proceeded to a finer titration. TliLs is described by Dean and Webb (1926) very much as follows : They prepared an initial 1 :100 dilution of horse serum (antigen) using a 100 c.c. volumetric flask. Ten tubes were placed in a rack and numbered for convenience 10 to 1 from left to right. A dilution suitable for tlie test was prepared from the initial 1 in 100 dilution: in tliis case 1 in 400 was used. Of this 1 in 400 dilution, quantities varying from 1 c.c. to 0.1 c.c. were jiipetted into each of the ten tubes. The difference betwceji eacli tube was therefore 0.00025 c.c. of horse scrum. The volume in each tube was made up to 1 c.c. l)y adding an appropriate volume of saline solution. One cubic centimeter of a 1 in 40 dilution of anti- serum was added to every tube. Thus the volume of antiserum in each tube was 0.025 c.c. Controls were also included. The rack containing the tubes in a single line was incubated at room tem- perature and observed constantly. The progress of precipitation was watched against a dark background properly illuminated by a shielded electric light. The formation of precipitate was observed 232 IMMUNOLOGY with the naked eye and with a reading glass. Tliere appeared at once in tubes 7 to 2 a cloudy opalescence; after five minutes the opalescence was obviously more marked in the center tubes of the rack and tubes 6 and 5 were leading. After twenty-five minutes tubes 6, 5, and 4 were ahead of the others. Tube Five Contains Optbial Proportions. — In tube 5 discrete particles formed earlier than in any other tube; within thirty minutes the particles of tube 5 were larger than those of tube 6 or tube 4. Particles were present in all the tubes after forty-five minutes. The largest particles were in tube 5, next largest in tube 4, then in tubes 6 and 3. Tube 5, the first tube to show distinct particles, contained 0.00125 c.c. of horse serum. The proportion of antigen to antibody in this tube was 1 to 20, i.e., as 0.00125 is to 0.025. In general, they found that strong or quickly acting anti- sera could be used in a dilution of 1 in 40. Unit Suggested by Dean and Webb. — In regard to applying this test to a quantitative estimation of antibody, Dean and Webb (Dean, 1931) reason that it is possible to express the antibody content in units if the ratio figure depends on the antibody con- tent of the serum. To do this they assume that a unit of anti- body is contained in the volume of antiserum which forms distinct particles most rapidly with 0.00001 c.c. of normal horse serum. To illu.strate the significance of the results, they state that if the optimal antigen-antiserum ratio is found to be 1 :200 it indicates that 0.00001 c.c. of horse serum would react with 0.002 c.c. of anti- serum (200 times its volume). Therefore 0.002 c.c. of antiserum contains one unit of antibody and one cubic centimeter Avould contain 500 units of the latter. (Dean, 1931, p. 430.) From this it is obvious that a low antigen-antiserum ratio figure indicates that the antiserum is rich in antil^ody while a high figure indicates the reverse. Precipitins Used to Estimate Haptens and Proteins. — ^Heidel- berger (1933) has reviewed tlie recent literature on precipitins and calls attention to the use of methods, based upon Dean and Webb's optimal proportions procedure, in the estimation of pro- teins and also specific polysaccharides. Reason for Coexistence of Antigen and Antibody in Blood. — Dean (1931) suggests that perhaps the reason that antigen and PRECIPITINS 233 antibody, at times, eoexist in the blood without the formation of a precipitate is the absence of optimal ratio of concentrations of each. Nature of Precipitate. — It is generally conceded (Dean, 1931, Zinsser, 1931) that the major portion of the precipitate is made np of serum globulin from the immune serum. Effect of pH on the Reaction.^ — The etfect of pH on the forma- tion of immune precipitates has been investigated by Hirsch (1923). Using sheep serum and antisheep serum he found that precipitation occurs over a wide range, i.e., pH 6 to 9.4 with the maximum precipitation occurring near the acid end of the range. Mixtures having an acidity greater than pH 6 showed nonspecific precipitation, while precipitation was inhibited when the reac- tion was more alkaline than pH 9. Neutralization of the mix- tures restored the ability to give specific precipitates. Effect of Salts on the Reaction. — The effect of varying the con- centration of sodium chloride used in the diluent was studied by Nuttall (1904), Dean and Webb (1926), Hektoen (1928), Baier (1933) and others. Dean and Webb found that the optimal pro- portions of antigen to antiserum did not vary when concentra- tions of sodium chloride varying from 0.02 to 1.0 per cent were used although the speed of the reaction was affected by the con- centration of the salt. In their opinion a concentration of 0.2 per cent NaCl was the most favorable. As the concentration of salt was increased, the reaction was slower. Hektoen recom- mends that where immune rooster serum is used sodium chloride concentrations of 1.8 per cent be employed to eliminate non- specific reactions. Recently Downs and Gottlieb (1932) have studied the effects of different electrolytes upon the formation of precipitates. They* found that "certain salts in molar solution inhibit the formation of a precipitate from horse serum and anti- horse rabbit serum, but others do not. All the salts studied in- hibit the formation of a precipitate from crystalline egg albumen and its antiserum when present in molar solution." They state that some additional factor besides the valence is of importance in determining precipitation. They found that the thiocyanates showed the maximum inhibitory eff'ect. In regard to the peptizing effect of the thiocyanate, they say, ' ' The thiocyanate ion can there- ♦Downs, C. M., and Gottlieb, S. : J. Infect. Dls. 51: 460, 1932. 234 IMMUNOLOGY fore be considered to be effective in peptization and ineffective in precipitation of colloids because it does not disturb the aqueous layer stabilizing the hydrophilic particle." Bancroft (1926) ex- pressed the belief, however, that hydration is not sufficient to ac- count for the Hofmeister series, but that the ions act by shifting the equilibrium aniono- the various pol^^neric forms of water. Suppression Phenomenon of Landsteiner. — Landsteiner and Van der Scheer* (1924) call attention to a phenomenon previously studied by Landsteiner: i.e., that "A single precipitin will regu- larly react with other substances if their chemical structure is sufficiently near to that of the homologous antigen." They (1931) have also shown that frequently these chemically similar substances will react with precipitin serum without the forma- tion of a visible precipitate and thus prevent homologous antigen from uniting with the precipitin. This "suppression phenome- non" has been used iDy many in immunochemical studies and is referred to in later chapters. Heidelberger and Kendall (1933) have suggested that the reason visible precipitates do not form in many instances is due to the solubility of rabbit serum globulin. (See Chapter XIX.) Haptens. — Attention has been called to the hapten factor in specificity. Type differentiation of red cells is due to the presence of specific chemical substances, nonprotein in nature, coupled with the cellular protein. Type specificity of pneumococci is due to nonprotein substances (polysaccharides) that are coupled to the pneumococcus protein. In a later chapter, attention is called to the work of Landsteiner and others who created new antigens by coupling various chemical substances to proteins. The type specific polysaccharides of the pneumococcus and the various chemical substances that are coupled either naturally or experimentally to proteins to create specific antigens have been called haptens by Landsteiner. Naturally oc- curring haptens are very important factors in antigenic specificity as will be seen from a later discussion. It is interesting to note that immune serum will react not only with the protein-hapten com- bination but also with the hapten fraction alone, with the formation of specific precipitates or the fixation of complement or the sup- pression of specific reactions. ♦Landsteiner and Van der Scheer: J. Exper. Med. 40: 91, 1924. precipitins 235 Haptens Yield Precipitates With Homologous Immune Serum. — Antibodies are not produced when the hapten fraction alone is injected but are produced as a rule when the hapten-protein complex is used for immunization. For example, Type II pneumo- coccus immune serum will agglutinate Type II pneumococci, form a specific precipitate when mixed with a solution of Type II pneu- mococci or when mixed with a protein-free solution of Type II, type specific polysaccharide. As previously mentioned in this chapter, Heidelberger and Kendall have used the quantitative precipitin test to determine the amount of type specific polysac- charide present in a solution as well as in the standardization of antisera. Reasons for Diluting Immune Serum in Agglutinin and Anti- gen IN the Older Precipitation Tests. — It will be observed from the descriptions given in this chapter of various older methods of performing the precipitin test and of using it for quantitative estimations of antigen or antibody that invariably the latter is used in relatively low dilutions, while tlie antigen is highly diluted. This may seem strange since in previous chapters, where examples of bacterial agglutination are given, it is stated that the suspension of bacteria is kept constant and various dilutions of immune serum are used to ascertain the greatest dilution (titer) giving agglutina- tion. Zinsser (1930, 1931) gives an excellent discussion of these ap- parently contradictory procedures and offers an explanation that is held by a large number of immunologists. In the first place it is quite generally believed that serological tests involving agglutina- tion, precipitation or flocculation are essentially alike so far as the mechanisms involved are concerned. While the underlying mecha- nisms are discussed in the chapters on colloids, agglutination, complement fiLxation, and flocculation, it would seem desirable to sketch briefly a few commonly held concepts. When bacterial immune serum is added to an homologous suspension of bacteria, it is thought that bacterial cells become to some extent filmed with antibody-globulin and that agglutination results from certain changes in membrane potentials and in cohesive forces, electrolytes being necessary for the reaction. It is found quite practicable to use high dilutions of the immune serum and yet have enough serum globulin per cubic centimeter to film the bacteria. It is thought 2d6 IMMUNOLOGY that a similar mechanism operates in tlie precipitin reaction. Here the antigen is a protein in colloidal solution which, because of the smallness of colloidal particles, permits of great surface exposure per unit of mass. The significance of this can best be appreciated by the following example : If a cube of solid material one centimeter on each side, i.e., 1 c.c. in volume and hence having a surface area of 6 sq. cm., is divided into 1,000,000,000,000,000 smaller cubes each being 0.0001 mm. on the side, the total surface would be increased from 6 square centimeters to 6,000 square centimeters. If, however, the original cube is divided into still smaller cubes, each having a side length corresponding to the minimum dimensions of colloidal particles, tlie total surface ex- posed would be increased from the original 6 sfjuare centimeters to 14.83 acres. Zinsser (1930) estimates that if a bacterial cell were divided into particles the size of the antigenic particles in colloid solution, the total surface exposed would be increased 10,000 times. It Avill thus be seen that in the precipitin reaction the antibody globulin is being filmed upon colloidal particles or aggregates of antigen and because of the colloidal state of the antigen, the surface to be filmed is relatively much greater than in the case of the filming of bacteria for agglutination. For this reason the antigen can be highly diluted, l)ut the antibody globulin can be diluted only moderately if it is to film the antigenic particles adequately. The method suggested by Cannon et al. reduces the surface of antigen by filming it onto collodion particles and tlnis permits of a real titration of the antibody. It is a more rational method than the ring technique. References Baier, .J. G.: Quantitative Studies on Precipitins. Reprint from Physiol. Zool. 6: 91, 1933. Bancroft: Colloid Symposium Monograph, New York, Chemical Catalogue Co. 4: 29, 1926. Barenberg, L. H., Lewis, J. M., and Messer, W. H.: Measles Prophylaxis: Comparative Results with Use of Adult Blood, Convalescent Serum, and Immune Goat Serum, J. A. M. A. 95: 4, 1930. Bayne-Jones, S. : The Titration of Toxins and Antitoxins by the Floccula- tion Method. Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 759. Bordet, J.: Studies in Immunity, Bordet and Gay, New York, 1909, John Wiley & Sons, Inc. Bordet: Cited in Kraus and Levaditi, Hand, der Tek. u. M«th., Jena, 1907, Gustav Fischer. PRECIPITINS 237 Calmette, A., and Massol, L.: Cited by Dean, 1931. Cannon, P. R., and Marshall, C. E.: An Improved Serologic Method for the Determination of the Precipitative Titers of Antisera, J. Im- munol. 38: 365, 1940. Creigil and Tulloch: Med. Research Council (London) Spec. Rept. Series, 156 (1931). Dean, H. R.: The Precipitation Reaction. A System of Bacteriology (V. 6 — Immunity), 1931, p. 424, Medical Research Council, London. Published by His Majesty's Stationery Office. Dean, H. R., and Webb, R. A.: The Influence of Optimal Proportions of Antigen and Antibody in the Serum Precipitation Reaction, J. Path. & Bact. 29: 473, 1926. The Determination of the Rate of Antibody (Precipitin) Production in Rabbit's Blood by the Method of Optimal Proportions, Ibid. 31: 89, 1928. Downs, C. M., and Gottlieb, S. : Precipitin Reaction. II. Effect of Certain Electrolytes on the Formation of Precipitates, J. Infect. Dis. 51: 460, 1932. Fornet, W., and IVrUller, M.: Praktische und theoretische Prazipitinunter- suchungen, Ztschr. f. Hyg. u. Infektionskr. 66: 215, 1910. Gruber, H., and Durham, H. E. : Eine neue Methode zur raschen Erkennung des Choleravibrio und des Typhusbacillus, Miinchen. med. Wchnschr. 43: 285, 1896. Heidelberger, M. : Immunochemistry. Annual Review of Biochemistry, Stanford University Press 2: 513, 1933. Heidelberger, M., Kendall, F. E., and Soo Hoo, C. M.: Quantitative Studies on the Precipitin Reaction. Antibody Production in Rabbits Injected With an Azoprotein, J. Exper. Med. 58: 137, 1933. Heidelberger, M., and Kendall, F. E.: A Quantitative Study of the Pre- cipitin Reaction Between Type III Pneumococcus Polysaccharide and Purified Homologous Antibody, J. Exper. Med. 50: 809, 1929. Hektoen, L.: Biologic Tests for Medicolegal Purposes, New England J, Med. 199: 120, 1928. Hektoen, L., and Welker, W. H.: Further Observations on Precipitin Re- action of Bence-Jones Protein, J. Infect. Dis. 34: 440, 1924. Hirsch, E. F.: Hydrogen-ion Studies. VII. Hydrogen-ion Concentration Range of Precipitin Reaction (Sheep Serum), J, Infect. Dis. 32: 439, 1923. Kolnier, J. A.: Infection, Immunity and Biologic Therapy, ed. 3, Phila- delphia, 1923, W. B. Saunders Co. Kraus, R. : Ueber specifische Reactionen in keimfreien Filtraten aus Cholera, Typhus, und Pestbouillonon Culturen, erzeugt durch Homologues-serum, Wien. klin Wchnschr. 10: 736, 1897. Kraus, R.r Ueber specifische Niederschlage (Prazipitine), Handbuch Path- ogenen Microorganismen, Kolle und Wassermann 4: 592, 1904. Landsteiner, K., and von Eisler: (Cited by Kraus, 1904.) Landsteiner, K., and Van der Scheer, J,: On the Specificity of Serological Reactions with Simple Chemical Compounds (Inhibition Reactions), J. Exper. Med. 54: 295, 1931. Landsteiner, K.: Cell Antigens and Individual Specificity, J. Immunol. 15: .589, 1928. Landsteiner, K., and Van der Scheer, J.: On the Specificity of Agglutinins and Precipitins, J. Exper. Med. 40: 91, 1924, Aforgan, H. J.: The Inhibition Zone in Precipitin Reactions with the Soluble Specific Substance of Pneumococcus, J. Immunol. 8: 449, 1923. Morgenroth, J. : Zur Kenntniss der Lebenzyme und ihrer Antikorper, Centralbl. f. Bakteriol. 27: 721, 1900. 238 IMMUNOLOGY Nicolle, C: Eecherclies sur la substance agglutinee, Ann. Inst. Pasteur 12: 161, 1898. Nuttall, G. H. F.: Blood Immunity and Kelationships, Cambridge Uni- versity Press, London, 1904. (Macmillan, New York, Agent.) Nuttall: (1901 & 1902) See Nuttall: Blood Immunity and Kelationships, London, 1904, Cambridge University Press. Obermayer, F., and Pick, E. P.: Ueber die chemischen Grundlagen der Arteigenschaften der Eiweisskorper, Wien. klin. Wchnsehr. 19: 327, 1906. Opie, E. L.: The Eelation of Antigen to Antibody (Precipitin) in the Cir- culating Blood, J. Immunol. 8: 55, 1923. Parker, J. T.: Zone Phenomenon in Complement Fixation with "Residue" Antigens, J. Immunol. 8: 223, 1923. Schur, H.: See Kraus, 1904, p. 630. Tchistovitch, Th.: ifitudes sur 1 'immunisation contre le serum d'anguilles, Ann. Inst. Pasteur 13: 406, esp. 413, 1899. Uhlenhuth, P., and Weidanz, O.: Teehnik und Methodik des biologischen Eiweissdifferenzierungsverfahrens (Praxipitinnathode) mit besonderer Berucksichtigung der forensischen Blut- und Fleishuntersuchung. Handbuch der Teehnik und Methodik der Immunitatsforschung, Kraus and Levaditi, J«na, 1909, Gustav Fischer, p. 721. Weil: (1916) Cited in Coca: Essentials of Immunology, Baltimore, 192.'^, Williams & Wilkins Co. Widal, F.: Serodiagnostic de la fi^vre tvphoide, Bull, et m6ra. Soe. mod. d. hop. 6: 26, 1896. Zinsser, H.: Resistance to Infectious Diseases, New York, 1931, The Macmillan Co. Zinsser, H.: Notes on the Quantitative Relations of Antigen and Anti- body in Agglutination and Precipitation Reactions, J. Immunol. 18: 483, 1930. Supplementary References Berthelsen, K, C: Studies of the Flocculation Reaction Time in the Course of Immunization and the Quantitative and Qualitative Changes of the Proteins, J. Immunol. 21: 43, 1931. Boyden, A., and Baier, J. G.: A Rapid Quantitative Precipitation Tech- nique, J. Immunol. 17: 29, 1929. Cox, H. C, and Manwaring, W. H. : Parenteral Denaturization of Foreign Proteins. VII. Test-tube Synthesis of "Hybrid" Specificities, J. Im- munol. 22: 237, 1932. Cromwell, W. H.: Quantitative Relations Between Antigen and Antibodj^ in the Precipitin Reaction, J. Infect. Dis. 37: 321, 1925. Cromwell, W. H., and Centeno, J. A.: The Reaction of the White Blood Cells to Specific Precipitates, J. Immunol. 17: 53, 1929. Downs, C. M., and Goodner, K.: Effect of Certain Substances on the Precipitin Reaction, J. Infect. Dis. 38: 240, 1926. Downs, C. M.: Antigenic Properties of Tissue Fibrinogen, J. Infect. Dis. 37; 49, 1925. Donally, H. H. : The Question of the Elimination of Foreign Protein (Egg White) in Woman's Milk, J. Immunol. 19: 15, 1930. Eagle, H.: Specific Agglutination and Precipitation. II. Velocity of the Reaction, J. Immunol. 23: 153, 1932. Felton, L. D. : Dissociation of the Specific Protein Precipitate of Anti- Pneumococcus Horse Serum and a Comparison with a Protein Isolated by Chemical Means from This Immune Serum, J. Immunol. 22: 453, 1932. PRECIPITINS 239 Francis, T., Jr.: The Identity of the Mechanism of Type-Specific Ag- glutinin and Precipitin Eeactions with Pnenmococcus, J. Exper. Med. 55: 55, 1932. Green, R. G., and Halvorson, H. O.: Surface Energj- as the Controlling Factor in Agglutination and Dispersion, J. Infect. Dis. 35: 5, 1924. Gordon, M. H.: Flocculation Test for Smallpox, Brit. Med. Res. Council Spec. Rept. Series 98 (1928). Hazen, E. L.: The Relation of the Bacterial Precipitin Reaction to the Ramon Flocculation Phenomenon, J. Immunol. 19: 393, 1930. Hektoen, L.: The Specific Precipitin Reaction of the Normal and Calarac- tous Lens, J. Infect. Dis. 31: 72, 1922. Hektoen, L.: The Precipitin Reactions of Extracts of Various Animal Parasites, J. Infect. Dis. 39: 342, 1926. Hektoen, L., Fox, H., and Schulhof, K.: Specificness of the Precipitation Reaction of Thyroglobulin, J. Infect. Dis. 40: 641, 1924. Hektoen, L., and Cole, A.: The Preparation and Precipitin Reactions of Egg Albumen and Blood Proteins of the Domestic Fowl, J. Infect. Dis. 40: 647, 1927. Hektoen, L., and Schulhof, K.: On Specific Erythroprecipitins (Hemo- globin Precipitins), J. Infect. Dis. 31: 32, 1922. Hektoen, L., and Schulhof, K.: Further Observations on Lens Precipitins; Antigenic Properties of Alpha and Beta Crystallins, J. Infect. Dis. 34: 433, 1924. Hektoen, L., and Welker, W. H.: The Precipitation Reaction of Fibrinogen, J. Infect. Dis. 40: 706, 1927. Hektoen, L., and Welker, W. H.: Precipitin Reactions of Serum Proteins, J. Infect. Dis. 35: 295, 1924. Hektoen, L., and Manly, L. S.: Specific Precipitin Reactions, J. Infect. Dis. 32: 167, 1923. Hirsch, E. F.: Hydrogen Ion Studies. VI. Hydrogen Ion Changes on Precipitation of Human Serum by Immune Serum, J. Infect. Dis. 30: 666, 1922. Hitchcock, C. H.: Classification of the Hemolytic Streptococci by the Precipitin Reaction, J. Exper. Med. 40: 445, 1924. Hitchcock, C. H.: Precipitation and Complement Fixation Reaction with Residue Antigens in the Nonhemolytic Streptococcus Group, J. Exper. Med. 40: 575, 1924. Hooker, S. B., and Boyd, W. C: A Quantitative Aspect of the Hypothetical Incorporation of Injected Antigen in Resulting Antibody, J. Im- munol. 21: 113, 193L Jacobs, J.: Serological Studies on lodizated Sera. Precipitins and Precip- itinogens, J. Immunol. 23: 361, 1932. Jacobs, J.: Serological Reactions of Azoproteins Derived From Aromatic Hydrocarbons and Diaryl Compounds, J. Exper. Med. 20: 353, 1937. Jones, L., and Fleisher, M. S.r Serum Sickness in Rabbits. II. Precipitino- gen Precipitins in Relation to Appearance of the Reaction, J. Exper. Med. 55: 79, 1932. Jones, L.: The Reaction of Cow's Milk to Blood Serum Precipitin, J. Exper. Med. 43: 451, 1926. Jungeblut, C. W.: A Specific Flocculation Reaction Occurring Between Alcoholic Extracts of Pneumococci and Antipneumococcus Sera, J. Exper. Med. 45: 227, 1927. Kiyotsuna, Sasaki: Precipitation Test for the Sexes of Fowl Blood Serum, with Special Reference to Egg Laj-ing, J. Immunol. 23: 1, 1932. Landsteiner, K., and Van der Scheer, J.: On the Influence of Acid Groups on the Serological Specificitv of Azoproteins, J. Exper. Med. 45: 1045, 1927. 240 IMMUNOLOGY Landsteiner, K. : Serological and Allergic Eeactions With Simple Chemical Compounds, New England J. Med. 216: 1199, 1936. Manwaring, W. H., Marino, H. D., Azevedo, J. L., and Torbert, H. C: Parenteral Denaturization of Foreign Proteins II, Denaturization Eate in Immune Dogs, J. Immunol. 15: 351, 1928. Nai, D.: On the Nature of Thermoprecipitins, J. Immunol. 19: 255, 1930. Perry, E. B.r Precipitin, Lvsin and Agglutinin Tests with Bile, J. Infect. Dis. 41: 21, 1927. Powell, H. M. : Precipitins and Their Applications, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 824. Eoberts, E. F.: Extirpation of the Antigenic Depot and Antibody Forma- tion, J. Immunol. 20: 291, 1931. Eosenow, E. C: The Precipitation Eeaction in the Identification of Scar- latinae Streptococcus Infection, J. Infect. Dis. 39: 141, 1926. Eothen, A., and Landsteiner, K. : Adsorption of Antibodies by Egg Albumen Films, Science 90: 65, 1939. Schilling, G. S.: The Chemical Nature of the Constituent in Fowl Serum Eesponsible for Nonspecific Eeactions, J. Immunol. 16: 439, 1929. Tuft, L., and Eamsdell, S. G.: The Antibody Eesponse in the Human Being After Injection with Normal Horse Serum, J. Exper. Med. 50: 431, 1929. Van Der Scheer, J., and Landsteiner, K. : Serological Tests With Amino Acids, J. Immunol. 29: 371, 1935. Zinsser, H.r Note on the Quantitative Eelations of Antigen and Antibody in Agglutination and Precipitation Eeactions, J. Immunol. 18: 483, 1930. Zinsser, H., Enders, J. F., and Fothergili, L. D. : Immunity, ed. 5, Eesistance to Infectious Diseases, New York, 1939, The Macmillan Co., pp. 244-255. CHAPTER XIV TOXINS AND ANTITOXINS Toxins Defined. — In earlier chapters antigens have been de- fined as colloids (thought to be invariably proteins or protein- hapten complexes) which when injected into the blood stream, body cavities or tissues of a suitable animal, stimulate the pro- duction of specific reacting substances (antibodies). Toxins are antigenic poisons (Coca, 1925). Their antigenic property sets them off from all other poisons since it is well established that neutralizing substances are never produced against cyanide, strychnine, morphine, ptomaines, endotoxins, etc., but only against true toxins. Attention has also been called to the various kinds of toxins found in Nature, all antigenically different from each other. Among these are the phytotoxins (plant toxins), ricin, robin, and abrin ; the zootoxins, such as snake venom and scorpion toxin ; and finally the bacterial toxins in which the medical stu- dent is primarily interested. True Toxins. — Only a few bacteria are known to produce anti- genic poisons or toxins. The first bacterial toxin discovered was that of C. diphtheriae. This important contribution was made by Roux and Yersin (1888, 1889). Two years later Kitasato (1891) discovered tetanus toxin. Since then, true toxins have been de- scribed for the following bacteria: B. pyocyaneus (Wassermann, 1896) ; CI. hotulinum (Kempner, 1897) ; Streptococcus scarlatinae (Moser, 1902; GabritschcAvsky, 1906; Dick and Dick, 1923; Dochez and Sherman, 1924) ; Streptococcus erysipeJatis (Birkhaug, 1925) ; B. dysentcria Shiga (Todd, 1903; Olitsky and Kligler, 1920) ; Vi- hrion septique (Robertson, 1916) ; CI. ivelchii (Bull and Pritchett, 1917) and also toxins of some strains of Staphylococcus aureus (Parker, 1924). Perhaps the student can best appreciate the important facts about these various toxins and their respective antitoxins by discussing a few of them separately and also with reference to the particular toxemic disease with Avhieh each is associated. 241 242 IMMUNOLOGY Etiological Agents of Diphtheria and Tetanus Discovered. — In 1883, Klebs recognized what we now know to be C. diphtheriae, in sections of a diphtheritic membrane. The following year (1884) Loeffler confirmed Klebs' observation and obtained the organism in pure culture. In studying its virulence for mice, rats, rabbits, guinea pigs, chickens, etc., he found that mice and rats are quite refractory to infection while rabbits and guinea pigs are exceed- ingly susceptible. He was very much impressed with the fact that when an animal dies following the local injection of the organism the organisms are not, as a rule, generally disseminated throughout the body but remain localized at the point of inocu- lation. Loeffler concluded that the disease and death of these animals was probably due to some toxic substance produced by the bacteria. In 1888, 1889, Roux and Yersin discovered that sterile filtrates of broth cultures of C. diphtheriae are exceedingly poisonous and can produce the classical symptoms, pathological changes and death in susceptible animals. Within the next two years Kitasato (1889-1891) isolated the tetanus bacillus in pure culture and discovered tetanus toxin. Behring and Kitasato (1890) immunized animals with diphtheria and tetanus toxin respectively and demonstrated specific neutral- izing substances (antitoxins) in their blood. In other words, they showed that these toxins are antigenic. They were the first in- dividuals to produce active immunity against a soluble toxin. Ac- cording to Dean (1913) Behring did the pioneer work in diphtheria antitoxin and collaborated with Kitasato in producing tetanus antitoxin. Invasive Power of C. Diphtheriae. — It is now known that C. diphtheriae possesses relatively little invasive power. In a pre- vious chapter attention has been called to recent work on the variation in invasive power of different strains of C. diphtheriae (Wells, 1932, Feierabend and Schubert, 1929, Ivanic, Dimitrijevic- Speth and Javanovic, 1923), Apparently some strains that are relatively poor toxin producers possess more invasive power than the majority of strains of C. diphtheriae. It is thought that this may account for the failure of antitoxin treatment in a few cases treated early where satisfactory results might have been expected. Effect of Toxin on Lower Animals. — It is interesting to note that the relative immunity to diphtheria toxin which Loeffler observed TOXINS AND ANTITOXINS 243 in the mouse and the rat is probably due to tissue insusceptibility. They do not possess antitoxin, and no other substance has been found that will explain their refractoriness to diphtheria toxin. According to Coca (1925) toxin circulates when injected into the blood stream of the rat without being either detoxified or combined with the tissues. Coca, Russell, and Baughman (1921) as well as Glenny and Allen (1922) have shown that exceedingly large doses are toxic. It should be remembered that it is impossible to prepare pure toxin and that the toxin injected is present in the filtrate of a broth culture that is at least seven to ten days old. This contains autolysed products of bacteria, by-products of metabolism other than toxin as well as the latter and also various ingredients of culture media. It is to be expected that large amounts of such a complex pharmacological product might pro- foundly affect many physiological mechanisms that may be in- volved in the maintenance of a refractory state to one ingredi- ent, toxin. Symptoms in Susceptible Animals. — If a susceptible animal such as a rabbit or guinea pig is given a subcutaneous injection of a fatal dose of either a virulent culture or of toxin alone, one observes a fairly definite train of symptoms. There is always an incubation period of several hours which varies with the virulence of the organism or amount of toxin and the size and age of the animal. The significance of the incubation period is unknown. It is generally assumed that this represents the time necessary for the toxin to reach and be bound by the tissues. Richet, accord- ing to Zinsser (1931) suggests that the toxin is not poisonous until acted upon by the body. The selective action of toxin has been ex- plained by assuming a difference of chemical affinity for or of solubility in the various tissues of the body. Pathology in Lower Animals. — After twelve, fifteen, or twenty hours or even several days, where the dose is small, the animals develop an inflammatory edema and necrosis at the point of in- oculation. This is destined to show definite extension. Simul- taneously with the development of the reaction at the point of inoculation, the animal also shows clinical evidence of illness. It becomes quiet, the coat is roughened, there is some tempera- ture change and weight loss as well as loss of appetite. Death may be made to occur within four or five days if the proper dose 244 IMMUNOLOGY is given. Autopsy shows hyperemia of the kidneys and adrenal glands, numerous small petechial hemorrhages scattered through- out the bod}^ and an increased amount of tluid in the serous cavities. The latter represents a serous exudate. Effect on Dogs, Sheep, and Birds.— Roux and Yersin (cited by Loeffler, 1913) studied the effect of toxin in sheep, dogs and birds as well as guinea pigs and rabbits. All were found susceptible to its action. They noted that in many cases where the dogs did not die they developed paralysis. Filtrates of 7- to 10-day-old broth cultures produced the same clinical and pathological picture as that produced by the injection of virulent organisms. Dean (1913) refers to an interesting observation of Martin (1898) that an organism may be toxicogenic and yet nonvirulent. This ob- servation indicates that to produce diphtheria the organisms must establish themselves and by the injury of tissues create conditions favorable to the absorption and distribution of toxin. Diphtheria in Man. — Diphtheria is usually classified as one of the diseases of childhood. Nursing infants rarely contract diph- theria either through lack of exposure or as a result of passive immunity obtained from the mother. Many adults are susceptible. The incubation period is usually twenty-four to forty-eight hours. While the primary focus of infection may be in a wound or on the mucous membrane of the eye or vagina, it is usually in the nose, throat or trachea. The local inflammatory response to infection is characterized by redness, edema and the outpouring of an inflammatory exudate rich in fibrin which forms a character- istic membrane. This latter, according to Mallory* (1913), "may appear white, dirty white, brownish, grayish or almost black in color." It is made up of fibrin, bacteria, leucocytes, desquamated epithelial cells and cellular debris. The breath of the patient has a peculiar odor which is almost of diagnostic importance. In laryn- geal diphtheria the patient is not only injured by the toxin but the membrane found in the trachea may mechanically obstruct breath- ing and lead to suffiocation. There is great variation in the amount of membrane formation in diphtheria. In some cases it may not be observed while in others it is quite extensive and readily noticed. ♦Mallory in The Bacteriology of Diphtheria by Nuttall and Graham-Smith, Cambridge Univer.sity Press. TOXINS AND ANTITOXINS 245 Symptoms. — Clinically, tJic patient exhibits only a moderate fever, a rapid pulse and more or less prostration. His first com- plaint may be onl}^ that of a sore throat. If a blood count is made, it usually reveals a moderate leucocytosis. In malignant cases there may be extensive enlargement of the regional lymph glands, extreme discomfort and prostration. Pathology in Man. — Mallory (1913) gives an excellent discus- sion of the pathology of diphtheria. He says that the local lesions are essentially the same in that they show "degenerative changes in the epithelial cells and underlying tissues combined with an extensive fibrinous exudate from the blood vessels." Broncho- pneumonia, myocarditis and paralysis of the soft palate are not uncommon complications of untreated diphtheria. For further information the student is referred to Mallory 's excellent dis- cussion of autopsy findings on a fairly large series of fatal cases of diphtheria. Causes of Death in Diphtheria.— Meyer and Gottlieb (1914) consider that diphtheria toxin is a specific vascular poison. The toxin also acts centrally upon the nervous system. Thus the direct injury of heart muscle, vascular system and adrenal glands combined with the effect upon the nervous system (vagus, phrenic, etc.) is thought to explain the profound drop in blood pressure and cardiac death seen in fatal cases. Source and Nature of Diphtheria Toxin. — The true bacterial "exotoxins" are generally described as secretory products of the bacterial cell. According to this conception they resemble in some respects the extracellular enzymes that are formed and liberated bj^ the cell. Dernby (1926) regards diphtheria toxin as a higher degradation product of proteolysis. Wells (1929) sug- gests that bacterial toxins may be toxic radicals attached to pro- teins. According to Eaton (1938) the theory that diphtheria toxin is formed by enzymatic degradation of proteins in the cul- ture media is becoming less tenable in the light of present re- search. He also says that there is no experimental evidence to sup- port the theory that toxins are conjugated proteins containing a physiologically active prosthetic group. Different strains of C. diphtheriae produce qualitatively the same toxin although there are definite quantitative differences even under optimum condi- tions. 246 IMMUNOLOGY Some strains of C. diphtheriae produce toxin of such potency that 0.001 c.c. will kill a 250 gram guinea pig within four or five days, while other strains produce toxins of exceedingly low potency; i.e., the corresponding minimum lethal dose is 0.10 c.c. or more. Maver (1930), working in Jordan's laboratory, carried out an extensive investigation of toxin production of C. diphtheriae in synthetic media.* She reports that "the simple mono-amino- acids, such as alanine, phenylalanine, valine and especially glycine, were more effective in stimulating growth in a synthetic medium than the more complex mono-amino-acids that were tried." She found that a modification of Braun and Hofmeier's synthetic medium containing glycine, a fourfold amount of cystine and asparagin or ammonium succinate instead of sodium asparate was quite satisfactory. One strain of C. diphtheriae grown in this medium yielded a toxin of which 0.1 c.c. constituted a minimum lethal dose and 0.0001 c.c. a skin test dose. When broth containing toxin is subjected to dialysis, the toxin dialyzes slowly but relatively more rapidly, according to Wells, than proteins are observed to dialyze. The toxin is precipitated by protein precipitants such as alcohol, ammonium sulphate, etc., which indicates that it is either a protein or closely associated with the proteins. In this connection it is interesting to note that proteolytic enzymes are said to destroy them. Toxins may be preserved for long periods of time if kept at a low temperature and protected from light. Both ultraviolet light and roentgen rays destroy them. Effect of Heat, Drouth, and Chemicals on Toxins. — When a solution of diphtheria toxin is boiled for five minutes both its toxic and antigenic properties are destroyed. The toxin is slowly reduced in potency when heated to 45° C. and more rapidly de- stroyed when exposed to temperatures of 60°, 70° and 80° C, respectively. Dried toxin withstands 100° C. but is destroyed at 150° C. Roux and Yersin (1889) found that small amounts of acid remove the toxic property and that the latter is restored when the acid is neutralized providing the neutralization is ef- fected within a certain ascertained period of time. Wellsf (1929) *Muller, J. H., and Miller, Pauline A., describe a new gelatine-hydrolysate medium that favors the production of diphtheria toxin of high potency, J. Immunol. 40: 21, 1941. tWells, H. G. : Chemical Aspects of Immunity, Reinhold Publishing Corpora- tion. Toxins and antitoxins 247 says that in this respect they "behave like peptids in which acids transform cyclic groups to open chains and alkalies restore the cyclic structure. ' ' Eaton (1938) says that toxins may be affected by physical and chemical agents in three ways: (1) they may be denatured or coagulated; (2) their toxic and biologic properties may be revers- ibly masked by such agents as soaps and lipids; (3) aldehydes, oxidizing agents and halogens may convert the toxin to toxoid. Toxoid or Anatoxin. — Pick (1908) has given an excellent re- view of the early literature relative to the effect of physical and chemical agents upon antigens including the toxins. It was recog- nized quite early that iodine, formaldehyde and certain other chemicals detoxify toxins without destroying their antigenic prop- erty. Ehrlich (1903, 1910) noted that when diphtheria toxin is allowed to age its toxic property is diminished although its antigenic property is retained. He named the deteriorated toxin thus produced toxoid. This latter term is now applied to toxin that has been detoxified by formaldehyde or other chemicals that do not destroy its antigenic property. Many unsuccessful attempts were made to prepare a satisfactory diphtheria toxoid for use in immunization before Ramon (1923, 1925) finally succeeded. He suggested that toxin detoxified by adding formaldehyde and in- cubating for a sufficient length of time at 37° C. be called "anatoxin." Since the term "anatoxin" might be confused with "antitoxin" his suggestion has not been adopted. To avoid any confusion the term "toxoid" is now generally used to signify a detoxified toxin intended for use in immunization. The observa- tion that toxoid is antigenic but nontoxic led Ehrlich to conclude that the toxic and antigenic fractions of the toxin molecule are not identical. His conclusions are borne out by the more recent work on toxoids formed by adding known chemical agents. An excellent discussion of the mechanism of detoxification of diphtheria toxin by formaldehyde is given by Eaton (1937, 1938). Method of Measuring Toxin and Antitoxin.— Ehrlich (1903, 1910) and his colleagues carried out an extensive investigation 248 IMMUNOLOGY of toxins and antitoxins. He devised methods for the measure- ment of each and formulated a theory to explain the phenomenon of toxin neutralization by antitoxin. Ehrlich's original method of determining the units of toxin and antitoxin are discussed briefly in Chapter XI. In 1922 Bamon introduced a flocculation metJiod for the titration of toxins and antitoxins. Since then, many papers have appeared in which both the mechanism of the reaction and the significance of the results are discussed. Two of the most important publica- tions bearing upon the subject are those of Bayne- Jones (1924, 1928). He says that Danysz (1902) observed the formation of a flocculent precipitate in neutral mixtures of ricin and antiricin. Similar results were obtained by Calmette and Massol (1909) with mixtures of venoms and antivenoms. Nicolle, Cesari and Debains (1920) stratified antitoxin over toxin-gelatin mixtures and noted the formation of a precipitate in certain tubes. Ramon (1922) added a uniform amount of toxin and diminishing amounts of antitoxin to a series of tubes and noted that a flocculent pre- cipitate formed first in the tube containing equivalent propor- tions of toxin and antitoxin. This tube he called the ''indicator tube." If the strength of the antitoxin, i.e., units per cubic centimeter, is known, one can readily calculate the amount of toxin per unit of antitoxin in the indicator tube. The amount of toxin which brings about the most rapid formation of a floc- culent preeipitate when mixed with one unit of antitoxin is called by Ramon the Lf or flocculating dose of toxin. A flocculent pre- cipitate occurs later in other tubes containing either an excess or an insufficient amount of antitoxin but the tube in which the flocculent precipitate first forms contains a balanced mixture of toxin and antitoxin. In the following protocol illustrating the Ramon flocculation technique it will be observed that 3.0 c.c. of toxin are added to each of 6 small test tubes. Antitoxin having a strength of 300 units per cubic centimeter was added in amounts varying from 0.18 to 0.06 c.c. The contents of the tubes were mixed and the tubes were placed in a water-bath having a tempera- ture of 54° C. In this particular experiment they were observed at five-minute intervals for one hour. The results are tabulated in Table VII. TOXINS AND ANTITOXINS 249 Table VII Standardization of Toxin Diphtheii?. toxin — strength to be determined. Diphtheria antitoxin contains 300 units per e.c. Temperature of bath — 54° C. TUBE 1 TUBE 2 TUBE 3 TUBE 4 TUBE 5 TUBE 6 toxin 3.0 c.c. 3.0 c.c. 3.0 c.c. 3.0 c.c. 3.0 c.c. 3.0 c.c. antitoxin 0.18 0.15 0.12 0.10 0.08 0.06 Time: 5 min. SI. C. i ^l.C. Sl.C. Sl.C. 10 min. C C C c __ __ 15 min. c C C c Sl.C. 20 min. c C P c c 25 min. c P F p c 30 min. c F F F c 35 min. c F F F c 40 min. p F F F c 45 min. F F F F c 50 min. F F F F c 55 min. F F F F p 60 min. F F F F F SI. C. 81. C— Slight ; turbidity 1 unit of antitoxin = ( ).00333 c.c. C— Cloudy 1 = : tube 3 (Indicator tube) P — Granular precipitate 0.12 ^ 0.00333 = 36.0 units of A.T. in tube 3 F — Floeculent precipitate 3.0 - 36.00 = 0.0833 c.c. (L, d ose of toxin) I — Indicator tube 1.0 c.c. of toxin is equivalent to 12.0 units of -- — No change antitoxin Lf Dose of Toxin. — It will be observed from an inspection of the above protocol that a floeculent precipitate appeared first in tube 3 to which had been added 0.12 c.c. of antitoxin. The antitoxin contains 300 units per cubic centimeter, therefore 0.12 c.c. represents 36 units. Since 3.0 c.c. of toxin brings about the most rapid flocculation of 36 units of antitoxin, one unit of the latter will be flocculated by 0.0833 c.c. of toxin. This amount of toxin constitutes an Lf or flocculating dose. If the Lf dose of toxin is known and the strength of antitoxin is unknown, the latter can be determined by a similar experiment. In practice an experiment similar to the above can be used as a trial titration. In the final titration the difference in the antitoxin content of any two adjacent tubes in the series may be reduced to 0.01 or 0.001 c.c, the range being determined by the trial titration. Ramon (1922) found that purified antitoxin consisting for the most part of pseudoglobulins is devoid of flocculation power when mixed with toxin. He and also Bayne- Jones confirmed the ob- 250 IMMUNOLOGY servation of Renaux (1924) that a nonflocculating antitoxin can be titrated by the flocculation test if it is mixed with a standard flocculating serum. The results obtained indicate the combined neutralizing value of the mixture and from this the strength of the unknown can be calculated. Neither Bayne- Jones (1928) nor Ramon noted the Danysz phenomenon in any of their experiments. The former reports (1928) that the optimum temperature for con- ducting the test is 45°-55° C. The speed of flocculation increases with the temperature up to 55° C. Above this temperature the results are irregular. At 0° C. to 5° C. the speed of precipitation is sufficiently slow to permit setting up the te$t and allowing the tubes to remain in the refrigerator overnight without a flocculent precipitate forming. Definite results are then obtained by incubat- ing at 45° to 50° C. Eaton (1936) presents an interesting report on the flocculation reaction with purified diphtheria toxin. He offers evidences that the flocculation rate of purified toxins is unclianged when tlie toxins have not been altered by the methods used in purification. pH of Toxin. — The pH of the toxin can vary, according to Bayne-Jones, between 6.8 and 8.4 without affecting the results of the flocculation test. Toxoid, as well as toxin, readily produces flocculation of antitoxin. The Lf dose of toxin represents its antigenic or combining power rather than its toxic strength. In the case of a fresh toxin containing practically no toxoid the Lf dose will equal the Lo dose of toxin. As the Lo dase of toxin is converted gradually to toxoid, the toxicity for guinea pigs dimin- ishes but its flocculation value remains the same. This is true even when it is completely converted to toxoid. Reliability of R.vmon Test, — Since the Ehrlich method of titrating toxin determined the toxic rather than the antigenic strength of a toxin, it is evident why the Ramon flocculation rather than the Ehrlich method is used to measure the strength of toxoid which is nontoxic but antigenic. The test is, however, not always a satisfactory index of the antitoxic property of a serum, since the sera of some horses flocculate slowly and yet possess a high antitoxic content. On the other hand, a rapidly flocculating standard antitoxic serum is considered quite reliable in determin- ing the flocculating dose of toxoid. TOXINS AND ANTITOXINS 251 111 regard to the antigens and antibodies involved in the floc- culation reaction, there has been much controversy as to whether it is a bacterial precipitation or a toxin-antitoxin flocculation phenomenon. Bayne- Jones (1928) states that he has reviewed and confirmed the conclusion of H. Schmidt (1926) that toxin- antitoxin flocculation occurs independently of bacterial precipitins. Some of the reasons upon which he bases his conclusion may be summarized as follows : 1. The flocculating and agglutinating titers of an antitoxic serum bear no relationship to each other. 2. Bacterial agglutinins may be demonstrated in the super- natant fluid of the indicator tube after flocculation is complete. 3. According to Moloney and Weld (1925) diphtheria bacilli are separable into groups by means of agglutinating sei-um but all produce a toxin that is flocculated by one antitoxin. 4. When bacterial precipitin is mixed with toxin, the latter is not removed and furthermore the presence of the precipitin does not interfere with the subsequent flocculation of toxin by anti- toxin. 5. Various controls also offer further evidence pointing to the specificity of the toxin-antitoxin reaction. Theories of Toxin-Antitoxin Mechanism. — In regard to the mechanism of diphtheria toxin-antitoxin neutralization, there have been three theories that have received extensive consideration. They were formulated by Ehrlich (1898, 1903, 1910), ArrheniiLS and Madsen (1907) and Bordet (1909), respectively. Ehrlich. — Ehrlich (1910) says that when he began the study of diphtheria toxin-antitoxin neutralization he regarded the toxin as a simple chemical substance but that his experimental results led him to think of it as a very complex compound of toxic and nontoxic antigenic substances. He concluded that the diphtheria bacillus secretes two antigenic toxic substances : one he called toxin and the second, which possesses less affinity for antitoxin, he called toxone. To toxin he ascribes the acute symptoms and death, while toxone produces slowly developing emaciation, paralysis and death of the experimental animal. In detei*mining an L^. dose of toxin, which theoretically would be 101 M.L.D.'s, he found that it is always larger than the theoretical figure, being very frequently 120, 130 or more M.L.D.'s. This led him to believe that in every 252 IMMUNOLOGY diphtheria toxin there develop toxoids with different affinities for antitoxin and that one of these, which he called protoxoid, has a greater affinity for antitoxin than has toxin. The fraction of toxoid possessing the same affinity as toxin for antitoxin he called syntoxoid. To other toxoids with lesser affinities he gave other names. Thus he built a concept of a toxin spectrum Avhich is exten- sively discussed in his studies (1910). He conceived of the union of toxin and antitoxin as resembling the reaction between a strong acid like sulphuric acid and a strong l)ase as, e.g., sodium hydroxide. These react completely in one direction and with only an appreciable degree of reversibility (Wells, 1929). Arrhenius and Madsen. — Arrhcnius and IMadsen (1907) studied the reaction between tetanolysin and antitetanolysin and concluded that the reaction between toxin and antitoxin resembled that between a weak acid such as boric and a base such as am- monia. When the latter react an equilibrium is established and there is present a measurable amount of free acid, base and the neutral salt. This theory is apparently accepted by Clenny (1931). Bordet's Theory of Adsorption. — The third thcoiy, that of Bordet, assumes that as antitoxin is added to toxin, there is a partial neutralization of each molecule of toxin by antitoxin in- stead of the neutralization of progressively large amounts of the toxin until finally all is neutralized by antitoxin. He does not deny the existence of toxoids but considers that Ehrlich's toxone is merely partially neutralized rather than a separate toxin. In his opinion the union of toxin and antitoxin is an adsorption phenomenon in which the mechanism of union is similar to that in other antigen-antibody reactions. Wells (1929) considers that Bordet's theory comes nearer explaining the ''zone phenomenon" and the "Danysz effect" than either of the other two theories. Zone Phenomenon. — The "zone phenomenon" is perliaps best illustrated by the trypanocidal effect of immune serum injected into rats infected with trypanosomes. Taliaferro and Johnson (1926), Johnson (1929) and Coventry (1930) find that if one starts with a dose of immune serum that destroj's the trypano- somes and injects a series of animals with progressively larger TOXINS AND ANTITOXINS 253 doses he finds zones of action and of inaction occur. Thus Coventry (1930) states that when she injected rats infected with T. lewisi with varying doses of trypanocidal immune serum (expressed in c.c. per 100 gram body weight) slie found that 1.1 c.c. had no effect, 1.5 c.c. was markedly trypanocidal, 1.9 c.c. had no effect, 2.3 c.c. only slightly trypanocidal, while 2.7 c.c. was markedly trypanocidal. Other Examples of the Zone Phenomenon. — Other examples of what is commonly called the zone phenomenon are frequently observed in antigen-antibody reactions. Larsen and Nigg (1928) observed that in the Wassermann test for syphilis in individuals liaving both sypliilis and leprosy they frequently observed more complement fixation in the tubes containing smaller amounts of patient's serum tlian in the one containing the maximum amount. When one works with bactericidal sera one notes quite fre- quently that there are fewer bacteria destroyed in the stronger concentrations of immune serum than in high dilutions. Corre- sponding exami)les of the zone phenomenon can also be dem- onstrated for precipitins and agglutinins. The zone phenomenon is quite characteristic of colloidal reactions. Danysz Effect. — ^Another phenomenon Avhich is apparently a colloidal reaction is called the "Danysz effect." Danysz noted that when lie added an excess of toxin to its antitoxin the amount of uncombined toxin varied according to the way he added the toxin to the antitoxin. If he added it in fractional amounts wdth proper time intervals, there was more unneutralized toxin than if the toxin were added to the antitoxin in one operation. A similar phenomenon has been described for other antigen-antibody mixtures. Bordet considers this an adsorption phenomenon and compares it to the adsorption of dye by filter paper. If one tears a piece of blotting paper into very small pieces and adds them one at a time to a solution of dye, they will take up more dye than if the blotting paper is added as one piece (see Wells, 1929, and also Bordet, 1909). Thus it wdll be seen that there is considerable evidence indicat- ing that colloidal phenomena play an important part in antigen- antibody reactions, l^'or a more extensive discussion of the physi- cal chemistry of toxins and antitoxins the student is referred to a short report by Maver (1928). 254 IMMUNOLOGY While active immunization against diphtheria in the experi- mental animal began with the production of antitoxin by Behring, it was not possible to apply it to man until a safe and dependable antigen was produced and some method devised to determine on a large scale the susceptibility of children and adults to diphtheria. The history of the research which led ultimately to the develop- ment of a satisfactory susceptibility test in man is of considerable interest and importance. In 1909, Homer and Sames introduced the intracutaneous method of titrating toxin and antitoxin. At the present time it is uni- versally employed in determining the virulence of C. diphtheriae and is used frequently in the titration of toxin. According to Glenny (1921) it is a convenient guide in following the trans- formation of toxin to toxoid. M.R.D. — Romer adopted two units of toxin, the M.R.D. (min- imum reacting dose) and the Lr (limit of reaction) dose, respec- tively. Glenny (1931) has defined the M.R.D. as the least amount of toxin which when injected intracutaneously into a guinea pig will produce, within 36 hours, an area of hyperemia that is at least 5 mm, in diameter. He defines the Lr dose of toxin as the least amount of toxin which when added to one unit of antitoxin will yield a mixture of such toxicity that 0.2 c.c. will produce a minimal skin reaction when injected intracutaneously into a guinea pig. Other doses of toxin such as Ly/lOO or Lr/500 refer to the least amount of toxin which when mixed with 1/100 or 1/500 unit of antitoxin, respectively, will give a minimal skin reaction in a guinea pig. Whether one employs an end point represented by a faint area of hyperemia, or by an area of hyperemia with resulting necrosis, is of importance since they represent different amounts of toxin. Glenny and his associates prefer the former wliile Romer made use of both end points. Romer (1909) found that 1/500 of an M.L.D. will produce hyper- emia only, while 1/250 of an M.L.D. will cause hyperemia fol- lowed by necrosis within 72 hours. Glenny, Pope and Wadding- ton (1925), using an improved intracutaneous technique, conclude that 1 M.L.D. is equal to 1,000 M.R.D. 's. Glenny (1931) says that more recent work in which a faint area of hyperemia 4 to 5 cm. in diameter is emploj'ed indicates that 1 M.L.D. is equal to 2,000 M.R.D. 's. TOXINS AND ANTITOXINS 255 Schick Test for Susceptibility. — In 1913 Schick announced that b}^ means of the Romer technique he could determine susceptibility or immunity to diphtheria. He found that susceptible individuals do not possess sufficient circulating antitoxin to neutralize 1/40 or 1/50 of a guinea pig M.L.D. of toxin when it is injected into the outer layers of the skin (intradermally) of the forearm. A positive reaction indicating susceptibility develops witliin IS to 26 hours at the site of inoculation and persists for 7 to 15 days. Occasionally delayed reactions are encountered. The positive reaction manifests itself as an area of hyperemia (redness) one centimeter or more in diameter. Also there may be some swelling and induration. As the reaction subsides scaling manifests itself and there remains a brownish pigmentation. A negative reaction indicates that the individual's blood contains at least 1/40 of a unit of antitoxin per cubic centimeter. The test is performed by injecting into the outer layers of the skin a toxin solution of such strength that 0.1 c.c. or 0.2 c.c. contains 1/40 or 1/50 of a guinea pig M.L.D. Since some individuals are hypersensitive to toxin (see NeiU, Fleming, Sugg and Gaspari, 1930), it is quite desirable to run controls with toxin heated to 60°-75° C. for thirty minutes. Pseudoreactions commonly disappear within 2 or 3 days (Banzhaf, 1928) and occasionally they are confusing. The time of disap- pearance and lack of pigmentation are frequently used as criteria in interpreting a reaction as negative rather than the use of a control. Recently Taylor and Moloney (1939) recommended that the Schick toxin be prepared from "fresh" toxin less than one year old and free from preservatives. They also suggest that the new Schick toxin have twice the toxicity and lower combining power than the standard Canadian dose. They insist that this will in- crease the percentage of interpretable reactions. Their claims seem to be substantiated by Cameron and Gibbard (1941). Variation in Susceptibles in Urban and Rural Populations. — According to data collected by Park (1919, 1933) and others, the number of susceptible individuals in a population varies in dif- ferent age groups and also with the opportunity afforded by the environment for acquiring immunity from mild contact infec- tion. He found that at birth 15 per cent of the infants born in 256 IMMUNOLOGY the cities are positive as contrasted with 40 per cent of those born in rural communities. Susceptibility in the other aoe oroups he tabulates in Table VIII. Table VIII* AGE YEARS PER CENT PER CENT SCHICK POSITIVE SCHICK POSITIVE IN IN CITY RURAL POPULATION 1- 2 60-70 80 2- 3 60-45 70 3- 6 45-40 60 6-10 40-30 55 10-16 21-16 50 16-30 20-12 45 ♦Parks and Williams : Pathogenic Microorganisms, Lea & Febiger, publishers. Susceptibility in Young Adults. — In a series composed of medical students at the University of Kansas and falling within the age groups of nineteen to twenty-three years, there were 28 per cent Schick positive individuals. This represents a mixed group made up of students coming from cities and rural communities. It will be noted from the data in the above table that ages of greatest susceptibility are the second, third and fourth years of childhood. Susceptibility Determined by First Dose of T.A.T. — In 1923, Park noted that susceptible individuals react to a subcutaneous injection of 1 c.c. of a toxin-antitoxin mixture, having a standard toxicity, with a reaction similar to that obtained by the Schick test. Thus a susceptibility test and the first of three immunizing injections are combined. It is obvious that this has distinct ad- vantages. Since the reaction depends upon the presence of diph- theria toxin, one can readily appreciate why toxoid, which is nontoxic, cannot be used in any susceptibility test, although it is a good immunizing agent. The test suggested by Park is read on the fifth or six day just as is done with a Schick test. The pseudo- reactions are usually gone by the third day. Park says that the highest percentage of pseudoreactions develop in older children and adults. Controls are usually not run with this test. Active and Passive Immunity. — As mentioned earlier in this chapter, interest in active and passive immunity to diphtheria began with Behring's and Kitasato's discovery that antitoxins can TOXINS AND ANTITOXINS 257 be produced by immunizing animals with toxin. Behring and others realized that the reason a patient contracts diphtheria is that he lacks circulating antitoxin and that the rational treat- ment consists in the administration of the latter. It also follows from this observation that susceptible individuals may be tem- porarily protected from diphtheria by the administration of anti- toxin. The presence of circulating antitoxin produced by the body is an example of active immunity, while the protection which results when this antitoxin is injected into a susceptible animal is an example of passive immunity. Passive Immunity. — In the treatment of diphtheria with anti- toxin, there are a number of important facts that must be con- sidered by the physician. He should remember that the disease is due to the effect of toxin upon the tissue cells and that the anti- toxin he injects acts only as a neutralizer of toxin and cannot undo any injury to tissue cells that toxin has already produced. It is be- lieved that one large dose of antitoxin will neutralize more toxin than the same amount given in several doses with appreciable inter- vals of time intervening. These facts warrant the administration of a large dose of antitoxin as early as possible in a case that is clin- ically diphtheria even before the report on the culture is available and in some cases when the laboratory findings are negative. Improved methods of purifying diphtheria antitoxin have been reported by Pappenheimer and Eobinson (1937), Pope (1938) and Northrop (1941). Northrop says the new antitoxin is about 20 times as active as the original antiserum. Dosage of Antitoxin. — As to what constitutes an adequate ini- tial dose of antitoxin Park and Williams (1933) recommend that for children under fifteen years of age, 3,000 to 5,000 units be given in mild cases, 5,000 to 10,000 in moderate cases, 10,000 to 20,000 in severe cases and 15,000 to 30,000 in malignant eases. For older children and adults the dosage is practically doubled for each type of case. Method of Administering Antitoxin. — In regard to the method of administering the antitoxin, they recommend that it be given intramuscularly in mild and moderate cases, both intramuscu- larly and intravenously in severe cases and intravenously only in malignant cases. 258 IMMUNOLOGY Prophylactic Dose of Antitoxin. — When it is desired to pro- tect a susceptible individual who has l)eeii exposed to diphtheria, it is customary to administer 500 or 1,000 units of antitoxin sub- cutaneously or intramuscularly. This will protect for two to four weeks depending upon the rate of elimination from or de- struction witliin the bodj\ Not infrequently Schick te.sts are made to ascertain whether the contacts are susceptible or immune and only the former given a prophylactic injection of antitoxin. Active Immunity. — As previously mentioned in this chapter, active immunity to diphtheria toxin was produced first by Behr- ing and Kitasato (1890). Interest in active immunization against toxins resulted immediately in numerous publications. Ehrlich (1892) reported the successful immunization of mice with ricin, abrin and robin. He found that the otTspring of immunized fe- male mice possess passive immunity to toxin. This he attributed to antitoxin obtained largely through the mother's milk. Im- munity was not transmitted from tlie father to tlie offspring. Two years later Ehrlich and Iliibener (1894) immunized guinea pigs with tetanus toxin and obtained similar results. Wernicke (1895) treated guinea pigs with toxin and then antitoxin and finally with several injections of toxin. He observed that immune females transmit antitoxin to the offspring but he was apparently in some doubt as to the mechanism involved. It remained for Anderson (1906) and Theobald Smith (1907) to show definitely that the offspring of an immune female guinea pig owes its passive immunity to antitoxin obtained through the mother's milk. Such immunity persists for about three months. Active Immunization of Horses With T.A.T. — In 1897 Park (1903) began immunizing horses with toxin-antitoxin mixtures. It should be remembered that the injection of toxin-antitoxin mixtures formed a part of Ehrlich 's method of determining a unit of antitoxin and of standardizing toxins. The first careful and extensive investigation of diphtheria toxin-antitoxin mixtures as antigens was made by Theobald Smith (1909) and Smith and Brown (1910). They found that partially neutralized or even overneutralized mixtures of toxin could be used in producing an active immunity to diphtheria. They suggest that the antitoxin "smuggles" the toxin into the body where it is slowly liberated with resulting antitoxin formation by the tissues of the body. TOXINS AND ANTITOXINS 259 They also noted marked variation in the degree of immunity which develops in guinea pigs. Active immunity when once established persists throughout the life of the guinea pig. Theobald Smith recommended that the method be used to immunize children to diphtheria. Behring, who Avas the first to produce active immunity in the lower animals to diphtheria toxin and to immunize a child pas- sively with antitoxin, was also the first to use toxin-antitoxin mix- tures to immunize children against diphtheria. This was in 1913 shortly before Schick published his susceptibility test, Behring 's work was interrupted by the war. Susceptibility Tests in New York. — In 1914 Park, Zingher and Scrota determined the susceptibility of a large number of indi- viduals and during the next year immunized about 10,000 infants, children and adults. They were the first to recognize the value of the Schick test in studying the immunizing effect of diphtheria toxin-antitoxin injected into human beings. Park (1932) says that by 1917 they had determined that 80 to 85 per cent of those receiving their 31^^ preparation at weekly intervals were immune and that in a majority of the cases the immunity lasted several years. In 1918 they started an immunization program which con- templated the immunization of all children in New York City. Park's Pioneer Work on Active Immunization.— In their earlier work they noted a small percentage of severe reactions to the 3L^ toxin-antitoxin mixtures. They also found that the best immunizing mixture is one having such a toxicity that one human dose will kill a 250 gram guinea pig in about four weeks. IMix- tures containing less toxin are poor immunizing agents. They according!}^ adopted a toxin-antitoxin mixture containing 0.1L+ dose of toxin per dose (usually 1 c.c.) in place of those containing 3L^. to 6L^ doses of toxin. Preparation of T.A.T. — According to Banzhaf (1928) this is usually prepared in 40 liter lots. Only toxin that has become stable through ageing and with a residual toxin content of at least 5L^ doses per cubic centimeter is employed. Concentrated and purified antitoxin containing at least 2,000 units per cubic centimeter is selected for the partial neutralization of the toxin. To prepare a 0.1 L^ toxin-antitoxin mixture, they place 4,000 L^ 260 IMMUNOr.OGY doses of toxin in a 2 liter flask and add 3,000 units of diluted antitoxin to the toxin. After mixing:, the contents of the flask is then added to enough 0.8 per cent saline containing 0.5 per cent phenol to make the desired volume of 40 liters. The toxin-antitoxin mixture thus prepared is then sterilized by filtration and its toxic property and immunizing value tested by guinea pig inoculation. "When a properly balanced mixture is injected into standard weight guinea pigs, one should find that 0.25 c.c. gives no symptoms, 0.5 c.c. may give slight paralysis after 18 days with recovery, 1.0 c.c. paralysis in 14 to 20 days and death Avith complete paralysis in 20 to 30 days. Five cubic centimeters should kill a standard weight guinea pig in from four to seven days. The pigs that sur- vive are tested five weeks later to determine their resistance to diphtheria toxin. Those that received 0.25 c.c. and 0.50 c.c. of T.A.T. should witlistand 5 ]\I.L.D. 's and 10 M.L.D. 's of diphtheria toxin respectively. Advantages of New Toxin-Antitoxin IMixture, — This new preparation causes fewer local reactions, since it contains less toxin and bacterial protein tluin the earlier mixtures employed. It is also safer and the first injection will serve for children under eight years of age both as a substitute for the Schick test and to initiate the production of antitoxin. In older individuals the reaction does not correlate so well with the Schick test. This is due to the greater toxicity and larger content of bacterial protein in the toxin- antitoxin mixture to which many older individuals are sensitive and give pseudopositive reactions. Park (1932) says that since about 1929 they have been using diphtheria antitoxin obtained from goats in the preparation of toxin-antitoxin mixtures. This is done to avoid sensitizing those injected to horse serum. Results Determined by Schick Test. — The i-esults of toxin- antitoxin immunization have been determined by means of the Schick reaction and by observing the incidence of diphtheria in school populations and other groups of immunized individuals. In Park's series, the Schick test indicates that only 30 to 40 per cent become immune within 3 weeks after the first injection, approximately 50 per cent at four weeks, 70 to SO per cent within six weeks and 80 to 85 per cent within 12 weeks. TOXINS AND ANTITOXINS 261 Harrison (1932) reports the results he obtained by retesting the children in the Washington schools, who had been immunized against diphtheria. He states that 64 per cent were successfully immunized by three injections of toxin-antitoxin. From a review of the literature he concludes ''that 70 per cent successful im- munizations from 3 injections would be a fair estimate of the general effectiveness of toxin-antitoxin mixture." He suggests that the wide variation in effectiveness reported by various workers is probably due to the use of deteriorated toxin-antitoxin mixtures and the variation in the strength of the toxin used in the Schick test. Owing to the slowness with which immunity develops, it is obvious that toxin-antitoxin cannot be used in the treatment of diplitheria nor to give immediate protection such as is afforded by passive immunization with antitoxin. Detoxified. Toxin.— The early work on the use of detoxified toxin in immunization experiments on the lower animals is re- ferred to by Pick (1908), Madsen (1908) and Loeffler (1913), Dean (1913), Glenny (1931) and Zinsser (1931). They review the literature and give excellent discussions of active immunity to diphtheria produced by both toxin-antitoxin mixtures and de- toxified toxins or toxoids. Apparently Behring (1890) and Frankel (1890) working in Koch's laboratory succeeded in im- munizing animals with toxoid. The former treated toxin with iodine trichloride, while the latter attenuated it by heat (60° C). According to Rosenau and Anderson (1908), Burchard (1895) and Anderson (1907) showed that formalin would detoxify tetanus toxin. It remained, however, for Glenny and Sudmersin (1921) to determine experimentally for the first time that diphtheria toxin, completely detoxified by the action of formaldehyde, retains its antigenic property. According to Park and Schroder (1932) Ramon "adopted the suggestions of Glenny as to the use of formalin, and of Loewenstein of Vienna as to the value of nontoxic toxoid in tetanus" and developed a satisfactory nontoxic diph- theria toxoid. He uses only a toxin of high potency to which he adds sufficient formalin (according to Glenny, 1931) to make a concentration of 0.3 to 0.4 per cent. The formalized broth culture containing toxin and bacteria is incubated for one month at 37° C. It is then sterilized by filtration and its toxicity de- termined. It should be detoxified to the extent that 6.0 cubic 262 IMMT'XOLOGY centimeters produce no symptoms, either local or general, when injected siibcutaneously into a 300 gram guinea pig. Banzhaf (1928) recommends filtering the toxin before adding formalde- hyde. Moloney and Weld (1925) report that broth cultures of C. diphtheriae are more readily detoxified by formalin than the filtered toxin. Larson and Nelson (1924) and Larson, Halvorson, Evans and Green (1925) have detoxified toxin with sodium ricinoleate. They report successful immunization witli nontoxic soap-toxin mixtures. Apparently phenol is not a satisfactory preservative for toxins. Glenny states that any condition such as the shaking of dilutions which favors the local concentration of phenol at the air-liquid surface results in definite reduction in the antigenic value of the toxin solution, iMoloney and Weld (1925) report that toxoids ob- tained by treating phenol-preserved toxin witli formaldehyde fre- (lUf'utly possess no antigenic value. Toxoid Specifications of U. S. Public Health Service.— Accord- ing to Harrison* (1932) all toxoid manufactured or sold in tiie United States must comply with the specification supplied l)y the National Institute of Health of the U. S. Public Health Service. He says that these specifications may be roughly stated as follows : "The toxin before detoxification must contain not less than 400 M.L.D.'s or 5L^ doses. Detoxification must be so complete that 5 human doses when injected into guinea pigs must show no sign of early or late diphtheria poisoning. The antigenic efificiency must be such that the initial human dose will immunize 80 per cent of guinea pigs in 6 weeks to such a degree that 5 j\LL.D.'s of toxin will fail to kill in 10 days." Park (1932) states that a good toxoid should contain at least 8 antigenic or fiocculation units per c.c. It is generally agreed that toxoid is a better im- munizing agent than toxin-antitoxin. Harrison (1932) recom- mends that at least 2 doses of 1.0 c.c. each be given with an in- terval of from three to four weeks. The importance of this interval of time is supported by the experimental work of Glenny and Pope, Waddington and Wallace (1925). Wlien toxoid is diluted, Park states that at least 4 antigenic units should be pres- ent in the dose used for immunization. With 3 injections of *Harrlson, W. T. : Am. J. Pub. Health i^: 17, 1932. TOXIXS AND ANTITOXINS 263 such a toxoid he olMained 94 per cent successful immunizations against diphtheria. He says that Volk of Pontiac was successful in immunizing- 83.8 per cent with two injections and 47 per cent with one injection of the .same toxoid. The objection to the Ramon toxoid is that severe reactions are produced frequently in older cliildren and adults. Foi* this reason Park adopted toxoid for the immunization of pi'c-scliool children and toxin-antitoxin for school children and adults. Bigler and Werner (1941) recom- mend for infants and young children combined immunization against tetanus and diphtheria, using two injections of 1 c.c. or three injections of 0.5 c.c. of the combined toxoids. They prefer intervals of three months or more between injections. Inunction as Method of Immunization. — Park has also tried out immunization with toxoid by inunction using toxoid mixed with lanolin after Loewenstein. Apparently 4 or 5 rubbings made at weekly intervals immunized about 70 per cent of the susceptibles (children) on whom it was used. The only advantages are in those cases in Avhich consent for injection of toxoid cannot be ob- tained or "in institutions where a nurse can apply it to the chil- dren as they enter" (Park and Schroder, 1932). Alum Toxoids. — Glenny (1931) states that he and his associ- ates reported in 1926 and 1928 that the addition of 0.01 to 0.1 per cent of potassium alum to toxoid increases the antigenic response. Park and Schroder (1932) state that they have had good results altliough their series is short and the period of ob- servation less than one year at tlic time of their report. They report guinea pig experiments that yielded interesting results. One large injection of alum toxoid (0.5 c.c.) or two doses ol" Ys c.c. each at weekly intervals produced inununity in 80 per cent of a series of guinea pigs as compared with 50 per cent and 20 per cent, respectively, for corresponding doses of toxoid with- out alum administered to a second series of animals. In 1931 Glenny and Barr recommended that alum toxoid precipitates be used as antigens. They state that from 1 to 2 per cent of alum will precipitate all of the toxoid; the amount of alum required varies "with the batch of toxoid. They recommended the resuspension of these precipitates in saline containing 0.5 per cent phenol. They noted that when amounts of alum varying from 3.5 to 10.0 per cent are used, the purity of the precipitate 264 IMMUNOLOGY increases, wliile the yield decreases. Since alum toxoid is slowly absorbed and remains in the body for a long period of time, while the Ramon toxoid is rapidly eliminated, they believed that it may be possible to immunize with one dose of alum toxoid. Theo- retically tlie latter should be able to supply both the initial and the secondary stimulus, whereas the Ramon toxoid is eliminated before the appearance of antibodies when a secondary stimulus is necessary for maximum antibody production and therefore a second and perliaps a third injection of Ramon toxoid is necessary. This M'ork of (rlenny and Barr has been followed up by Wells, Graham and Havens (1932) and more recently l)y Graham, Mur- phree, and Gill (1933). The latter report that "a single injec- tion of from 5 to 10 units of precipitated toxoid has rendered 171 or 92.4 per cent of 185 strongly Schick positive children Schick negative. "Of 613 children, 592 or 96.6 per cent were Schick negative when tested from two to four months after a single injection. The original immunity status was unknown, but 72 per cent w^ere pre-school children." Ramon (1940) regards the single injection of toxoid as ineffective. Active-Passive Immunity. — ^Ramon (1940) has reviewed the work he and his associates have done since 1925 on what they term active-passive immunity to tetanus or diphtheria toxins respec- tively. Ramon reports that active immunity can be produced by injecting one or, better, two doses of toxoid sometime after the simultaneous injections of the specific antitoxin and toxoid. They say that this procedure is not only of value in prophylactic immunization but that they are recommending it as a new treat- ment of acute tetanus or diphtheria. Recommendations of New York City Department of Health.* — As a result of extensive experience in diphtheria immunization of children, the New York City Board of Health (1940) has made recommendations which may be summarized as follows : 1. Because of the passive immunity acquired from the mother it is unwise to begin immunization of the child before the ninth month of age. *New York Department of HeaUh, Quarterly Bulletin 8: 2, 1940. TOXINS AND ANTITOXINS 265 2. Two doses of alum precipitated toxoid produce as high an active immunity as three doses of plain toxoid. Since the fine precipitate is the active immunizing agent a technique should be employed that assures its administration. Be- cause of the occurrence of annoying local reactions follow- ing the use of precipitated toxoid the Department of Health is recommending that three doses of plain toxoid be employed instead of the two doses of precipitated toxoid. 3. The Department recommends that the interval between im- munizing injections be one month. 4. Experience has shown that a variable proportion of chil- dren successfully immunized at the age of nine months "will be found to have very little immunity at the age of four or five years." To bring up the immunity of such children to a protective titer, a single injection of toxoid is sufficient. It is advised that all children immunized in infancy be given a single injection of toxoid shortly before entering school. This is done routinely in the Department's child health sta- tions ; the Schick test is omitted. Children not previously im- munized are given the full course of injections. 5. Schick tests are given after an interval of two or three months following the completion of a course of immunizing injec- tions. To avoid errors in interpretation the sites of injection are examined from the fifth to the seventh day. A positive reaction is indicated by a persistence of redness or a bro\vnish discoloration at the spot, 6. Dosage of plain toxoid (three injections). a. For children ages 6 years or less three injections con- sisting of 0.5 c.c, 1.0 c.c, 1.0 c.c. respectively. b. For children over 6 years of age three injections con- sisting of 0.25 c.c, 1.0 c.c, and 1.0 c.c respectively. When a reaction occurs, the subsequent injection should be either the same or less depending upon the severity of the reaction. If the Schick test remains positive after the three injections of toxoid are giveJi, the series of immunizing injections should be repeated. If the Schick test is still positive the Department feels that further injections are inadvisable. 266 IMMUNOLOGY Summary. — A few of the important points brought out in this chapter as well as some additional information may be sum- marized as follows: 1. Diphtheria is essentially a toxemic disease. To produce it the organism, C. diphtherioe, must be able to establish itself and create favorable conditions for the production and absorption of toxin. While all strains produce qualitatively the same toxin, they differ iti the amount they can produce, in their invasive power, and perhaps in their oxygen requirements. 2. Man seems to be the one naturally susceptible host, although the disease can be produced experimentally in guinea pigs, dogs, and many other animals. Mice and rats are refractory to experi- mental intoxication with diphtheria toxin. Their resistance seems to depend upon tissue insusceptibility rather than antitoxin. Im- munity in man is due largely to antitoxin although tissue resis- tance to invasion and adsorption of toxin may play some part in natural immunity. 3. The units of measurement of toxin are the minimal lethal dose (M.L.D.), minimal reacting dose (M.R.D.), limit of reaction dose (Lr), limes nul or threshold dose (Lo), limes Tod or death dose (LJ, flocculating dose (Lf) and the Schick skin test dose (1/50 M.L.D.). These are defined and discussed. Ehrlich intro- duced the units that involve death or protection against death as end points while Eomer introduced a method of measuring toxin in terms of the amount necessary to produce a skin reaction. Modifications of his technic have formed the bases of the skin test units such as the M.R.D., Lr and Schick test dose of toxin. The technique of the Ramon flocculation test and method of dc- tennining the Lf dose of toxin and the mechanism of the reaction are also discussed. 4. Diphtheria toxin like all true toxins is an antigenic poison. It is probably secreted by C. diphtherme. According to Eaton it does not appear to be either a cleavage product from the media nor a toxic radical attached to a protein. When in solution it is relatively thermolabile, being destroyed when heated to 60° C. for thirty minutes. Dried toxin with- stands 100° C. but is destroyed at 150° C. TOXINS AND ANTITOXINS 267 5. Toxin is neutralized but not destroyed by antitoxin. When some batches of toxin-antitoxin mixtures are frozen, they become more toxic (Banzhaf, 1928, p. 749). When a mixture of toxin- antitoxin is injected into the tissues of man or any suitable ani- mal, the toxin is liberated slowly and stimulates the production of antitoxin. 6. Three theories as to the mechanism involved in toxin neu- tralization liy antitoxin are given. Elirlicli conceived of the reaction as similar to that between a strong acid and a strong base. To explain many clinical and experimental phenomena he postulates the secretion of two tox- ins, a toxin and a toxone. These differ in their avidity for anti- toxin and in the symptoms they produce. In his opinion the toxone causes the late paralysis observed. He also assumes that these toxins readily deteriorate into toxoids that differ in their affinity for antitoxin. His conclusion that the toxic and antigenic properties arc not interdependent is borne out l)y subsequent work. A second theory is that of Arrhenius and ]\Iadscn. They re- gard the reaction as similar to that between a weak acid and a weak base. This theory is apparently accepted by Glenny (1931) and his associates. A third theory is called the adsorption theory of Bordet. When antitoxin is added to toxin he believes that instead of neutraliz- ing a fraction of the toxin molecules present, it partly neutralizes every molecule of toxin. According to this theory Ehrlich's toxone is only a partly neu- tralized toxin. Bordet explains the zone phenomenon and the "Danysz effect" from tlie standpoint of physical chemistry. Wells (1929) and others seem to feel that Bordet 's theory more nearly explains all of the observed phenomena than any other theory. Ehrlich's original conception that it is analogous to the reaction between a strong acid and a strung base seems to be generally abandoned. 7. The history of the development of passive and active im- munity to diphtheria is discussed. Passive immunization is em- ployed in the treatment of clinical diphtheria and to give imme- diate protection to susceptible individuals who have been ex- 268 IMMUNOLOGY posed recently. In the treatment of diphtheria one large dose should be administered as early in the disease as possible. This is preferable to the administration of the same amount of anti- toxin in divided doses. After toxin is bound to tissue, it is difficult to neutralize. The function of antitoxin is to neutralize toxin; it cannot restore injured tissue to normal nor destroy the diphtheria bacillus. 8. It has been ascertained by means of the Schick test that the greatest percentage of susceptibility in a population occurs in the children of preschool age. Toxin-antitoxin, Ramon toxoid, alum toxoid and alum precipi- tated and resuspended toxoid are all used in active immuni- zation against diphtheria. At the present time the antitoxin present in toxin-antitoxin mixtures is obtained from goats. This avoids the possibility of sensitizing an individual to horse proteins. Toxoid is apparently superior to toxin-antitoxin mixtures as an immunizing agent. It has the additional advantage of not con- taining serum protein of any kind. The Ramon toxoid gives severe reactions in many older children and adults but seems to be nontoxic for children under 8 years of age. Toxoid cannot be used to determine susceptibility. The student should bear in mind that active immunity develops slowly ; i.e., within three to six weeks and hence active immuni- zation is of no value in treatment or immediate prophylaxis. Harrison reports that 64 per cent of the susceptible school chil- dren of Washington were successfully immunized with toxin- antitoxin. Park, on the other hand, reports that about 80 per cent are successfully immunized by toxin-antitoxin. In some series he has been even more successful, the percentage reaching 85 or even 90. It will be several years before one can say defi- nitely how much better toxoid and treated toxoid are than toxin- antitoxin. There is considerable evidence indicating that tlie best immunizing agent is alum toxoid or precipitated toxoid, while the Ramon toxoid is second and toxin-antitoxin third. The quality of the preparation used, the dosage, number of doses, time interval intervening between injections and also between the first injection and the date of retesting are important factors to be considered in evaluating data. Individuals who possess a TOXINS AND ANTITOXINS 269 small amount of antitoxin are said to develop immunity more rapidly than those who do not have some immunity. In all im- munization work one recognizes that the body must be stimulated by the antigen over a relatively long period of time. Where an antigen is rapidly absorbed and perhaps much of it destroyed or eliminated, it is necessary to give several injections, and care must be used as to the time interval between injections. This varies with the physiological capacity of the body to respond. The first injection is called the primary stimulus. The subse- quent injections are given just far enough apart to keep the anti- body content of the blood rising. 9. Ramon's work in active-passive immunity is discussed briefly. Whetlier or not his new method of treating tetanus or diphtheria will find general acceptance will depend upon a more extensive clinical trial. References Anderson, J. : Transmission of Kesistance to Diphtheria Toxine by Female Guinea Pig to Her Young, J. Med. Res. 15: 24, 1906. Arrhenius, S., and Madsen, T. : Immunochemistry, New York, 1907, The Macmillan Co. Banzhaf, E. J.: The Preparation and Purification of Toxins, Toxoids, and Antitoxins, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 745. Bayne- Jones, S. : The Titration of Toxins and Antitoxins by the Floccula- tion Method, J. Immunol. 9: 481, 1924. Also, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 759. Behring, E. von, and Kitasato, S. : Ueber das Zustandekommen der Diph- therie-Immunitat und der Tetanus — Immunitat bei Thieren, Deutsche med. Wchnschr. 16: 1113, 1890. Bigler, J. A., and Werner, Marie: Active Immunization Against Tetanus and Diphtheria in Infants and Children, J. A. M. A. 116: 2355, 1941. Birkhaug, K. E.: Toxin Production of S. Erysipelatis, Proc. Soc. Exper. Biol. & Med. 23: 201, 1925. Toxin Production of Streptococcus Erysipelatis, Ibid., p. 291. Bordet, J.: Studies in Immunity (Translated by Gay), New York, 1909, John Wiley & Sons, Inc. Bull, C. G., and Pritchett, I. W.: Toxin and Antitoxin of and Protective Inoculation Against Bacillus Welchii, J. Exper. Med. 26: 119, 1917. The Prophylactic and Therapeutic Properties of the Antitoxin for Bacillus Welchii, Ibid., p. 603. Identity of the Toxins of Different Strains of Bacillus Welchii and Factors Influencing Their Production in Vitro, Ibid., p. 867. Calmette, A., and Massol, L.: Les precipitines du serum antivenimeux vis-a-vis du venin de cobra, Ann. Inst. Pasteur 23: 155, 1909. Cameron, G. D. W., and Gibbard, J.: Report of a Trial of a New Schick- toxin, J. Immunol. 40: 47, 3941. 270 IMMUNOLOGY Coca, A. F. : Essentials of Immunology, Baltimore, 1925, Williams & Wilkins. Coca, A. F., Baughman, W. E., and Eussell, E. F. : Reaction of Eat to Diphtheria Toxin With Observations on Teclmic of Eoemer Method of Testing Diphtheria Toxin and Antitoxin, J, Immunol. 6: 387, 1921. Coventry, F. A.: The Trypanocidal Action of Specific Antiserums on Try- panosoma Lewisi in Vivo, Am. J. Hyg. 12: 366, 1930. Danvsz, J.: Des Toxines avec Leurs Antitoxines, Ann. Inst. Pasteur 16; 331, 1902, Dean, G.: The Bacteriology of Diphtheria by Nuttall and Graham-Smith, London, 1913, Cambridge University Press, Sec. V, p. 449. Dernby, K. G.: Nature of Diphtheria Toxin, J. Gen. Physiol. 8: 311, 1926. Dick, G. F., and Dick, G. H.: Experimental Scarlet Fever, J. A. M. A, 81: 1166, 1923. Dochez, A. E., and Sherman, L.: The Significance of Streptococcus Hemolyticus in Scarlet Fever, J. A. M. A. 82: 542, 1924. Eaton, M. D.: The Flocculation Reaction With Purified Diphtheria Toxin, J. Immunol. 30: 361, 1936. Eaton, M. D.: Chemical IModifications of Purified Diphtheria Toxin. T. The Mechanism of Detoxification bv Formaldehvde, .T. Immunol. 33: 419, 1937. Eaton, M. D.: Eecent Chemical Investigations of Baclerial Toxins, Bacf. Eev. 2: 3, 1938. Ehrlich, P.: Ueber Immuuitiit durch Vererliung und Saugung, Ztschr. f. Hyg. II. Infect. 12: 183, 1892. Ehrlich, P., and Hiibener, W.r Ueber der Vererl)ung der ImmunitJit bei Tetanus, Ztschr. f. Hyg. 18: 51, 1894. Ehrlich, P.: Studies in Immunity, 1)y Ehrlich-Bolduan, New York, 1910, John Wiley & Sons, Inc. Feierabend, B., and Schubert, O.: Experimentelle Untersuchungen mit Stammen von maligner Diphtherie, Ztschr. f. Immunitiitsforsch. u. exper. Therap. 62: 283, 1929. Fitzgerald, J. G., Defries, R. D., Fraser, D. T,, Moloney, P. J., and McKin- non, N. E.: Experiences with Diphtheria Toxoid in Canada, Am. J. Pub. Health 22: 25, 1932. Frankel, F.: (1890) Cited by Dean, 1913, p. 502. Gabritschewsky, V. G.: Ueber Streptokokken vaccine und deren Ver- wendung l>ei der Durse Pferde und dem Scharlach des Menschen, Centralbl. f. Bakt., Orig. 41: 719, 1906. Ueber Streptokokken- erytheme und ihre Beziehungen zum Scharlach, Berl. klin. Wchnschr. 44: 556, 1907. Glenny, A. T., and Sudmersin, H. J.: Notes on the Production of Im- munity to Diphtheria Toxin, J. Hyg. 20: 176, 1921. Glennv, A. T., and Barr, M.: Alum Toxoid Precipitates as Antigens, J. 'Path. & Bact. 34: 118, 1931. Glennv, A. T.: A Svstem of Bacteriology, Immunity, Med. Res. Council, "London 6: 106,' 1931. Glenny, A. T., and Allen, K. : Action of Diphtheria Toxin on Mice, J. Hyg. 21: 96, 1922. Active Immunity to Diphtheria in Absence of Detectable Antitoxin, Ibid., p. 100. Schick Test of Diphtheria Toxin as a Secondary Stimulus, Ibid., p. 104. Graham, A. H., Murphree, L. E., and Gill, D. G. : Diphtheria Immuniza- tion with a Single Injection of the Precipitated Toxoid, J. A. M. A. 100: 1096, 1933. Harrison, W. T.: Advantages of Toxoid in Diphtheria Prophylaxis, Am. J. Pub. Health 22: 17, 1932. TOXINS AND ANTITOXINS 271 Ivanic, S. Z., Dimitrijevic-Speth, V., and Jovanovic, L.: Kruppstamme von nialigner Diphtheric mit uberwiegender A'irulenz bei geringer Toxigenitat, welche nieh durch antitoxisches, wohl aber durch ein von ihnen gewonnoncs antibaktcrielles .Serum iin Meerschweinchen paralysiert werden, (^entralbl. f. Bakt. 121: 432, 1921. Jolinson, T. L.: In Vivo Trvpanolysis With Special Keferenee to Zones of Inhibition, Relapse Phenomena and Immunological Specificity, Am, J. Hyg. 9: 260, 1929. Kenipner, W.: Weiterer Beitrag zur Lehrc Aon der Fleischvergiftung. Das Antitoxin des Botulisraus, Ztschr. f. Hyg, 26: 481, 1897. Kitasato, S.r Experimentelle Untersuehungen iiber das Tetanusgift, Ztschr. f. Hyg. 10: 267, 1891. Klebs, E.r Ueber Diphtheria, ihre parasitare Natur Verhaltniss des localen Prozesses zur allgemeinen Infection, Contagiositat, Therapie, und Prophylaxie, Verhandlungen des Congresses f. Inn. Med. 2: 125, 1883. Larsen, N. P., and Nigg, C: Some Observations on the Wassermann and Kahn Reactions, J. Lab. & Clin. Med. 13: 843, 1928. Larson, W. P., and Nelson, E.: The Effect of the Surface Tension of the Medium Upon Bacterial Toxins, Proc. Soc. Exper. Biol. & Med. 31: 278, 1924. Larson, W. P., Evans, R. D., and Nelson, E.: Th« Effect of Sodium Ricinoleate upon Bacterial Toxins, and the Value of Soap-Toxin IMix- tures as Antigens, Proc. Soc. Exper. Biol. & Med. 22: 194, 1924. Larson, W. P., Halvorson, H. D., Evans, R. D., and Green, R. G.: Colloid Symposium Monograph, New York, 1925, Chemical Catalogue Co. Loeffler, F.: See Loeffler's Contribution in The Bacteriology of Diphtheria, London, 1913, Nuttall and Graham-Smith, Cambridge University Press, Sect. 1, p. 1. Longcope, W. T.: Cited by Coca. Essentials of Immunology, Baltimore, 1925, Williams & Wilkins Co. Madsen, T.: Allgemeines iiber baktcrielle Antigene — Toxine, der Anti- korper antitoxische Eigenschaften aufweisen, Kraus und Levaditi Hand, der Tek. u. Meth., 1908, Vol. I, p. 35. Mallory, E. B.: The Bacteriology of Diphtheria by Nuttall and Graham- Smith, London, 1913, Cambridge University Press, Sect. 5, p. 54. Martin, L.: ]898. Cited by Dean, 3913, p. 462. Maver. M. E.: The Growth and Toxin Production of Corynebacteriuni Diphtheriae in Synthetic Mediums, J. Infect. Dis. 47: 384, 1930. Maver, M. E.: The Physical Chemistry of Toxin and Antitoxin, Newer Knowledge of Bacteriology and Immunology, .lordan and Falk, (Chicago, 1928, University of Chicago Press, p. 739. Molonev, P. J., and Weld, C. B.: Diphtheria Toxin- Antitoxin Flocculation (Ramon Test), J. Path. & Bact. 28: 655, 1925. Moloney, P. J., and Taylor, E. M. : The Preparation of Stabilized Schick Toxin, J. Pub. Health 22: 38, 1932. Meyer, H. H., and Gottlieb, R. : Pharmacology, Clinical and Experi- mental; a Groundwork of Medical Treatment (author, trans, by J. T. Halsey), Philadelphia and London, 1914, J. B. Lippincott Co. Moser, P.: Ueber die Behandlung des Scharlachs mit einem Scharlach- Streptokokkenserum, Wien. klin. Wchnsehr. 15: 1053, 1902. Neill, J., Fleming, W., Sugg, J., Avery, R., Richardson, L., and Kane, B. : H^-persensitiveness to Diphtheria Bacterial Products. Comparison of Highly and Weaklv Toxicogenic Strains, .J. Imnninol. 18: 437, 1930. 272 IMMUNOLOGY Neill, J., Fleming, W., Sugg, J., and Gaspari, E. L.: Hypersensitiveness to Diphtheria Bacterial Products. Prevention of Hypersensitive Eeactions by Neutralization of the Toxin Previous to Intradermal Injection, J. Immunol. 18: 455, 1930. Nicolle, M., Cesari, E., and Debains, E.: Etudes sur la precipitation mutuelle des anticorps et des antigenes, Ann. Inst. Pasteur 34: 596, 1920. Northrop, J. H.: Purification of Diphtheria Anti-toxin, Science 93: 92, 1941. Olitsky, P. K., and Kligler, I. J.: Toxins and Antitoxins of Bacillus Dys- enteriae Shiga, J. Exper. Med. 31: 19, 1920. Olitsky, P. K., and McCartney, J. E.: Separation of the Toxins of Bacillus Dysenteriae Shiga, J. Exper. Med. 37: 767, 1923. Pappenheimer, A. M., Jr., and Eobinson, E. S. : A Quantitative Study of the Ramon Diphtheria Flocculation Reaction, J. Immunol. 32: 291, 1937. Park, W. H.: 1897. Cited by Park and Williams, Pathogenic Microor- ganisms, Philadelphia, 1933, Lea & Febiger, p. 406. Park, W. H., Zingher, A., and Serota, M. H.: 1914. Cited by Park and Williams, Pathogenic Microorganisms, Philadelphia, 1933, Lea & Febiger. Park, W. H.: See Park and Schroder, 1932. Park, W. H.: (1903) Cited by Park and Williams, Pathogenic Micro- organisms, Philadelphia, 1933, Lea & Febiger. Park, W. H.: (1919) The Schick Reaction and Its Practical Application, Monthly Bull. Dept. Health, New York City 9: 65, 1919. Also cited by Park and Williams, Pathogenic Microorganisms, 1933, Phila- delphia, Lea & Febiger. Park, W. H., and Schroder, M. C: Diphtheria Toxin-Antitoxin and Toxoid. A Comparison, Am. J. Pub. Health 22: 7, 1932. Park, W. H., and Williams, A. W. : Pathogenic Microorganisms, Phila- delphia and New York, 1933, Lea & Febiger, p. 403. Parker, J. T. : The Production of an Exotoxin by Certain Strains of Staphylococcus Aureus, J. Exper. Med. 40: 761, 1924. Pick, E. P.: Handbuch der Technik und Methodik der Immunitats- forschung, Kraus and Levaditi, Jena, 1908, Gustav Fischer, p. 331. Pope, C. G.: Disaggregation of Proteins by Enzymes, Brit. J. Exper. Path. 19: 245, 1938. Provitsky, O. R.r Ramon Flocculation Test for Determining Potency of Antiscarlatinal Serum, Proc. Soc. Exper. Biol. & Med. 22: 426, 1925. Degree of Immunization from Injection of Diphtheria Toxoid (a) of a Different Strength Toxoid (b) at Varying Intervals (c) of Treated Toxoids, Am. J. Pub. Health 22: 29, 1932. Ramon, G.: Floculation dans un melange neutre de toxine-antitoxine diph- teriques, Compt. rend. Soc. de Biol., Paris 86: 661, 1922. Sur une tech- nique de titrage in vitro du serum antidiphteriques. Ibid. 86: 711, 1922. A propos du titrage in vitro du serum antidiphterique par la flocculation. Ibid. 86: 813, 1922. Ramon, G. : La floculation dans les melanges de toxine et de serum anti- diphterique, Ann. Inst. Pasteur 37: 1001, 1923. Sur la toxine et sur 1 'anatoxine diphteriques pouvoir floculant et properietes im- munisantes. Ibid. 38: 1, 1924. Ramon, G. : Combined (Active-Passive) Prophylaxis and Treatment of Diph- theria or Tetanus, J. A. M. A. 114: 2366, 1940. Renaux, E. : Sur le floculation de la toxine diphterique par le serum anti- diphterique, Compt. rend, Soc. de Biol., Paris 90: 964, 1924. TOXINS AND ANTITOXINS 273 Robertson, M. : An Experiment on Vaccination with a Coccus Derived from Human Cases, J. Path. & Bact. 21: 173, 1916. Eomer, P. H.: Ueber den Nachweis sehr kleiner mengen dcs Diphtherie- giftes, Ztschr. f. Immunitatsforsh. u. exper. Therap. 3: 208, 1909. Romer, P. H., and Sames, T.: Zur Bestimmung sehr kleiner mengen Diph- therieantitoxins, Ztschr. f. Immunitatsforsh. u. exper. Therap. 3: 344, 1909. Rosenau, M. S., and Anderson, .T. F.: The Standardization of Tetanus Anti- toxin, Hygienic Lab. Bull., 1908, p. 43. Roux, E., and Yersin, A.: Contribution a 1 'etude de la diphterie. Etude experimentale du B. diphterique, Ann. Inst. Pasteur 2: 629, 1888. Contribution a 1 'etude de la diphterie. Etude experimentale du B. diphterique, Ibid. 3: 273, 1889. Schick, B.: Die Diphtherietoxin— Hautreaktion des Menschen als Vorprobe der prophvlaktischen Diphtherieheil-seruminjektion, Miinchen. med. Wchnschr.'eO: 2608, 1913. Schmidt, H.: Zur kenntnis der Natur der Diphtherie-toxin-antitoxin-flock- ung, Ztschr. f. Imnmnitatsforscli. u. exper. Therap. 48: 217, 1926. Smith, T.: Active Immunity Produced by So-called Balanced or Neutral Mixtures of Diphtheria Toxin and Antitoxin, J. Exper. Med. 11: 241, 1908. Smith, T., and Brown, H. R.: Further Studies on the Immunizing Effect of Mixtures of Diphtheria Toxin and Antitoxin, J. Lied. Research 23: 433, 1910. Smith, T.: The Degree and Duration of Passive Immunity to Diphtheria Toxin Transmitted bv Immunized Female Guinea Pigs to Their Im- mediate Offspring, .T.'Med. Research 16: 359, 1907. Taliaferro, W. H., and Johnson, T. L. : Zone Phenomena in "In Vivo" Trvpanolysis and the Therapeutic Value of Trypanolytic Sera, .J. Prev. Med. 1: 85, 1926. Taylor, E. M., and Moloney, P. J.: A New Schick-toxin, J. Immunol. 37: 223, 1939. Todd, C: On a Dysentery Antitoxin, Brit. M. J. 2: 1456, 1903. Wassermann, A.: Experimentelle unter.suehungen, iiber einige theoretische punkte der immunitatslehre, Ztschr. f. Hyg. 22: 263, 1896. Wells, J. R. : Intradermal Method of Virulence Testing of Corynebacterium Diphtheriae, Am. J. Pub. Health 22: 308, 1932. Wells, H. G.r Chemical Aspects of Immunity, New York, 1929, The Chem- ical Catalog Co., pp. 49 and 132. Welhs, D. J., Graham, A. H., and Havens, L. C: Diphtheria Toxoid Precipitated with Alum. Its Preparation and Advantages, Am. J. Pub. Health 22: 648, 1932. Wernicke, R.: 1895. Cited by Dean, p. 503, also by Smith, 1907, p. 360. Zinsser, H. : Toxin and Antitoxin, Resistance to Infectious Diseases, New York, 1931, The Macmillan Co., p. 147. Supplementary References Ando, K., and Nishimura, H. : On the Heat Stability of the Diphtheria Toxin, J, Immunol. 19: 465, 1930. Berthelsen, K. C: A Study of the Relationship of Surface Phenomena to the Reaction of Toxin and Antitoxin, With Toxin Produced in an In- fusion-Free Peptone Medium, J. Immunol. 21: 21, 1931. Berthelsen, K. C. : Studies of the Flocculation Reaction Time in the Course of Immunization and the Quantitative and Qualitative Changeg Proteins, J. Immunol, 21: 43, 1931. ijj LIBRARY [^ 274 IMMUNOLOGY Berthelsen, K. ('., and ^furdick, P. P.: The Distribution of Electrolytes in Serum During Immunization. J. Immunol. 21: 69, 1931. Bezi, I.: A Study of Action of Saliva and Extract of Tonsils Upon Diph- theria Bacillus and Diphtheria Toxin, J. Immunol. 22: 1, 1932. Bunnev, W. E.: A New Diluent for Diphtheria Toxin in the Schick Test. J. Immunol. 20: 71, 1931. Bunney, W. E.: The Action of Formaldehyde on Diphtheria Toxin, J. Im- munol. 20: 47, 19.11. Bunnoy, W. E., and White, B. : Advantages and Disadvantages of the Buf- 'fored Diluent for Diphtheria Toxin, J. Inuminol. 20: 61, 1931. Ecker, E. E., and Weed, L. A.: Studies on the Adsorption of Diphtheria Toxin to an Evolution From Magnesium Hvdroxide, J. Immunol. 22: 61, 1932. Freund, .1. : On tlie Meclianism of Toxin- Antitoxin Reaction. J. Immunol. 21: 127, 1931. Freund, .!., and Bonanto, Mary V.: Antitoxin-Formation After Intra- or Subcutaneous Injection of Plain or Alum Diphtheric Toxoid, J. Im- munol. 40: 437, 1941. Githens, T. S. : Factors Influencing the Antigenic Power of Toxin-Antitoxin Mixtures, .1. Immunol. 22: 197, 1932. Gerlough, T. D., and WHiite, W. : Purification of Concentrated Antitoxins, J. Immunol. 22: 331. 1932. Hagen, E. L.: The Relation of the Bacterial Precipitin Reaction to the Ramon Flocculation Phenomenon, .1. Immunol. 19: 393. 1930. Johlin, .T. M., and Rigdon, R. H. : The Detoxification of Staphylococcal Toxin by Adsorption on Organic Liquids, J. Immunol. 41: 233, 1941. Kirkbride. M. B., Berthelsen, K. C, and Clark, R. F. : Comparative Studies of Infusion and Infusion-Free Diphtheria Toxin in Antitoxin Produc- tion and in Standardization by the Flocculation, Subcutaneous, and Intracutaneous Test, J. Immunol. 21: 1. 1931. Leonard, B. F.. and Varlev, .1. R.r The Effect of Alum on Horses Used for the Production of Diphtheria Antitoxin. .1. Immunol. 23: 261, 1932. Locke, A., and Main, E. R.: The Relation of Copper and Iron to Produc- tion of Toxin and Enzyme Action, J. Infect. Dis. 48: 419. 1931. Locke, A., Main, E. R.. and Miller, F. A.: The Production of Diphtheria Antitoxin, J. Infect. Dis. 41: 32, 1927. Locke. A., and IMain. E. R.r (Cited by Sanderson and Yol.) Mishulow, L., aTid Xrumwiede, C: (Cited by Sanderson and Yol.) Morton. H. E. : Corvnebacterium Diphtheriae. A Correlation of Recorded Yariations Within the Species, Bact. Rev. 4: 177, 1940. Morton, H. E. : Corjiiebacterium Diphtheriae. II. Observations and Disso- ciated Studies — the Potentialities of the Species, ,T. Bact. 40: 755, 1940. Myers, H. R.: A Contribution to the Studv of the Etiology of Serum Disease, .1. Immunol. 22: 83. 1932. Ncill, .1. M., Gaspari, K. L.. and Sugg, J. Y.: Diphtiieria Antibodies in the Urine of a Child After Intramuscular Injection of Antitoxic Hor.se Globulin, J. Immunol. 20: 187, 1931. Xeill, J. M., Gaspari, E. L., and Mosley, R. A.: Loss of Immune Substance From the Body. I. Diphtheria Antitoxin in Human Urines, J. Immunol. 20: 347, 1931." Neill, J. M., Gaspari, E. L., Mosley, R. A., and Sugg, J. Y. : Loss of Im- nuine Substances From the Body. III. Diphtheria Antitoxin in Human Sweat, J. Immunol. 21: 101, 1931. Neill, J. M., Gaspari, E. L., Richardson. L. Y., and Sugg, J. Y. : Diphtheria Antibodies Transmitted From Mother to Child, J. Immunol. 22: 117, 1932. TOXINS AND ANTITOXINS 275 Neill, J. M., Sugg, J. Y., and Eichardson, L. V.: Anaphylaxis to Diphtheria Toxin. Passive Transfer Experiments, J. Immunol. 22: 131, 1932. Provitzky, O. R. : Diphtheria Toxoid, Preparation and Dosage, J. Immunol. 20: 247, 1931. Provitzky, Olga R. : Diphtheria Toxin, Importance of Old and New Factors in Its Production, J. Immunol. 16: 421, 1929. Ramon, G., and Lemetayer, E. : Method for Increasing and Prolonging the Production of Antitoxin in Horses, J. Immunol. 22: 125, 1932. Reiner, L.r Comparison of the Combining, Antigenic and Toxic Properties of Chemically Altered Diphtheria Toxoid and Toxin, ,T. Immunol. 24: 213, 1933. Reiner, L. : Adsorption of Diphtheria Toxoid by Cellulose Derivatives From Iron Hydroxide Gel, J. Immunol. 24- 221, 1933. Reiner, L.: The Purification and Concentration of Diphtheria Toxoid by Means of Electrodialysis, J. Immunol. 22: 439, 1932. Richardson, L. V.: Diphtheria Antibodies Transmitted to the Offspring of Immune Guinea Pigs, J. Immunol. 22: 351, 1932. Robinson, E. S., and White, B. : Effect of Exposure to Low Temperatures on Diphtheria Toxin-Antitoxin Mixtures. Second Communication, .1. Im- munol. 15: 381, 1928. Sanderson, E. 8., and Yoi, J. H. : Observations on the Proposed Gold Chloride Titration for Determining the Toxicity of Diphtheria Toxin, J. Im- munol. 16: 429, 1929. Siebenmann, ('.: Comparison of Different Diphtheria Antitoxin Serums With Regard to Their Rate of Flocculation, .T. Immunol. 23: 285, 1932. Sugg, J. Y., Ricliardson, E. V., and Neill, .7. M.r Hypersensitiveness to Dipli- theria Bacterial Products. Inhibition of the Anapliylaxis of Anti- toxic Immune (Actively Sensitized) Guinea Pigs, .T. Immunol. 20: 25, 1931. Sugg, J. Y., Richardson, L. V., and Neill, J. M. : Transmission to the Third Generation of Antitoxin Derived by Active Immunization of the First Generation, J. Immunol. 20: 255, 1931. Sugg, J. Y.: Diphtheria Antibodies Transmitted From Mother to Child, J. Immunol. 22: 117, 1932. Sugg, J. Y., Richardson, L. V., and Neill, J. M.: Anaphylaxis to Diphtheria Toxin. Sensitization by Placental Transmission of Antitoxin, J, Im- munol. 22: 401, 1932. Surgeon General : Active Immunization Against Tetanus by Vaccination With Tetanus Toxoid, Circular Letter No. 34, J. A. M. A. 116: 2857, 1941. Wadsworth, A.: The Action of Bacterial Toxins, J. Immunol. 26: 81, 1934. Wadsworth, A. B., and Wheeler, M. W. : The Attenuation and Toxin Produc- tion of the Diphtheria Bacillus. Attenuation of Diphtheria Bacillus; Svnthetic Mediums, Factors Affecting Growth and Toxin Production, J. Infect. Dis. 42: 179, 1928. Wadsworth, A. B., and Hoppe, E. N. : The Neutralization or Destruction of Diphtheria Toxin by Tissue, J. Exper. Med. 53: 821, 1931. White, B., Bunney, W. E., and Malcolm, W. G.: An Improved Diluent for Diphtheria Toxin in the Schick Test, J. Immunol. 22: 93, 1932. CHAPTER XV TOXINS AND ANTITOXINS (CONTINUED) CONVALESCENT AND IMMUNE SERA Introduction. — There are a number of diseases other than diph- theria in which the physician may find it desirable to use a specific immune or convalescent serum for diagnosis, prophylaxis or treat- ment. Among these diseases are scarlet fever and some other streptocDceus infections, pneumococcus pneumonia, meningococ- cus meningitis, measles, poliomyelitis, tetanus, botulism, gas gangrene and tularemia. It is the purpose of this chapter to bring out a few important immunological facts relative to each of the diseases mentioned. Scarlet Fever. — Scarlet fever is a toxemic and infectious dis- ease which is moderately contagious. It is quite generally con- ceded that Dick and Dick (1923) established a beta hemolytic streptococcus as the causal agent. The disease is characterized by fever, leucocytosis, angina (sore throat), a diffuse erythema that can be blanched by scarlet fever antitoxin or by pooled convalescent serum, desquamation, and the development of a lasting immunity following recovery. The subject of scarlet fever is treated so extensively in a mono- graph by Dochez (1924) and in all of the standard texts of Pathogenic Bacteriology that only a few salient facts will be mentioned in this chapter. The evidence which points to the hemolytic streptococcus as the etiological factor may be summarized as follows : 1. Beta hemolytic streptococci are invariably found associated with the disease. 2. Dick and Dick (1923) experimentally reproduced typical cases of scarlet fever in man with pure cultures of these strepto- cocci and apparently satisfied the postulates of Koch. 3. The organisms produce a soluble toxin, when properly cul- tured, that is capable of producing fever, rash, leucocytosis, desquamation, and antitoxin when injected in sufficient amounts into a susceptible human subject. 276 TOXINS AND ANTITOXINS 277 4. Hpet'ific antitoxin obtained by innnunizing horses will blanch the rash of scarlet i'ever apparently in the same way that pooled convalescent serum blanches it. This is called the Schultz-Charl- ton blanching test. 5. When streptococcus antitoxin is used in the treatment of severe cases of scarlet fever, there is a striking clinical improve- ment. Blake and Trask showed that there is a drop in tempera- ture and a rapid clearing up of the rash and other symptoms of toxemia. 6. Susceptibility to the toxin as indicated by the skin test de- vised by Dick and Dick can be made to disappear by the injection of an adequate number of immunizing doses of toxin. 7. According to the Dicks (1924) immunization Avith scarlatinal toxin produces immunity to scarlet fever. 8. They also maintain that susceptibility as indicated by the skin test (Dick test) correlates with susceptibility to scarlet fever. A review of the literature indicates that at the present there is a great deal of confusion and disagreement over the classifica- tion and identification of scarlet fever streptococci as well as the interpretation of studies relative to the efficiency of immuni- zation against and the determination of susceptibility to the dis- ease. Our experience with the Dick test (1926) led us to feel that in specific instances it is not as accurate a criterion of sus- ceptibility to scarlet fever as the Schick test is to diphtheria al- though it may reflect the percentage of susceptibility in a group at large with a reasonable degree of accuracy. To be of maximum value to the physician it must tell him specifically whether a child is or is not susceptible to scarlet fever. Subsequent investigations of toxins produced by different strains of hemolytic streptococci have revealed wide antigenic variations. Ando and others have shown that the toxic filtrates of streptococcus broth cultures contain two important substances which give rise to red skin reactions when injected intradermally. One of these is the true toxin and the second is a nucleoprotein derivative of the streptococcus. These discoveries explain many but not all of the discrepancies we observed in our studies of the Dick test. 278 ' IMMUNOLOGY The Dicks report statistics that indicate a very high percent- age of efficiency for the susceptibility test. Prophylactic Immunization. — In regard to the value of prophy- lactic immunization against scarlet fever, there is much disagree- ment. The results reported by the Dicks are apparently quite definite. They observed no cases of scarlet fever among 1,191 susceptible interns and nurses who had been immunized with toxin whereas they noted 37 cases of scarlet fever in a control group. Hektoen and Johnson (1934) report statistics from the Durand Hospital which support the findings of the Dicks. There are at present a great many who report results at variance with those just cited. Zinsser, Enders and Fothergill (1939) suggest that active immunization with the Dick toxin may be of value among nurses and interns but do not recommend large scale im- munization as a part of a public health program. The Dick Test and Toxin Valency. — While a negative Dick test indicates a certain amount of immunity to the toxins of scarlet fever streptococci, it is not a measure of immunity to infection with the organisms. It has been shown by Kirkbride and Wheeler (1924) and others that hemolytic streptococci isolated from cases of erysipelas are able to produce scarlet fever toxins. Downs and Stookey (1932) have shown that hemolytic streptococci from cases of puerperal sepsis produce toxins indistinguishable from those produced by scarlet fever streptococci. Similar results have been obtained for hemolytic streptococci from a number of pathologi- cal conditions (Wadsworth, 1934). Furthermore, Wheeler (1932), Wadsworth (1934), Trask and Blake (1933), and others have called attention to antigenic differences in the toxins of scarlet fever streptococci. Antitoxin specific for one strain may not neutralize the toxin produced by another. Since scarlet fever streptococci are of "questionable specific- ity," Zinsser and Bayne-Jones (1939) recommend that strains that possess as great an antigen valency as possible be selected for toxin production. They call attention to the Dochez N Y 5 strain which produces a toxin of "great antigenic valency." The Dicks have used a polyvalent toxin; i.e., one prepared by mixing the toxins of several strains to meet this requirement. TOXINS AND ANTITOXINS 279 At the present time it is possible to procure what is regarded as an efficient antistreptococciis antitoxin. It may be used for temporary protection lasting two or three weeks or as a therapeutic agent. Measurement op Antitoxin and Toxin. — The unit of antitoxin established by the federal government is described as follows : "One unit of antitoxin is the smallest amount of antitoxin which neutralizes 50 skin test doses of scarlatinal streptococcus toxin." (Zinsser and Bayne- Jones, 1939, p. 300.) Wadsworth* states that the skin test dose of toxin is "the least quantity of toxin, which, when injected intracutaneously into persons known to be susceptible to the toxin, will induce a reaction equal to that induced on the same persons at the same time by the injection of a skin test dose of the standard toxin supplied by the U. S. National Institute of Health (Hygienic Laboratory)." This dose is contained in a volume of 0.1 c.c. A positive reaction appears as a pink or red area at least one centimeter in diameter usually within six to twelve hours and begins to fade after about twenty-four hours. It is usually read after twenty-two to twenty-four hours. Schultz and Charlton Blanching Test. — In 1918 Schultz and Charlton reported that the serum of those convalescing from scarlet fever wdll blanch the rash of scarlet fever when injected intradermally. Their results have been extensively confirmed. Toomey and Nourse (1924) state that it is necessary to use pooled convalescent sera to obtain an efficiency of approximately 100 per cent in diagnosis. They recommend the intradermal in- jection of one cubic centimeter of serum and that the test be read not earlier than eight and preferably not until after twenty- four hours. Commercial antitoxin for use in the blanching test is equally satisfactory. Potency of Convalescent Serum. — The question arises not in- frequently as to the relative value of convalescent serum and com- mercial antitoxin as a diagnostic prophylactic or therapeutic agent. The interesting work of Rhoads and Gasul (1934) bears directly upon this question. They call attention to the meager- ness of reports of the use of convalescent serimi for the protec- tion of contacts since its therapeutic value was demonstrated by ♦Wadsworth: J. Immunol. 31: 255, 1931. 280 IMMUNOLOGY Weissbecker in 1897. The amount used for protecting contacts has varied. Neff (1922) obtained satisfactory results with doses varying from 15 to 30 c.c, but as a rule smaller doses have been employed. Rhoads and Gasul state that the therapeutic dose has varied from 10 to 240 c.c. and that there is at present a ''trend toward smaller doses." They determined the potency of twelve lots of pooled convalescent serum and found that it varied from 250 to 1,000 neutralizing units per cubic centimeter (one unit neutralized one skin test dose of toxin). The govern- ment requires that commercial scarlet fever antitoxin have a potency of 15,000 neutralizing units per cubic centimeter. This would indicate that so far as antitoxic potency is concerned the commercial antitoxin is definitely superior to convalescent serum. Park's Recommendations. — Park (1928) calls attention to the value of antistreptococcus antitoxin, convalescent serum and even normal serum or citrated normal blood in the treatment of severe cases of scarlet fever, especially where sepsis is present. For the purpose of treatment with serum or whole blood, he divides scar- let fever cases into two groups. Early Malignant Cases. — In group one he places the earh^ malignant cases seen between the first and the fourth day. In these cases there are observed delirium, restlessness, deep red petechial rash, marked adenitis and severe angina. While specific anti- toxin is of great value and may be used, it is also possible to inject intramuscularly convalescent serum or blood taken during the second or third week of convalescence and obtain excellent therapeutic results. The blood is obtained from the median basilic vein of the arm, citrated and injected into various mus- cles of the patient. Park suggests the triceps, vastus externus (thigh), soleus (calf of leg), and gluteal muscles. In young children he would administer 15 c.c. and in older children 30 c.c, in each of these places. The same sites may be used for subse- quent injections since the blood is rapidly absorbed. About six hours after the administration of the blood, the temperature begins to drop and reaches normal in twenty-four to thirty hours. There occurs also as a rule an early fading of the rash and a disappearance of other evidence of the toxemia. Ijate Septic Cases. — In the second group he places the later septic cases seen between the fifth and the eighth day of the disease. TOXINS AND ANTITOXINS 281 While the rasli may have faded, tliey have a high, septic tempera- ture (103° to 105° F.), the tonsils and fauces are extensively inflamed and covered with membrane, and there is a persistent cervical adenitis. This group also responds to antitoxin and convalescent serum or blood. Park states that whole normal blood has been used by Zingher in the treatment of eight cases belonging in this group. He found it to be of definite therapeutic value. Its administra- tion leads to a distinct lowering but not to a striking critical drop in tlie temperature and to a definite improvement in symp- toms. Immune-Transfusion. — In addition to the use of antitoxin, nor- mal and convalescent sera in the treatment of scarlet fever with sepsis, there has also been employed the blood of individuals actively immunized with bacterial vaccines. This procedure called immuno-transf\isio7i was originally recommended by Wright (1919) for the treatment of typhoid fever. It has not been used extensively. For a more extensive discussion of scarlet fever streptococci, their toxins and antitoxins, the student is referred to Zinsser and Bayne- Jones (1939) or some other standard textbook of pathogenic bacteriology. Erysipelas. — Another interesting disease caused by the beta hemolytic streptococci is erysipelas. It is an acute infection of the skin appearing as a red, swollen, inflamed area, diffuse at first and later showing a sharp line of demarcation. General febrile changes usually occur. Within a few days the redness and in- filtration may disappear and tlie skin appear normal or perhaps rcddisli brown, dry and desquamating. The disease lasts usually for one or two weeks. Except in debilitated individuals, the prognosis is usually good. The bacteria in the lesion are present in the lymph spaces of the corium. As the former advance, the area of inflammation follows. For an excellent discussion of the minute lymphatics of the living skin the student is referred to the work of Hadack and MacMaster (1933). Types of Lesions in Erysipelas. — A study of the inflammatory exudate in erysipelas shoM^s that it is rich in large mononuclear 282 IMMUNOLOGY phagocytic cells (elasmatocytes). While the inflammation leads to a general systemic reaction, it is not as a rule accompanied by suppuration. Various severe types of the disease may occur. They are, according to Kaufmann, given descriptive names sucli as erysipelas vcsiculosum, erysipelas pustulosum, erysipelas gan- grenosum and erysipelas phlegmonosum, respectively. The latter term applies to an inflammation (erysipelas) which becomes dif- fuse in both the skin and subcutaneous tissues with resulting sup- puration. The term indicates the transition to a phlegmon. Streptococci in Erysipelas. — Fehleisen (1883) not only con- sistently isolated streptococci from the lesions but also experi- mentally reproduced the disease in man. His work has been con- firmed repeatedly. He concluded that there is a distinct species of streptococcus responsible for the disease and accordingly named it Streptococcus erysipelatis. This conclusion lias led to a great deal of controversy. Birkhaug and othcT-s offer experimental support to the concept of Fehleisen, while Wheeler, Wadsworth, Williams, and others maintain that there is antigenic similarity between hemolytic streptococci from erysipelas, scarlet fever, and other pathological conditions. They are certain that there is no definite and strict correlation between the species of streptococcus and the pathological conditions it produces. Birkhaug and others have studied the soluble toxins produced by hemolytic strepto- cocci from erysipelas and have produced specific antitoxins of definite therapeutic value. McCann maintains that scarlet fever antitoxin is equally ef- fective in the treatment of the disease as are the antitoxins pro- duced by Birkhaug and others. At the present time it seems to be settled that a satisfactory antitoxin has been produced and is available in the treatment of erysipelas. To be effective it niusl be able to neutralize the toxin of the streptococcus involved in the particular case and also supply other specific antibodies. This indicates the importance of using toxins of wide antigenic valences in immunizing animals to produce antistreptococcus antitoxin. The other points at issue are at present unsettled. Puerperal Sepsis. — Puerperal sepsis or childbed fever may be due to hemolytic streptococci indistinguishable from those found in scarlet fever, or as Harris and Brown* (1929) and others have ♦Harris, J. W., and Brown, J. H. : Johns Hopkins Hosp. Bull. 44: 1, 1929. TOXINS AND ANTITOXINS 283 shown it may be caused by an anaerobic streptococcus first de- scribed by Schottmiiller in 1910. A review of the literature indi- cates that puerperal sepsis may be caused by organisms similar to those responsible for "gas gangrene." When the disease is caused by beta hemolytic streptococci, it is conceivable that an antistreptococcus antitoxin is of value if it can opsonize the bac- teria and neutralize the toxins of the particular hemolytic strep- tococcus causing the trouble. In the light of our present Iviiowl- edge one should not expect it to be of value in the treatment of the disease when the anaerobic streptococcus is the causal agent unless an antigenic relationship exists. Serum treatment of puer- peral sepsis has been quite unsatisfactory, very likely for the reasons given above. Streptococcus Septicemia. — At the present time there is no satis L'jietory specific scrum therapy for streptococcus septicemia. Frequent transfusions are used quite extensively with vaiying re- sults. Antistreptococcus serums are of apparent value in some cases. It seems that in streptococcus septicemia there is not only a deficiency of antibodies but there also exists a deficient activity and mobilization of the phagocytic cells of the body. Gay has shown that unless the phagocytic army is mobilized and active, the supplying of antibodies will be relatively ineffective. The value of chemotherapy in streptococcal and other bacterial infections is discussed later in this chapter. Immuno-Transfusion IN Septicemia. — Specific immuno-trans- fusion is recommended by Brady and Crocker (1932) in the treat- ment of streptococcus septicemia. Stevenson (1933) reports the use of nonspecific and specific immuno-transfusions in a case of hemolytic streptococcus septicemia, with satisfactory results. For the nonspecific immuno-transfusion normal individuals were given typhoid vaccine intravenously and put to bed for the moderate chill that followed in about one hour. Seven hours later 500 c.c. of a donor's blood was withdrawn, citrated, and injected into the pa- tient with septicemia. This type of treatment was repeated sev- eral times with beneficial results. During this period a vaccine was made from streptococci isolated from the patient and a nor- mal volunteer immunized. His blood was used in the final trans- fusion. There was observed a shift in the Schilling count from left to right after each transfusion. 284 IMMUNOLOGY It is obvious that this method of treatment has a limited appli- cation and that its efficiency is probably due to the antibody con- tent which Gay has shown is only one factor in the body's de- fense against streptococcus infections. The shift in the Schilling count may not be so significant as it would appear since there are many factors to be considered which affect the peripheral leuco- cyte count. Blood Banks. — The practice of citrating blood and storing it at 4° to 6° C. is quite common. Kolmer (1940) reviews the liter- ature in connection with his own experimental studies and con- cludes that blood is ''better preserved by the addition of glucose" as suggested by Rons and Turner (1916) or of dextrin as sug- gested by Maizels and Whittaker (1940).* Pneumococcus Pneumonia. — Pneumonia, both lobar and bron- cho- due to the pneumococcus, is one of the principal causes of death in the United States. Until 1929 it was generally accepted that there are three specific types of pneumococci and an hetero- geneous group called by Dochez and Gillespie (1913) Type IV. Olmstead (1917) discovered that the latter type is composed of a number of small groups rather than single strains. Recently Cooper et al. (1929, 1932), working in Park's laboratory, have divided Group IV into 29 new types. Additional types have been described by Kauffmann, M0rch and Schmith (1940) and by Walter, Guevin, Beattie, Cotler and Bucca (1941). Their results bring the total number of different pneumococcal types including subtypes to 55. A subtype is one that shows cross reaction with immune serum for another type. Park (1933) said that it is pos- sible to identify fifty or sixty types but only seven or eight are prevalent and therefore important. Types in Lobar and Bronchopneumonia. — Sutliff and Finland (1933) report six types as being responsible for 84.1 per cent of their cases of pneumococcus lobar pneumonia. They list them relative to their order of frequency as Types I, II, III, VIII, V, and VII. They report also the order of frequency of the ten types, e.g., Ill,' VIII, XVIII, X, V, VII, XX, II, XI, and XIV which were responsible for 81.1 per cent of their cases of pneumo- coccus bronchopneumonia. Their findings are quite similar to those of Avery et al. (1917) who pointed out that Types I and II ♦See also Studies on Preserved Human Blood, J. A. M. A. 114: 850, 858, 859, 1940. TOXINS AND ANTITOXINS 285 were responsible for 66.8 and Type III for 13.0 per cent of 454 cases of pneiiniococcus lobar pneumonia studied by them. History of Serum Treatment of Pneumonia. — The history of the serum treatment of pneumococcus pneumonia extends back to the beginning of the last decade of the nineteenth century. Ac- cording to Cole and Dochez (1915), the first to apply pneumo- coccus immune serum from animals in the treatment of lobar pneumonia were G. and F. Klemperer (1891). They used serum from highly immunized rabbits in the treatment of 18 cases and observed improvement in some and failures in others. Foa and Scabia (1892) and Jansson (1892) also reported some favorable results from the use of immune rabbit serum in lobar pneumonia. Washbourne (1897) and Payne (1897) employed immune sera from horses and donkeys respectively and noted improvement in some of their cases. Eyre and Washbourne (1899) reported that serum sent them by Payne was effective against four strains of pneumococei but ineffective against a fifth which they had. While tliere were a number of reports favorable to the serum treatment of lobar pneumonia, there w^ere a great many that were definitely unfavorable. Discovery of Types. — It was not until Neufeld and Handel (1910) and Dochez and Gillespie (1913) had demonstrated defi- nite types of the pneumococcus, and that immune serum is type specific, that any light was thrown upon the failures of serum treatment of lobar pneumonia. Neufeld and Handel called atten- tion to the importance of larger doses of serum in the treatment than had been used previously. They also introduced the mouse protection test as a method of determining the potency of im- mune serum. Discovery of Soluble Spp:cific Substance. — In 1917 two other important contributions to our knowledge of the etiological agent were made. Dochez and Avery discovered the soluble specific sub- stances and as previously mentioned Olmstead reported that T.ypc IV is made up of a number of groups rather than single strains of pneumococei. The soluble specific substances of Types I- XXXII are discussed by Brown* (1939). Concentration of Antibody. — Efforts had been made by Gay as early as 1915 to increase the protective property of specific im- ♦Brown, R. : Chemical and Immunologrical Studies of Pneumococcus Soluble Specific Substances of Types I-XXXII. J. Immunol. 37: 445, 1939. 286 IMMUNOLOGY mime serum by methods of concentration, but it was not until 1924 that a satisfactory method was devised. This was accom- plished by Felton whose concentrated antibody solutions of Types I and II sera are now used extensively. Efforts to produce an effective Type III antibody solution have been uniformly unsuc- cessful. Up to the present time the best results obtained in the serum or concentrated antibody treatment of pneumonia are in Type I and to a lesser extent in Type II infections. The introduc- tion of a type specific antipneumococcic rabbit serum promises bet- ter results in some types other than I. (Goodner, Horsfall and Dubos, 1937.) Result of Serum Treatment. — To be effective large intra- venous or intramuscular injections should be administered early in the disease ; i.e., before the fourth day. Statistics indicate that the mortality can he reduced 50 per cent by such treatment. The serum treatment of pneumonia has been most successful with severe cases Avhen combined with chemotherapy. An excellent dis- cussion of serum therapy and chemotherapy in pneumonia is given by Finland, Spring and Lowell (1940). Bullowa (1934) reports definite therapeutic value for Type VIII serum in pneu- monias due to the corresponding type of pneumococcus. This is interesting in vicAV of the close relationship of Type VIII and Type III pneumococci and the ineffectiveness of serum therajiy in pneumonia due to the latter. Enzyme Treatment of Type III Pneumococcus Infection. — While serum therapy has been found ineffective in the treatment of Type III pneumococcus pneumonia Dubos and Avery (1931) report that a bacterial enzyme, which they had previousl^y re- ported (1930) as possessing the property of destroying the capsu- lar polysaccharide of the Type III pneumococcus, is of thera- peutic value in the treatment of Type III pneumococcus infection in mice. Goodner, Dubos and Avery (1932) report that dermal infections (Type III pneumococcus) in rabbits undergo an early termination with recovery following the intravenous injection of adequate amounts of the enzyme. The infection is fatal in un- treated animals. Whether this method can be used to treat suc- cessfully Type III pneumococcus infection in man remains to be determined. It has long been known that the capsule is associ- ated with virulence, and as a result of the extensive work of TOXINS AND ANTITOXINS 287 Avery, Goebel, Heidelberger and others upon the chemical na- ture of the type specific polysaccharides, it has been established that they are definitely correlated also with virulence. Appar- ently when the polysaccharide of Type III pneumococcus is de- stroyed by the enzyme tlie organisms are deprived of their most important weapon of offense and can he destroyed rapidly l)y tlie body. In the case of serum therapy in pneumococcus pneumonia part of the antibodies injected unite with circulating polysac- charide and thus the amount of the former that is available for opsonification is reduced. In a subsequent paper Dubos (1940) discusses in more detail the enzyme mentioned above and also re- ports the discovery of a bactericidal, or at least a bacteriostatic, agent (Gramicidin) in extracts of a sporulating soil bacillus. It acts upon gram-positive but not upon gram-negative bacteria and is quite toxic for the animal body. Chemotherapy in Pneumococcal and Other Bacterial Infec- tions.— It is generally agreed that 1932 marks the beginning of a renaissance in chemotherapy. In that year, Domagk, director of the pathological laboratory in Elberfeld, Germany, discovered that prontosil has marked therapeutic value in streptococcal septicemia in mice. In 1933, Foerster treated successfully a case of staphylo- coccal septicemia in a ten-month-old infant with prontosil. Domagk (1935) introduced a second therapeutic azo dye which he called prontosil soluble and reported additional success in the treatment of both experimental and clinical streptococcus infec- tions. It remained, however, for Colebrook and Kenny, and But- tle, Gray and Stephenson in England, Love, Bliss and Marshall, and Mellon, Gross and Cooper in America to arouse interest among clinicians and to discover that the sulfanilamide portion of pronto- sil is the active chemotherapeutic agent. It is interesting to note that sulfanilamide was first prepared by Gelmo in 1908 in the course of his investigations of azo dyes. In 1938 Whitby reported that a new sulfonamide drug, sulfapyridine, synthesized in Eng- land by Ewins and Phillips, was of therapeutic value in pneumo- coccal and staphylococcal infections in mice. In 1939 suLfa- thiazole and its methyl derivatives were prepared by Fosbinder and Walter, and Lott and Bergeim independently. According to Goodman and Gilman (1941) over 1200 organic compounds con- taining sulfur have been synthesized and studied. New com- 288 IMMUNOLOGY pounds are being produced and investigated quite rapidly. At the present time sulfathiazole, sulfapyridine, and in certain cases sulfanilamide are being used extensively in the treatment of many types of bacterial infection and as prophylactic chemotherapy in war wounds and a few other conditions. As a rule these drugs are given orally although the sodium salt of sulfanilamide may be injected. It should be emphasized that these drugs are not a cure- all, that toxic reactions may follow their administration, and that their employment requires clinical supervision and laboratory de- termination of blood levels, etc. Sulfathiazole and sulfapyridine are being employed in conjunction with type specific sera in the treatment of pneumonia. According to Goodman and Gilman (1941), sulfathiazole and sulfapyridine are apparently of value in pneumococcal pneumonia, bacteremia and empyema ; in a great variety of staphylococcal, hemolytic streptococcal, meningococcal, and gonococcal infections. According to Hamilton (1941), and Hamilton and Wasson (1941), certain strains of Lancefield's group D streptococci were refractory to sulfathiazole. Major (1940) has reported apparent cures in two cases of strepto- coccus endocarditis treated early in the disease. None of the sulfanilamide compounds, according to Carey (1940), seem to be of value in rheumatic fever, diphtheria, virus disease, H. influenzae or typhoid-dysentery infections. This is interesting since Buttle and associates reported (1937) that sulfanilamide protects mice against E. typhosa and S. paratyphosi. Kolmer (1940) found sulfanilamide protected mice against Br. abortus but not against Br. suis and was only moderately protective against Br. nielitensis. Carey (1940), however, states that sulfanilamide was used in three cases of Br. abortus infection without any demonstrable effect. According to Buttle (1940), the sulfonamide compounds are in- effective against CI. oedenuitiens but very effective against CI. welchii. For a more extensive discussion of the chemistry, pharmacology, mode of action, indication and contraindication of these compounds, the student is referred to the publications of Mellon, Gross and Cooper (1938), Goodman and Gilman (1941), Lockwood (1940), Carey (1940), and other papers listed in the references at the end of this chapter. TOXINS AND ANTITOXINS 289 Mening-ococcus Meningitis. — Meningitis is an iniiammation of tiie meninges or eoverings of the brain and spinal eord. While it may be caused by various species of pathogenic bacteria, serum therapy is of value only when the meningococcus is the causal agent. In every case of suspected meningitis, spinal fluid should be obtained and examined as early as possible since, if the meningo- coccus is found, quarantine can be established and specific poly- valent antimeningococcus immune serum administered as a thera- peutic agent. While there are a great many papers appearing relative to meningococcus toxins, there is no definite evidence that the meningococcus produces a true soluble exotoxin. Flexner (1906) developed the serum treatment of meningococcus menin- gitis. He and others have shown that when serum containing antibodies for the strain of meningococcus present in any par- ticular epidemic is used, the mortalitj^ is reduced from 70 per cent in untreated cases to 25 or 30 per cent in those receiving adequate and early treatment with specific serum. Cellular Response Due to Serum. — Occasionally a physician is called as a consultant in a case of suspected meningitis that has received antimeningococcus serum intraspinally before the spinal fluid was examined. The question arises as to the effect of the injected serum upon the cell count of the spinal fluid. Very little has appeared in the literature bearing upon this subject, l^uryea (1930) studied the reaction in two normal individuals to the intraspinal injection of 20 c.c. of concentrated serum prepara- tions. A corresponding amount of spinal fluid was withdrawn befoi'e the injections were given. Before treatment the cell count in one volunteer was four and in the other two cells per cubic millimeter. Within one hour the counts were increased to 3,800 and 4,640 per cubic millimeter respectively. At the end of twenty hours the cell count of the first was 180 and of the second 300 per cubic millimeter. Both neutrophiles and mononuclear cells were increased. Such phenomena should be borne in mind in interpreting the laboratory findings in treated cases. Use of Serum in Virus Diseases. — Convalescent serum is em- ployed frequently in the prevention and treatment of measles and aLso in the treatment of poliomyelitis. It is very important that serum of any kind intended for such purposes be sterile. A 290 IMMUNOLOGY number of fatal septicemias have resulted from the injection of serum containing virulent organisms. These may result from the presence of virulent organisms in the blood of the donor, acci- dental contamination may occur through carelessness or in some cases infection may be carried in from the skin of the patient. In a previous chapter attention is called to the importance of test- ing the donor's blood for syphilis before it is injected into a patient. Measles. — While there is some controversy over the etiology of measles, it appears to be well established that it is a virus disease. Rake and Shaffer (1940) report culturing the virus in the chorio- allantois of the fertile hen's egg. Park (1928) advises that con- valescent serum or plasma be emploj-ed prophylactically in such amounts that the child develops a mild form of the disease since this will confer a lasting immunity. If the child is completely pro- tected, it will possess a transient immunity that will last for two to four weeks or as long as sufficient serum remains in the body. Preparation of Convalescent Serum. — The method used at the Willard Parker Hospital for obtaining blood Ls described by Park (1928) somewhat as follows: A sterile 16-gauge Luer needle is inserted into a vein and the blood is collected in a sterile 500 c.c. bottle containing 20 c.c. of a 25 per cent sodium citrate solution and 0.3 gram of oxyquinolin sulphate as a preservative. The cells are allowed to settle and the plasma is removed, tested for sterility, and put up in 3 c.c. vials, 6 c.c. vials, and 30 c.c. bot- tles. The latter is for institutional use. A sample of uncitrated blood is obtained for the Wassermann test. Partial Rather Than Complete Protection Recommended. — In 1916 Park and Zingher reported satisfactory results in a short series of cases. They found that S c.c. of convalescent serum protected completely, whereas 4 c.c. modified the disease. Since then convalescent serum has been used extensively in New York City with very encouraging results. The great difficulty has been to obtain convalescent serum. Barenberg, Lewis and Messer (1930) compared adult whole blood, convalescent serum and Tunnicliff's immune serum relative to their protective value in measles. They found convalescent serum to be the most effective, although whole blood from adults who have previously had T0XIX8 AND ANTITOXINS 291 measles is of great value. It permits of the development of an attenuated form of the disease and is usually available. They found that 6.0 c.c, of convalescent serum completely protected 73 per cent of the childi-cn who received it, wliile the remainder came down with mild attacks. Whole blood from adults wlio had previously had measles was used in amounts of 30 c.c. Of 56 children so treated after exposure to measles 77 per cent de- veloped a mild form of measles. The Tunnicliff serum was ad- ministered but failed to protect or to modify the disease. Importance of Epidemiological Factors. — Karelitz and Schick (1935) are of the opinion that epidemiologic factors must be taken into consideration in measles prophylaxis. They review the literature bearing upon this subject and report tlic results of their studies. They find that in homes where good hygiene is practiced there is a greater percentage of patients showing pro- tection from the administration of convalescent immune seruni than in homes where j)oor hygienic conditions are found. I'sK of Pooled Placental Blood. — Largely to overcome the dif- ficulty of obtaining a serum having protective value in measles, ►Salazar de Sanza (1932) and Dulitskiy (1932) suggest the use of pooled placental blood or serum. They report quite favorable results from their respective studies of its potency, although they encountered some difficulty in sterilizing it without reducing its therapeutic value. As a result of the experimental work of McKhann and Chu (1933) these objections have been overcome and a concentrated extract, called immune globulin, is being pre- pared for clinical use by at least some of the manufacturers of biologic products. Poliomyelitis. — It is now quite well established that poliomye- litis is a virus disease. Park (1928) states that from the time that Flexner and Lewis (1910) and also Levaditi and Landsteiner (1910) discovered that serum from convalescent monkeys con- tains virucidal substances, it has been held that convalescent serum, when administered early, is of prophylactic and thera- peutic value. This was also the opinion of Aycock et al. as late as 1929. In 1931 there occurred an extensive epidemic of polio- myelitis in New York. Park carried out an extensive and con- trolled experimental investigation of tlie ^•alue of convalescent 292 IMMUNOLOGY serum when administered in the preparalytic stage. The treat- ment did not affect the incidence of paralysis nor the mortality rate. An excellent review of the literature bearing upon the serum treatment of poliomyelitis is given by Harmon (1934). While convalescent serum is not an effective therapeutic agent so far as the prevention of paralysis and death is concerned, it was shown by Flexner to be of definite value when administered prophylactically. There is no promise, at the present time, that active immuniza- tion can be effected by means of a vaccine. This subject is dis- cussed quite extensively by Zinsser, Enders and Fothergill, 1939, p. 767. For those interested in the extensive research relative to the virucidal property of normal, convalescent and immune sera as well as the inactivating effect of anterior pituitary and other substances of endocrine origin, a supplementary list of references is appended. The student is also referred to an excellent brief review of the subject of serum therapy in poliomyelitis given in an editorial (1934). The editor calls attention to the fact that while statistical evidence indicates that serum used therapeutically may not prevent paralysis or death, nevertheless, symptomatic; improvement is reported almost universally following the ad- ministration of such sera. On the other hand Zinsser, Enders and Fothergill (1939, p. 771) state that it is their opinion that the intraspinal injection of serum may increase the edema of the cord, and therefore be definitely harmful in some cases. Mumps. — Another virus disease which apparently can be pre- vented by the injection of specific convalescent serum is epidemic parotitis or mumps. It has an incubation period of about 18 days and is exceedingly contagious. Park (1928) reviews the work of Hess (1915) and Regan (1925) and is inclined to at- tribute definite ])rophylactic value to convalescent serum. Vaccinia and Variola. — There are a few reports in the litera- ture such as those of Blackfan, Peterson and Conroy (1923), Weech (1924) and Mitchell and Ravenel (1924) which indicate that convalescent serum is of value in the prevention of variola. Downs and Stookey (1932, 1933), working in this laboratory in- vestigated the prophylactic and therapeutic value of specific im- mune serum when administered to rabbits before and after in- TOXINS AND ANTITOXINS 293 fection with the Armstrong vaeeine virus. This virus is uni- formly fatal to normal rabbits. When administered before or at the time of infection, the animals are protected, but the serum is ineffective after the virus is once established within the tissues of the body. Tetanus. — Tetanus is a disease of great antiquity. The causal relationship of CI. tetani to the disease was established by Kitasato in 1889. Specific antitoxins were produced by Behring and Kitasato in 1890. The organisms arc present in the intestinal contents of most wanii-])loodod animals, iiicliuling man, and hence have a wide dis1ril)iition in nature. They possess little or no in- vasive power and gain entrance to the tissues through mechanical injury. Once within the tissues they cannot esta])lish tliemselves unless anaerobic conditions prevail. It is generally agreed that they remain localized at the point of entry, multiply to a certain extent, and secrete two soluble toxins; one is hemolytic and the other brings about the characteristic symptoms of tetanus. While there are several serological types, all tetanus bacilli produce the same toxin. It is generally accepted that local tetanus and general tetanus are both due to the action of the toxin on nerve cells in the cord and brain. As to how the toxin reaches the cord and brain there are three variants of a neural transport theory. According to one, the toxin is transported in the nerve fibrils or axones of the motor nerves by some process of "protoplasmic streaming." A second explanation that is widely accepted assumes that the toxin reaches the central nervous system along the endo- or perineural lymphatic vessels of the motor nerves. According to a third theory, the toxin is conveyed to the central nervous sys- tem in the tissue spaces of the nerves. Reason for Ineffectiveness of Tetanus Antitoxin in Treat- ment.— In previous chapters the units of tetanus toxin and anti- toxin are given. Evidence is also offered which shows that tetanus antitoxin is of definite value in preventing the disease. In the treatment of general tetanus, large doses of antitoxin are admin- istered. While the antibodies neutralize the circulating toxin, it appears from statistical studies that such treatment does not materiallv affect the outcome of the disease. When symptoms of 294 IMMUNOLOGY general tetanus appear, it is evident that toxin has ah-eady com- bined with nerve cells of the central nervous system. Wasser- mann and Takaki discovered that brain tissue forms a very firm combination with toxin, which fact may account for the ineffec- tiveness of the serum treatment of general tetanus. In evaluating statistical studies relative to the efficiency of serum and other treatments of tetanus, it should be remembered that there is a definite relationship between the observed incuba- tion period and the mortality rate of the disease. When the in- cubation period is short, i.e., four to six days, the mortality is ap- proximately 90 per cent, while it is only 50 per cent when the incubation period is ten days or longer. The longer incubation period permits the body to develop active immunity which is not possible when the incubation period is short. There are perhaps a number of other factors that affect the outcome of the disease. In the preceding chapter attention is called to a new method of treating acute tetanus reported by Ramon (1940). He has called it combined (active-passive) treatment. A more extensive clinical application will determine its value. Those interested in the methods of preparation of commercial antitoxin are referred to an excellent discussion of the subject in a paper by McCoy (1928) and to the more recent publications of Predtechensky (1931) and of Sneath (1934). An interesting dis- cussion of the present status of tetanus is contained in a paper by Miller and Rogers (1935). Reference to improved methods of antitoxin production are mentioned elsewhere in this chapter and in a preceding chapter. Gas Gangrene. — There are five pathogenic anaerobes, any one alone or any combination of which may gain entrance into a wound and produce either gas and local necrosis or a fulminating gangrene with severe systemic manifestations. In the latter case death may follow within a few hours. Such tyj)es of infection are designated as "gas gangrene." The five anaerobes listed in the order of their frequency of occurrence in such conditions are : CI. welchii, CI. septicum, CI. oedematiens {B. novyi), CI. fallax, and CI. Jiistolyticum. It is generally agreed that the first four of these organisms produce soluble toxins, but there is some disagree- ment in the case of 67. Imtolyticiim. Zinsser and Bayne-Jones TOXINS AND ANTITOXINS 295 (1939, p. 629) state definitely that it does not, while Robertson (1929), Peterson and Hall (1923), and others report that it pro- duces a true toxin that is very unstable. The disagreement results from a difference in the conception of the properties of soluble toxins. They all agree that whole cultures injected intramuscularly into guinea pigs produce a rapid digestion of the tissues. Robert- son and others state that the digestion is due to an extracellular en- zyme that is antigenic and for this reason feel that it comes within the proper definition of a true toxin. Infection, Factors Affecting the Germination of Spores. — In natural infection where soil containing spores of various patho- genic anaeobes, along with spores and vegetative forms of other bacteria, gains entrance to a wound, the question arises as to the factors favoring or discouraging the germination of the spores. It is thought that the normal oxygen tension in the tissues is un- favorable to germination. In a wound the presence of dead tissue and a low oxygen tension are probably the most important conditions favoring germination of the spores and the vegetative multiplication of the pathogenic anaerobes. Under these condi- tions, the bacteria begin to produce toxin quickly, and this leads to the production of more necrosis and better conditions for bac- terial growth. Aerobic and facultative anaerobic contaminants present in the wound aid materially in reducing the oxygen ten- sion, and certain chemical tissue debilitants carried in with the infectious agents favor necrosis. Importance of Removal of Devitalized Tissue. — Early in the World "War gas gangrene presented a serious medical and surgical problem. It was soon discovered, however, that early surgical I'emoval of all devitalized tissue from a Avound and the control of surgical closure by bacteriologieal examination reduced the incidence of the disease to a very low level. The serum treatment of gas gangrene did not receive much attention until after Bull and Pritchett (1917) had shown that CI. ivelchii produces a soluble toxin. Previous to that time it was well established that CI. aepticum and CI. oedematiens are capable of producing specific antigenic poisons. For a recent discussion of infection by gas- forming anaerobes the student is referred to a j^aper by Reeves (1935). 296 IMMUNOLOGY Summary of Important Factors Relative to Gas Gangrene. — Some of the important facts that have been established by clini- cal and experimental investigations of gas gangrene may be sum- marized as follows: 1. There are three of these anaerobes, i.e., CI. welcMi, CI. septicum and CI. oedematiens, that are regarded as the most impor- tant ones in gas gangrene. There is at the present time a poly- valent antitoxin capable of neutralizing all three toxins avail- able conmiercially. 2. Incubation Periods for Various Toxins. — When the toxin of either CI. welchii or CI. septicum is injected in sufficient amounts intravenously into rabbits, death occurs within five to fifteen minutes. In other words, there is no incubation period fol- lowing the intravenous injection of adequate doses of these toxins. On the other hand, the toxin of CI. ocdematiens never kills acutely but only after a recognizable incubation period. Zinsser and Bayne- Jones (1939) state that when 0.01 c.c. of a potent toxin of this organism is injected intravenously into a 300 or 400 gram guinea pig, death results within forty-eight hours. When lethal doses of these respective organisms are injected into the thigh muscles of guinea pigs, there is an incubation period of several hours in each case. CI. welcMi produces deatli within twenty-four to sixty hours, CI. septicum within twelve to twenty-four hours, and CI. oedcnuitiens within twenty-four to forty-eight hours. Kettle (1919) states tliat the absence of a cellular response such as is seen in pyogenic infections is characteristic of the lesions due to these anaerobes. While there is not a collection of phagocytic cells in the muscle tissue, he says there may be a few neutrophiles in the subcutaneous and connective tissue. 3. Tetanus in Wounds. — Spores of CI. tctani not infrequently gain entrance to wounds in the same material that contains spores of the anaerobes responsible for gas gangrene. It is, therefore, advisable to administer tetanus antitoxin simultaneously with the polyvalent antitoxin for the latter organisms. 4. Tissues Affected. — Gas gangrene was regarded at one time as a disease of muscle tissue only, but it has since been found that the toxins are not specific for muscle but act on other tissues as well. Gas gangrene of the abdominal wall following major oper- ations has been reported by Orr (1934) and others. TOXINS AND ANTITOXINS 297 5. Invasive Power. — While CI. tetanus has no invasive power and, therefore, remains localized at the point of entry, the or- ganisms causing gas gangrene rapidly invade the tissues and blood stream and may become widely distributed in the body, 6. In experimental gas gangrene the areas of gangrene produced by pure cultures of CI. welchii, CI. sejyticimi, and CJ. ocdemati- ens exhibit characteristic differences according to Robertson (1929) that can be recognized grossly and microscopically. Difference in Pathological Picture. — CI. welchii and CI. septicum produce relatively large amounts of gas in the tissues while gangrene caused by CI. oedcmatiens shows little, if any, crepi- tation. The edema fluid due to CI. welchii is slightly pink in color, that of CI. septicum deep red, while CI. oedematiens produces an extensive colorless gelatinous edema which, when the infection is very rapid, may become pink. In CI. welchii infection the muscles are friable, soft, and have a sodden digested appearance, and a sour rancid odor is evolved. There is no blackening and no sug- gestion of putrefaction. In gangrene due to CI. septicum the skin and muscles are not friable nor do they appear digested. 7. Effect of Toxins on Heart and Adrenals. — The effect of these toxins upon the heart and adrenals has been studied ex- tensively. Acute death in cases of gas gangrene is said by Robert- son (1929) to be due to heart failure and the exhaustion of the su]irarenal glands. The latter, also, occurs in fatal gangrene due to CI. septicum. Robertson and Dale (1920) analyzed the toxic action of CI. septicum by means of a kymograph. Robert- son cites this work and quotes Dale's opinion that the toxin exerts both a pressor action and also a poisoning of the heart muscle. The action of the toxin of CI. oedematiens has not been studied extensively. 8. Therapeutic Value of Antitoxins. — To be effective, poly- valent antitoxin should be administered before symptoms have appeared or very early in the disease. As a therapeutic agent it is injected intravenously, intramuscularly, and infiltrated lo- cally. Unless the serum contains antitoxins for each of the an- aerobes causing the gangrene, it will be ineffective since the one or more whose toxins are not neutralized will still be active. The effect of sulfanilamide and related compounds upon CI. welchii, CI. 298 nrMT'X0T>0GY tctuni, and other anaerobes is discussed earlier in this chapter. For further information the student is referred to papers listed under References to Chemotherapy. Botulism. — The interesting point about CI. botulinum is that there are tliree strains of tliis anaerobe that produce toxins anti- genieally unlike. While antitoxins are available commercially for types A and B and usually are administered, they are appar- ently ineffective in the treatment of the disease. Tularemia, — In 1934 Foshay reported upon the treatment of tularemia with specific immune serum obtained from goats that had received repeated injections of formalized suspensions of P. tularensis. The early promising results obtained with this serum have subsequently been disappointing. References''^ Abel, J. J.: On Poisons and Disease and Some Experiments With the Toxin of Bacillus Tetani, II, Science 79: 121, 1934. Ayeock, W. L., Luther, E. H., and Kramer, S. D. : Technique of Convales- cent Serum Therapy in Poliomyelitis, J. A. M. A. 92: 385, 1929. Avery, O. T., Chickering, H. T., Cole, R., and Dochez, A. E.: Acute Lobar Pneumonia, Prevention and Serum Treatment, Monographs of the Rockefeller Institute for Medical Research, 1917, No. 7. Banzhaf, E. J.: The Preparation and Purification of Toxins, Toxoids and Antitoxins, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 745. Barenberg, L. H., Lewis, J. M., and Messer, W. H. : Measles Prophylaxis: Comparative Results With the Use of Adult Blood, Convalescent Serum, and Immune Goat Serum, J. A. M. A. 95: 4, 1930. Blake, F. G., and Trask, J. D.: The Treatment of Scarlet Fever With Anti- toxin, Boston M. & S. .L 193: 659, 1925. See also Blake, Trask and Lynch: J. A. M. A. 82: 712, 1924. Bliss, E. A., McClaskey, Wm. D., and Long, Perrin H.: A Study of Pneumo- coccus Carriers, J. Immunol. 27: 95, 1934. Brahdy, M. B., and Lenarsky, M.: Acute Epidemic Poliomyelitis Complicat- "ing Pregnancy, J. A. M. A. 101: 195, 1933. Brahdy, M. B., and Lenarsky, M. : Difficulty in Swallowing in Acute Epidemic Poliomyelitis, J. A. M. A. 103: 229, 1934. Brodie, M. : Active immunization in Monkey Against Poliomyelitis With Germicidally Inactivated Virus, Science 79: 594, 1934. Brodie, M., and Elvidge, A. R.: The Portal of Entry and Transmission of the Virus of Poliomyelitis, Science 79: 235, 1934. Bull and Pritchett: (1917) See Robertson and O'Brien, 1929. Bullowa, Jesse: Therapeutic Pneumococcus Tj^e VIII (Cooper) Serum, J. A. M. A. 102: 1560, 1934. Cecil, R. L., and Plummer, N.: Pneumococcus Type I Pneumonia, J. A. M. A. 95: 1547, 1930. Cole, R. I., and Dochez, A. R. : Forchheimer 's Therapeusis of Internal Dis- eases, Vol. 5, New York, 1915, D. Appleton Co., p. 472. *See separate list of references to Cliemotherapy at end of general list for cliapter. TOXINS AND ANTITOXINS 299 Cooper, G., Edwards, M., and Rosenstein, C: The Separation of Types Among the Pneumococci Hitherto Called Group IV and the Develop- ment of Therapeutic Antiserums From These Types, J. Exper. Med. 49: 461, 1929. Cooper, G., Rosenstein, C, Walter, A., and Peizer. L. : Tlie Further Separa- tion of Types Among the Pneumococci Hitherto Included in Group IV and the Development of Therapeutic Antisera for These Types, J. Exper. Med. 55: 531, 1932. Dick, G. F., and Dick, G. H.: Therapeutic Results "With Concentrated Scarlet Fever Antitoxin: Preparation, Standardization and Dosage of Antitoxin, J. A. M. A. 84: 803, 1925. Scarlet Fever Antitoxin, Ibid. 82: 1246, 1924. Dick, G. F., and Dick, G. H.: (1939) Cited bv Zinsser and Bavne-Jones, p. 297. Dick, G. F., and Dick, G. H.: Experimental Scarlet Fever, J. A. M. A. 81: 1166, 1923. The Etiology of Scarlet Fever, Ibid. 82: 301, 1924. Dick, G. F., and Dick, G. H.: A Skin Test for Susceptibility to Scarlet Fever, J. A. M. A. 82: 265, 1924. Dochez, A. R.: Etiology of Scarlet Fever, Medicine 4: 251, 1925. Dochez, A. R., and Gillespie, L. J.: A Biologic Classification of Pneumo- cocci by Means of Immunity Reactions, J. A. M. A. 61: 727, 1913. Downs, C. M., and Stookey, P.: Some Observations Concerning Erythem- atous Eruptions Simulating Scarlet Fever Developing in the Puer- perium, Am. .T. ()]>st. & (iynec. 23: 735, 1932. Dubos, R. .1. : The Effect of Specific Agents Extracted From Soil Microorgan- isms Upon Experimental Bacterial Infections, Ann. Int. Med. 13: 2025, 1940. Dulitskiy, S. O. : Prophylaxis of Measles by Means of Serum of Adults and of Placental Serum, Jurnal P. Rannemu Detskomu Vozrastu, Moscow 12: 317, 1932. Abstract in J. A. M. A. 100: 1733, 1933. Duryea, A.: Reaction to Antimeningococcus Serum, J. A. M. A. 95: 1582, 1930. Eberson, F. : Experimental Study of an Antipoliomyelitic Serum From Monkeys Artificially Immunized With an Organism Cultivated From Poliomvelitis Nervous Tissue, J. Immunol. 24: 433, 1933. Editorial: Serum Therapy in Poliomyelitis, J. A. M. A. 103: 262, 1934. Editorial: A Poliomyelitis Vaccine, J. A. M. A. 103: 264, 1934. Editorial: The Hemolytic Streptococci in Rheumatism, J. A. M. A. 100: 260, 1933. Editorial: Active Immunization With Tetanus Toxoid, J. A. M. A. 101: 934, 1933. Editorial: Refined Antipneumococcus Serum, J. A. M. A. 96: 1505, 1931. Falk, G. K., McGuire, G., and Rosenstein, C: Studies on Antibodies. Yl. Dried Antimeningococcus Serums, J. Immunol. 22: 445, 1932. Felton, L. D. : Dissociation of the Specific Proteins Precipitate of Anti- pneumococcus Horse Serum, and a Comparison With a Protein Isolated bv Chemical Means From This Immune Serum, J. Immunol. 22: 453. 1932. Felton, L. D., and Kauffman, G.: The Lipoidal Content of Antipneumococcic Horse Serum, J. Immunol. 24: 543, 1933. Felton, L. D.: Pneumococcus Antibodies — ^What Are They? Science 79: 277, 1934. Ferry, Newell S.: Meningococcus Antitoxin. I. Prophylactic and Thera- peutic Tests on Guinea Pigs,* J. Immunol. 23: 315, 1932. Ferry, Newell S.: ileningococcus Antitoxin. II. Therapeutic Tests on Monkeys, J. Immunol. 23: 325, 1932. Ferry, Newell S.: Meningococcus Toxin and Antitoxin. III. Further Tests on Monkeys, J. Immunol. 26: 133, 1934. 300 IMMUNOLOGY Ferry, Newell S., and Schornack, Philip J.: Meningococcus Toxin and Anti- toxin, IV. Further Tests on Guinea Pigs and Eabbits, J. Immunol. 26: 143, 1934. Ferry, N. S., Norton, J. F., and Steele, A. H.: Studies of the Properties of Bouillon Filtrates of the Meningococcus: Production of a Soluble Toxin, J. Immunol. 21: 293, 1931. Finland, M., Spring, W. C, and Lowell, F. C: Specific Treatment of the Pneumococcic Pneumonias: An Analysis of the Eesults of Serum Therapy and Chemotherapy of the Boston City Hospital From July, 1938 to June, 1939, Ann. Int. Med. 13: 1567, 1940. Finland, Maxwell, and Sutliff, W. D.: The Specific Serum Treatment of Pneumococcus Type II Pneumonia, J. A. M. A. 100: 560, 1933. Flexner, Simon: Modes of Infection, Means of Prevention, and Specific Treatment of Epidemic Meningitis, J. A. M. A. 49: 639, 721, 817, 1917. Flexner, S., and Lewis, P. A.: (1910.) Cited by Park in Newer Knowledge of Bacteriology and Immunology by Jordon and Falk. Citation p. 941. Foshay, L. : Tularemia, Treated by New Specific Antiserum, Am. J. M. Sc. 187: 235, 1934. Foshaj'-, L.: Aids in Diagnosis and Treatment of Tularemia, J. Med. 15: 186, 1934. Gage, I. M.: Gas Bacillus Infection. A Frequently Unnoticed Source in Civil Life. With Report of Four Cases, Am. J. Surg. 1: 177, 1926. Goodner, K., and Dubos, R.: Studies on the Quantitative Action of Specific Enzyme in Type III Pneumococcus Dermal Infection in Rabbit.s, J. Exper. Med. 56: 521, 1932. Goodner, K.: The Effect of Pneumococcus Autolysates Upon Pneumococcus Dermal Infection in the Rabbit, J. Exper. Med. 58: 153, 1933. Goodner, K., and Stillman, E. G.: The Evaluation of Active Resistance to Pneumococcus Infection in Rabbits, J. Exper. Med. 58: 183, 1933. Goodner, K. : A Test for the Therapeutic Value of Antipneumococcus Serum, J. Immunol. 25: 199, 1933. Goodner, K., Dubos, R., and Avery, O. T.: The Action of a Specific Enzyme Upon the Dermal Infection of Rabbits With Type III Pneumococcus, J. Exper. Med. 55: 393, 1932. Goodner, K., Horsfall, F. L., and Dubos, R. J.: Type-Specific Antipneunio- cocci Rabbit Serum for Therapeutic Purposes. Production, Processing and Standardization, J. Immunol. 33: 279, 1937. Gordon, J. E.: Immunotransfusion in Scarlet Fever, J. A. M. A. 100: 102, 1933. Hartlev, G. A., Millice, G. S., and Jordan, P. H. : Undulant Fever Menin- gitis, J. A. M. A. 103: 251, 1934. Hcffron, R., and Anderson, G. W. : Two Years' Study of Lobar Pneumonia in Massachusetts, J. A. M. A. 101: 1286, 1933. Hektoen, L., and Johnson, C: The Prevention of Diphtheria and Scarlet Fever in Nurses, J. A. M. A. 102: 41, 1934. Henderson, Y., and Greenberg, L. A.: Gas Analysis With an All-Glass SjTinge for Pneumonia Tents, J. A. M. A. 96: 1474, 1931. Henry, J. N., and Johnson, G. E.: Acute Anterior Poliomyelitis in Phila- delphia: A Comparative Study of the 1916 and 1932 Epidemics, J. A. M. A. 103: 94, 1934. Hooker, S. B., and Follensby, E. M. : II. Different Toxins Produced by Hemolytic Streptococci of Scarlatinal Origin, J. Immunol. 27: 177, 1934. Hooker, S. B., and Follensby, E. M.: Some Properties of Two Active Sub- stances Contained in Certain Scarlatinal Streptococcus Filtrates, Pre- liminary Report, J. Immunol. 15: 601, 1928. TOXINS AND ANTITOXINS 301 Howitt, B. F., Shaw, E. B., Thelander, H., and Limper, M.: The Immuniza- tion of Goats and Slieep to Poliomyelitis Virus : Clinical Application of Their Serums, J. A. M. A. 96: 1280, 1931. Hunt, L. W.: The Treatment of Scarlet Fever With Antitoxin, J. A. M. A. 101: 14-44, 1933. Hurxthal, L. M. : Sterile Meninjfitis Following Lumbar Puncture, J. A. M. A. 100: 1489, 1933. .Tudd, E. S.: War Surgery, The Journal-Lancet 38: 617-620, 1918. .Tungeblut, C. W.: The Immunological Characteristics of the Poliocidal Substance in Human Serum. Concentration, Thermo-stability, Ab- soiption and Specificity, J. Immunol. 27: 17, 1934. .Tungeblut, C. W., Meyer, K., and Engle, E. T.: Inactivation of Poliomye- litis Virus and of Diphtheria Toxin bv Various Endocrine Principles, .1. Immunol. 27: 43, 1934. KMufViiiann, F., Mffrcli, E., and Scliniitli, K. : On llic Serology of the Pneumo- coccus Group, J. Immunol. 39: .397, 1940. Karelitz, S., and Schick, B.: E])idemiologic Factors in Measles Prophylaxis, J. A. M. A. 104: 991, 193.^. Kettle: (1919.) See Robertson and O'Brien, 1929. Kirkbride, M. B., and Wheeler, M. W. : Comparison of Reactions in In- dividuals to Toxins Prepared From Three Strains of Scarlet Fever Streptococci, Proc. Soc. Exper. Biol. & Med. 22: 85, 1924. Kolmer, .1. A.: Studies on the Preservation of Human Blood, Am. J. M. Sc. 200: 311, 1940. Kolmer, J. A., and Rule, A. M. : Concerning Vaccination of Monkeys Against Acute Anterior Poliomyelitis. With Special Reference to Oral Immunization, J. Immunol. 26: .505, 1934. Levine, M. I., Neal, J. B., and Park, W. H. : Relation of Physical Char- acteristics to Susceptibility to Anterior Poliomyelitis, J. A. M, A. 100: 160, 1933. IFcCann, W. S.: The Serum Treatment of Erypsipelas, .1. A. M. A. 91: 78, 1928. McCoy, G. W.: Control and Standardization of Biological Products, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, Univer- sity of Chicago Press, p. 947. ;^^cKhann, C. F., and Chu, Fu Tang: Antibodies in Placental Extracts, J. Infect. Dis. 52: 268, 1933. Malcolm, W. G., and White, B.: Studies on Meningococcus I. Endotoxin, J. Immunol. 23: 291, 1932. Meader, F. M.: Scarlet Fever Prophylaxis, J. A. M. A. 94: 622, 1930. Mehlman, J., and Seegal, B. C. : A Comparison of the Sensitizing and the Therapeutic Effect of Rabbit and Horse Antipneumococcus Type I Serums in Albino Mice, J. Immunol. 27: 81, 1934. Miller, R. H., and Rogers, H.: Present Status of Tetanus, J. A. M. A. 104; 186, 1935. l^folitch, M.: Prevention of Scarlet Fever, J. A. M. A. 102: 2021, 1934. JFurdick, P. P., and Cohen, S. M. : A Note on the Fractionation of Anti- meningococcus Serum, J. Immunol. 24: 531, 1933. Neff, F. C: Scarlet Feyer Immunization, Arch. Pediat. 39: 250, 1922. Neufeld and Handel: (1910.) Cited by Dochez and Gillespie, 1913. Nevin, M. : Experimental Gas Gangrene, J. Infect. Dis. 25: 178, 1919. Olmstead, M.: An Antigenic Classification of the Group IV Pneumococci, From the Presbyterian Hospital, New York Citv, J. Immunol. 2: 425, 1917. Oram, Florence: Pneumococcus Leucocidin, .1. Immunol. 26: 233, 1934. Orr, T. G.: Gas Bacillus Infection of Abdominal Wall, .1. A. M. A. 102: 2081. 1934. 302 immuxologV Park, W. H., and Spiegel, E. G. : Complexity of the Scarlet Fever Toxin and Antitoxin, J. Immunol. 10: 829, 1925. Park, W. H., and Williams, A. W. : Pathogenic Microorganisms, Philadelphia, 1933, Lea & Febiger, p. 327. Park, W. H. : The Types of Pneumococci in Adults and Children and Their Significance, J. State M. 38: 621, 1930. See also Discussion of paper by Sutliif and Finland, J. A. M. A. 101: 1294, 1933. Park, W. H.r The Use of Human Serum From Convalescent Cases in Pre- vention and Treatment of Disease. Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 934. Park and Zingher: (1916.) Cited by Park, 1928." Peterson and Hall: (1923.) See Eobertson and O'Brien, 1929. Powell, H. M., and Jamieson, W. A.: Further Studies on the Meningo- coccus With Special Reference to the Shwartzman Reaction, J. Immunol. 23: 481, 1932. Predtechenskv. S. N.: Production of Potent Antitetanic Serum, J. Im- munol.'20: 143, 1931. Rake, G., and Shaffer, M. F.: Studies on Measles: I. The Use of the Chorio-allantois of the Fertile Hen's Egg, J. Immunol. 38: 177, 1940. Reeves, J. R.: Infections bv Gas-Forming Anaerobic Bacilli: An Analysis, J. A. M. A. 104: 526, 1935. Rhoads, P. S., and Gasul, B. M.: Convalescent Scarlet Fever Serum and Commercial Antitoxin: A Comparison of Their Protective Values, J. A. M. A. 102: 2005, 1934. Rhoads, P. S.: Skin Tests and Immunization Against Scarlet Fever and Diphtheria, .J. A. M. A. 97: 153, 1931. Riley, C. V., and Wilson, M. A.: A Comparison of the Toxicity of Various Meningococcus Preparations, J. Immunol. 23: 269, 1932. Ritter, J. A.: Streptococcic Septicemia of Hematogenous Origin in a New- born Infant, J. A. M. A. 101: 771, 1933. Robertson, M., and O'Brien, R. A.: Organisms Associated With Gas Gangrene. A System of Bacteriology 3: 225, 1929. Med. Research Council. His Majesty's Stationery Office, London. Rosenow, E. C: A Specific Reaction of Convalescent Serum on the Strep- tococcus Isolated in Studies of Poliomyelitis, J. Immunol. 23: 455, 1932. Salazar de Sanza, J.: Abstract in J. A. M. A. 100: 49, 1933. Schultz and Charlton: (1918.) Cited by Sherwood and Auchard, 1926. See Toomey and Nourse, 1924. Schultz, E. W., and Gebhardt, L. P.: Immune Serum Production in Polio- myelitis Refractory Animals, J. Immunol. 26: 93, 1934. Seegal, D., and Heidelberger, M. : Streptococcus Scarlatinae, J. A. M. A. 100: 186, 1933. Sherwood, N. P., and Auchard, V. M. : An Intensive Study of a Small Epidemic of Scarlet Fever at Lawrence, Kansas, J. Kansas State M. Soc. 26: 215, 1926. Shwartzman, G.: Phenomenon of Local Skin Reactivity to Pneumococcus, J. Immunol. 23: 429, 1932. Sides, G. M.: Local Skin Reactions Obtained by Intravenous Injection of Agar Following Intracutaneous Inoculation of Meningococcus Toxin, J. Immunol. 20: 169, 1931. Smillie, W. G. : The Epidemiology of Lobar Pneumonia. A Study of the Prevalence of Specific Strains of Pneumococci in the Nasopharynx of Immediate Family Contacts, J. A. M. A. 101: 1281, 1933. Sneath, P. A. T.: Development of Tetanus Antitoxin Following Administra- tion of Tetanus Toxoid, J. A. M. A. 102: 1288, 1934. TOXINS AND ANTITOXINS 303 Stephenson, R.: Nonspecific Immunotransfusions in Hemolytic Strepto- coccus Septicemia: Analysis of Blood Changes by the Schilling Method, J. A. M. A. 100: 100, 1933. Stillman, E. G., and Goodner, K. : Resistance to Pneumococcus Infection in Rabbits Following Immunizing Injections of Heat-Killed Pneumococcus Suspensions, J. Exper. Med. 58: 183, 1933. SutliflF, W. D., and Finland, M.: Type I Lobar Pneumonia Treated With Concentrated Pneumococcus Antibody (Felton). The Clinical Course, J. A. M. A. 96: 1465, 1931. Sutliff, W. D., and Finland, M.: The Significance of the Newly Classified Types of Pneumococci in Disease. Type IV to XX, inclusive, J. A. M. A. 101: 1289, 1933. Toomey, J. A., and Nourse, J. D.: Cited by Sherwood and Auchard, 1926. Trask, J. D., and Blake, F. G.: Heterologous Scarlet Fever, J. A. M. A. 101: 753, 1933. Wadsworth, A., Crowe, M., and Smith, L.: Absorption Spectra of Carbo- hydrates of the Pneumococcus. A Preliminary Note, J. Immunol. 26: 481, 1934. Wadsworth, A., and Brown, R. : Chemical and Immunological Studies of Pneumococus. II. The Ether-Soluble Fraction of Type I Pneumo- coccus, J. Immunol. 21: 255, 1931. Wadsworth, A.: The Action of Bacterial Toxins, J. Immunol. 26: 81, 1934. Walter, A. W., Guevin, V. H., Beattie, M. W., Cotler, H. Y., and Bucca, H. B.: Extension of the Separation of Types Among the Pneumococci: Description of 17 Types in Addition to Types 1 to 32 (Cooper). With Recommendations for Terminology of All Tj^es Reported Through 1940, J. Immunol. 41: 279, 1941. Warnshuis, F, C, and Kolk, B. V. : A Case of Gas Gangrene Treated by In- filtration of the Tissues With Permanganate Solution, J. A. M. A. 102: 1757, 1934. Wheeler, M. W. : Notes on the Antigenic Activity of Hemolytic Strep- tococci From Different Types of Infection, J. Immunol. 23: 311, 1932. Zinsser, H., and Bayne- Jones, S.: Textbook of Bacteriology, New York, 1939, D. Appl'eton Co., p. 300. Chapter on Streptococcus Infections and Scarlet Fever. Su2)pleme7itary References Gregory, K. K., West, E. J., and Stevens, R. E.: Epidemic Cerebrospinal Meningitis (Meningococcic), J. A. M. A. 115: 1091, 1940. Leonard, G. F., and Holm, A.: A Method for the Production of Staphylo- coccus Toxin and Toxoid, J. Immunol. 29: 209, 1935. Levinson, S. O. : Human Serum Transfusions, J. A. M. A. 115: 1163, 1940. Ravitch, M. M.: The Blood Bank of the Johns Hopkins Hospital, J. A. M. A. 115: 171, 1940. References to Chemotherapy Abernathy, T. J., Dowling, H. F., and Hartman, C. R.: The Treatment of Lobar Pneumonia With Sulfapyridine and Sodium Sulfapyridine, With Observations Upon the Effective Blood Levels, Ann. Int. Med. 13: 1121, 1940. Barlow, O. W., and Climenko, D. R. : Phannacologv of Sulfapyridine and Sulfathiazole, J. A. M. A. 116: 282, 1941. Bierman, W., and Baehr, G.: P^Texia and Chemotherapy in Bacterial Endo- carditis, J. A. M. A. 116:' 292, 1941. Bigler, J. A., Clifton, W. M., and Werner, M. : The Leukocyte Response to Sulfanilamide Therapy, J. A. M. A. 110: 343, 1938. 304 IMMUNOLOGY Blake, F. G.: Chemotherapy of Pneumococcal Infections With Sulfa- pyridine, Proc. Third Internat. Cong. Microbiol., 1940, p. 590. Bliss, E. A., and Long, P. H.: Observations Upon the Mode of Action of Sulphanilamide and Sulfapyridine, Proc. Third Internat. Cong. Micro- biol., 1940, p. 585. Buttle, G. A. H.: Chemotherapy of Infected Wounds, Lancet 1: 890, 1940. Burton, H., McLeod, J. W., McLeod, T. S., and Mayr-Harting, A.: On the Eelationships Between the Respiratory Activitives of Bacteria and Their Sensitiveness to Sulphanilamide, p-Hydroxylamino- and p-Nitro- benzenesulphonamide, Brit. J. Exper. Path. 21: 288, 1940. Carey, B. W.: Use of Sulfanilamide and Related Compounds in Diseases of Infancy, J. A. M. A. 115: 924, 1940. Carpenter, C. M. : Tlie Effect of Sulfanilamide and Related Compounds on Bacterial Toxins, Proc. Third Internat. Cong. Microbiol., 1940, p. 595. Carroll, G., Kappel, L., and Lewis, B.: Sulfathiazole: Clinical Investiga- tions, J. A. M. A. 115: 1350, 1940. Colebrook, L., Buttle, G. A. H., and O 'Moara, R. A. Q. : Tlie Mode of Action of p-Aminobenzenesulphonamide and Prontosil in Haemolytic Strepto- coccus Infections, Lancet 231: 1323, 1936.2 Dobson, L., Holman, E., and Cutting, W.: Sulfanilamide in Actinomycosis, J. A. M. A. 116: 272, 1941. Cowling, H. F., and Abernathy, T. J.: The Causes of Secondary Fever in Sulfapyridine-Treated Pneumonia, Ann. Int. Med. 14: 1815, 1941. Farrel, L. N.: Appraisal of Therapeutic Agents in Experimental Staphylo- coccal Infection, Brit. J. Exper. Path. 21: 302, 1940. Finland, M., Lowell, F. C, Spring, W. C, Jr., and Taylor, F. H. L.: Parenteral Sulfapyridine; The Intravenous Use of Sodium Sulfapyridine and a Report of Clinical and Laboratory Observations in the Use of a Glucose-Sulfapyridine Solution, Ann'. Int. Med. 13: 1105, 1940. Flippin, H. F., Reinhold, J. G., and Schwartz, L. : Sulfapyridine and Sulfa- thiazole Therapy in Pneumococcic Pneumonia, J. A. M. A. 116: 683, 1941. Foley, J. A., and Yasuna, E. R.: Sulfanilamide in the Treatment of Erysipelas, J. A. M. A. 115: 1330, 1940. Friedberg, C. K.: Sulfapyridine in Lol)ar Pneumonia, J. A. IM. A. 116: 270, 1941. Fuller, A. T., and Colelirook, L.: The Mode of Action of Sulphanilamide, Proc. Third. Internat. Cong. Microbiol., 1940, p. 582. Fuller, A. T., Colebrook, L., and Maxted, W. R.: The Mode of Action of Sulphanilamide, J. Path. & Bact. 51: 105, 1940. Fuller, A. T., and James, G. V.: Dosage of Sulphanilamide in Prophylaxis of Wound Infections, Lancet 1: 487, 1940. Garvin, C. F. : Sulfathiazole: Renal Complications, J. A. M. A. 116: 300, 1941. Gay, F. P., and Clark, A. R.: On the Mode of Action of Sulfanilamide in Experimental Streptococcus Empyema, J. Exper. Med. 66: 535, 1937. Goodman, L., and Gilman, A. : Sulfanilamide and Related Sylf onamide Drugs. The Pharmacological Basis of Therapeutics, New York^ 1941, The Macmillan Co., pp. 1002-1103. Hamilton, T. R.: Pathological Aspects of Streptococcic Infections in Human Blood With Reference to Sulfathiazole Effect on Growth Rates in Vitro, Thesis, Library of University of Kansas School of Medicine, Kansas City, Kan. Hamilton, T. R., and Wasson, Better Some Scientific Bases for Sulfonamide Therapy, J. Kansas M. Soc. 42: 219, 1941. Hamilton, T.R., and Wasson, Better Enterococcus Endocarditis Due to Streptococcus Fecalis, J. Kansas Med. Soc. (In Press). TOXINS AND ANTITOXINS 305 Horrel. W. E., and Brown, A. E. : Treatment of Septicemia, .T. A. M. A. 116: 179, 1941. Hoyne, A. L.: Epidemic Meningitis, .T. A. M. A. 115: 1852, 1940. Kennedy, P. C. : Agranulocytosis From Sulfathiazole, J. A. M. A. 116: 295, 1941. Lockwood, ,J. S.: Sulfanilamide in Surgical Infections, J. A. M. A. 115: 1190, 1940. Lockwood, J. S. : Observations on the Mode of Action of Sulfanilamide and Its Application to Surgical Infections, Ann. Surg. 108: 801, 1938, Lockwood, J. S. : Studies on the Mechanism of the Action of Sulfanilamide. III. The Effect of Sulfanilamide in Serum and Blood on Hemolytic Streptococci in A'itro, J. Immunol. 35: 155, 1938. Major, R. H. : Sulfanilamide Compounds in Endocarditis, Am. .1. M. Sc. 199: 6, 1940. Mellon, R. E., Gross, P., and Cooper, F. B.: Sulfanilamide Therapy of Bacterial Infections, Baltimore, 1938, Charles C Thomas. Nathanson, M. H.: Diffusion of Sulfonamide Compounds, J. A. M. A. 116: 280, 1941. Pelouze, P. S.. Barnes, E. W., Clark, A. L., Fox, O. F., Deakin, E.. Onstott, E. H., Usilton, L. J., and Vonderlehr, E. A. : Gonon liea in Male, J. A. M. A. 115: 1630, 1940. Plummer, N., and Ensworth, H. K.: Sulfapyridine in the Treatment of Pneumonia, J. A. M. A. 113: 1847, 1939. Ehoads, P. S., Hoyne, A. L., Levin, B., Horswell, R. G., Eeals, W. H., and Fox, W. W. : Treatment of Pneumococcic Meningitis, J. A. M. A. 115: 917, 1940. Eosenthal, S. M. : Some Nonsulfur Compounds of Interest in Bacterial Chemotherapy, Proc. Third Internat. Cong. Microbiol., 1940, p. 598. Schoeflfel, E. "W. : A Micro Bedside Test for the Determination of the Sulfanilamide Group Concentration in Body Fluids, J. A. M. A. 115: 122, 1940. Shaffer, P. A.: The Mode of Action of Sulphanilamide, Proc. Third Internat. Cong. Microbiol., 1940, p. 592. Smith, C. H., and Nemir, E. L. : The Sulfapvridine Treatment of Pneumonia in Children. J. A. M. A. 113: 1857, 1939. Spink, W. W., and Hansen, A. E.: Sulfathiazole, J, A. ]\r. A. 115: 840, 1940. Stirling, W. C: Sulfathiazole in Septicemia, J. A. M. A. 114: 118, 194(1. Sweeney, J. S., and Allday, L. E. : Granulocytopenia From Sulfanilamide With Unusual Blood Crisis and Eecovery, Ann. Int. Med. 13: 1241, 1940. Thompson, J. E. M.: The Ten Commandments for the Treatment of Com- pound Fractures, J. A. M. A. 115: 1855, 1940. Van Slyke, C. J., Wolcott, E. E., and Mahoney, J. F.: The Chemotherapy of Gonococcic Infections, J. A. M. A. 116: 276, 1941. Wagoner, S. C, and Hunting, W. F.: Sulfathiazole and Sulfapyridine in Pneumonia, J. A. M. A. 116: 267, 1941. Winters, W. L., Ehoads, P. S., Fox, W. W., and Eosi, E.: The Treatment of Pneumococcic Pneumonia With Sulfapyridine, Ann. Int. Med. 14: 1827, 1941. CHAPTER XVI SERUIM RP]ACTIONS When one administers an animal serum to a patient there is always the possibility that some form of "serum reaction" will result. The kind, amount, and condition of the serum, the method of its administrations, as well as the medical history, present con- dition, and inherent capacity of the patient to react, are all fac- tors to be taken into consideration by the physician. Serum reactions may be divided into (1) immediate reactions which occur within a few minutes or within a few hours after the serum is injected, and (2) delayed reactions (serum disease) which manifests itself only after an incubation period of several day.s or even weeks. Immediate Reactions. — Individuals who develop immediate re- actions may be classified into several groups as follows : 1. The asthenic child who presents a picture of status lymphaticus. Such individuals cannot withstand even mild shocks from almost any cause and may die following the injection of anti- toxin. They are even poorer risks if tliey present a history of asthma. Park and Williams (1933) state that practically all deaths from antitoxin administrations have fallen in this group. Fortunately the general practitioner rarely encounters cases of this type. 2. Nonspecific and anaphylactoid reaction : Lord and Heffron (1938) report that, in a series of 1,755 cases receiving intra- venous injections of scrum, 7 per cent developed symptoms of dififi- cult breathing, flushing of tlic face, cyanosis, abdominal or lumbar ]5ain, rapid and weak pulse and a few other symptoms. The same authors report 17.8 per cent of the series as developing thermal reactions. Not only febrile reactions but also severe chills occur quite frequently following the injection of antipneumococcie rabbit serum. In regard to the chill-producing factors present in serum. Good- lier, Horsfall and Dubos (1937) report that they can be removed for the most part by heating the rabbit serum to 58° C, for thirty 306 SKRIT.M lUvVCTIOXS 307 minutes and then adsorbing it at 4° C. with sterile washed kaolin for fifteen minutes. For a discussion of the treatment of the thermal reaction the student is referred to the reports of Bullowa (1937) and MacLeod (1939). The term anaphylactoid has been applied to phenomena in the lower animals characterized by anaphjdaetic-like symptoms, cardiac dilatation, hemorrhages and thrombosis. Apparently the symp- toms and pathological changes are nonspecific in that they do not depend upon a natural or acquired hypersensitiveness to a par- ticular substance. Karsner (1928), and Hanzlick and Karsner (1919-20), who have studied this reaction in the lower animals, noted it more frequently following intravenous than inti-aperi- toneal or subcutaneous injections. 3. Specific reactions: (a) A third group of individuals wlio may give a severe reaction to the injection of antiserum are those who are mtiuralh/ sensitive to such animal protein, just as others are sensitive to feathers, face powder, or ragweed pollen, and develop hay fever or asthma when the exciting agent is injected or i-eaches the mucous membrane of the nose or intestine or other ''shock organ." These people are said to be "atopically" sensi- tive. They react to much smaller doses of tlie serum and also react more violently than thase who have been made sensitive by a previous injection of serum. They may react violently to intra- dermal or subcutaneous injections and they cannot be desensitized quickly by administering small doses of the serum. Vaughan and Piper (1937) have collected thirty-five cases from the litera- ture in which death followed the administration of horse serum. In six of these the serum had been administered intracutaneously. Park and Williams (1933) state that about 1 in 10,000 persons develop severe reactions and alarming symptoms, and about 1 in 50,000 die within a short time after the subcutaneous or intra- muscular injection of antitoxin. According to Tuft (1937) the incidence of fatal serum reaction is about 1 in 70,000. (b) Another group giving immediate specific reactions to serum is made up of individuals who have acquired hypersensitiveness to horse proteins through a previous injection of horse serum. This may have been in the form of a previous injection of diph- theria, tetanus, Welchii or scarlet fever antitoxin, or from the injection of normal horse serum antipneumococcus, antistrepto- 308 IMMUNOLOGY eoccus or antimeningoeoccus sera. Mild sensitization may occur from the small amount of horse serum in toxin-antitoxin mixtures used in immunization (Hooker, 1924; Tuft, 1932). To avoid the latter source of sensitization, the manufacturers of T.A.T. are using antitoxin obtained from goats, thus avoiding the inclusion of horse serum in the mixture. Park's experience was quite exten- sive and extremely valuable. He found that the history of a previ- ous injection of horse serum is not a contraindication to the use of antitoxin properly administered. As previously stated statis- tical studies show that fatalities, when they occur, follow the first rather than the second injection of horse serum, and tlius would come under the first three groups mentioned above. Since tlie introduction of antipneumococcus rabbit serum by Ooodner, Horsfall and Dubos (1937) there has been a growing in- terest in acquired hypersensitiveness to rabbit serum. AVhile Horsfall, Goodner, MacLeod and Harris (1937) suggest the use of an intravenous test to detect allergy to rabbit serum, Warner (1939) and also MacLeod (1939) conclude that it should not be tried until the cutaneous and ophthalmic tests are made and found negative. In the modified intravenous test 0.01 c.c. of a 1 :10 dilution in 5 c.c. of saline is used for slow intravenous injection. A positive reaction is indicated if during the period of five minutes after administration there is a drop in the blood pressure of 15 mm. of mercury and an increase in heart rate of 15 beats per minute. The ophthalmic test consists in putting a drop of undiluted or diluted serum in the eye of the patient. It indicates mucous membrane sensitivity and may be negative when the skin test is positive. Rarely is the reverse true. If the skin tests are negative or weakly positive, undiluted serum may be employed for the ophthalmic test but when the skin test is strongly positive dilutions of 1:10 or 1:100 should be employed according to Tuft (1937). Brown (1938) and also Walzer (1939) report that rabbit serum is not irritating to the skin and therefore properly performed intracutaneous tests do not yield too many nonspecific reactions. They suggest using 0.01 c.c. of a 1 :100 or 1 :10 dilution of normal rabbit serum for the intracutaneous test. The ophthalmic test is said to be negative frequently in acquired hypersensitiveness to SERUM REACTIONS '309 animal scrum while the cutaneous test may be positive. A positive ophthalmic test suggests natural or atopic hypersensitiveness and is regarded by many as a contraindication to the administration of serum. Delayed Reactions. — 1. Serum Sickness in Man. — Besides the serum reactions described in the preceding paragraphs there is another phenomenon called serum sickness which may follow the administration of horse, hog, rabbit and perhaps other sera. It is not an immediate reaction but develops usually after several days or wdthin two or three weeks. It is characterized by headache, joint pains, fever and a rash. Edema of the face or other parts of the body may occur. Regional enlargement of the lymph nodes is noted quite commonly. The symptoms may last for periods varying from one day (accelerated type), to one week or longer. Relapses have been observed according to Zinsser, Enders and Fothergill (1939). Coca concludes that serum sickness can be produced in 100 per cent of all individuals if as much as 50 c.c. of normal horse serum is given intravenously. It may appear, how- ever, following the administration of much smaller doses. The student should remember that diphtheria antitoxin represents the pseudoglobulin fraction of horse serum that has been precipitated, washed and concentrated. Serum sickness in man is more readily produced with whole serum than with the pseudoglobulin fraction. Harten and Walzer (1939) state that most workers report that the administration of antistreptococcic and antipneumococcic sera gives a higher incidence and greater severity of serum reactions than diphtheria or tetanus antitoxin. It would appear from the reports of Bradshaw (1939) and Hutchinson (1939), that the incidence of serum sickness is greatly reduced if antitoxin is employed that has been subjected to a special method of treatment involving peptic digestion. Several of those who have perfected enzymatic methods for the treatment of immune serum are Parfentjev (1936), and Coghill, Fell, Creighton and Brown (1940) in America, Pope (1939) in Eng- land, and Grabar (1938) and Hanson (1938) in France. Parfentjev (1936) developed a process of peptic digestion of antitoxic sera under carefully controlled pH conditions which ac- complishes 70 to 80 per cent digestion of the plasma proteins. His 310 IMMUNOLOGY method of separating the pseudoglobulin antibody fraction is ac- complished by dialysis and "salting-out" of the pseudoglobulins. For a more extensive discussion of tlie method employed and its effect on serum proteins the student is referred to a report by Weil, Parfentjev and Bowman (1938). Coghill, Fell, Creighton and Brown (1940) report on a new concentration process for antitoxin which utilizes the enzyme- complex Taka-diastase. The enzyme is permitted to act on the immune horse serum for 3 to 5 daj'S at a pH of 3.5 to 4.5 and a temperature of 37° C. Concentration is then accomplished by a modified sodium sulfate (Na^SOi) salting out procedure. In all of these new methods of enz>nne treatment of immune serum the antitoxin concentration is not impaired while the biologic specificity and antigenic ]n-operties have been modified. Coghill, et al., say that in addition to " despeciation " their process also produces highly concentrated antitoxins that do not elicit serious reactions upon injection into either lower animals or man. 2. Serum Sickness in the Lower Animals. — In an excellent re- view of Serum Allergy, Harten and Walzer (1939) call attention to the production of serum disease in the horse by sera of cattle, man and rabbits and to serum disease in cattle produced by horse serum. Fleisher and Jones (1931, 1933, 1934, 1939) have pub- lished extensively upon serum sickness in rabbits. They describe the symptoms as elevation of body temperature, altered leucocyte picture, and redness and swelling of the ears. The incubation period was usually five or six days after a primary intravenous injection of horse serum. They noted variation in the ability of the sera of normal horses to produce "serum sickness" in rabbits. 3. Mechanism of Serum Sickness. — While Zinsser, Enders and Fothergill (1939) seem to accept as an established fact the anti- body theory of serum sickness proposed by von Pirquet and Schick (1905), Harten and Walzer (1939) state that an analysis of all available data does not support such a conclusion. In the theory of von Pirquet and Schick it is postulated that fol- lowing the injection of horse serum the antibody is formed gradually before the antigen is completely eliminated from the cir- culation and that the newly formed antibody reacts with the cir- SERUM HKACTIOXS -') 1 1 dilating antigen residue, causing the symptoms of serum sickne.ss. In support of this von Dungern demonstrated the coexistence of antigen and antibody in the blood stream of rabbits injected with horse serum. Longcope and Rackemann (1918) detected pre- cipitins for horse serum in the blood of patients suffering from serum disease and believed they had detected a correlation be- tween antibody production and serum sickness. Opposed to these findings according to Harten and Walzer (1939) are those of Coca (1932) that precipitins for horse serum may occur in man without the development of serimi sickness and are not present (Coca, 1933), (Tuft, 1933) in the blood of patients treated with normal horse serum even though serum sickness develops. Davidsohn (1929, 1930, 1933) carried out an extensive investi- gation of the titer of heterophile antibodies in the blood of in- dividuals who had received injections of normal or immune horse serum. He seems to think there is some relationship between the heterophile antigen in horse serum and serum disease. Doubt is cast upon such a conclusion by Powell, Jamieson and Kempf (1935) who found that the removal of heterophile antigen from horse serum did not alter its capacity to produce serum sickness. Davidsohn (1938) has also called attention to an increase in titer of /? isoagglutinin in patients developing serum sickness. There was no significant increase noted in a agglutinins. Atopic reagins, according to Tuft and Ramsdell (1929), appear irregularly in the blood of individuals receiving injections of therapeutic horse serum. Doubt is cast upon their importance by the irregularity of their occurrence and the lateness of their appearance in many cases of serum sickness. Since Zinsser, Enders and Fotliergill apparently accept as fairly well established the antigen-antibody theory of von Pirquet and Schick, it would seem desirable to review and discuss the evidence which they accept as justifying such a conclusion. This evidence may be summarized as follows : (a) The proteins in the serum injected are antigenic. Hooker (1923) described at least three antigenic fractions of horse serum. (b) The existence of an incubation period between the injection of horse serum and the appearance of serum sickness. 312 IMMUNOLOGY (c) The appearance of antibodies for horse serum proteins in the blood of many patients receiving serum injections and an apparent correlation of their appearance with the development of symptoms. (d) The shortening of the incubation period following subse- quent injections of horse serum. While the evidence supporting the theory of von Pirquet and Schick is very suggestive, it can only be regarded as circumstantial in nature. One should not lose sight of the fact that horse serum is foreign to man and that it, and perhaps to a lesser extent the fractions obtained from it, may possess pharmacological properties distinct from tlie antigenic ones. The toxic properties of antigenic substances are indicated by reactions of smooth muscle to toxic doses of horse serum and other antigens described by Dale (1913) and Sherwood and Stoland (1923) as distinct from antigenic prop- erties. The Brodie reaction (1900) in cats is usually ascribed to the presence of acetyl choline in normal serum. Incidentally, it has been shown in this laboratory that the Brodie reaction re- sembles the anaphylactic response in cats as determined by blood pressure changes (Kabler and Sherwood, 1938). These examples are given to emphasize the point that serum may have toxic as well as antigenic properties. Another example which may bear more directly upon serum disease is the pulmonary permeability and vagus irritability changes produced in dogs by the injection of horse serum. Manwaring, Chilcote and Hosepian (1923) who tirst described the change in pulmonary permeability that follows the injection of horse serum attributed it to anaphylactic sensitiza- tion. That the phenomenon is not an antigen-antibody reaction is indicated by the work of Sherwood and Stoland (1930) and Sto- land, Sherwood and Woodbury (1931). The evidence they presented may be summarized as follows : (a) The phenomenon is observed in animals that show no other evidence of sensitization to horse serum. (b) It can be demonstrated by perfusion with Locke's solution from which antigen is omitted. (c) It can be demonstrated in desensitized dogs. (d) The incubation period is shorter than in anaphylaxis. (e) The chronaxic studies indicate that following the injection of horse serum there develops an increa.se in irritability of the SERUM REACTIONS 313 vagus nerve which correlates with increased permeability of the tissues but not with the classical anaphylactic sensitization. That the existence of an incubation period is not in itself proof of the antigen-antibody mechanism of serum sickness is suggested by the fact that periods varying from hours to days elapse be- tween the injection of various drugs, tuberculin and toxic sub- stances and the appearance of symptoms. In drug allergy there is frequently an incubation period of 6 or more days and symptoms that in part at least may resemble serum sickness. As in serum sickness tlic incubation ])eriod is shortened by subsequent injections of tlie exciting agent. In the tuberculin type of skin reactions and in positive Schick tests there is an incubation period, so to speak, of 24 to 48 hours or longer. All efforts to show that drug allerg}^ and allergy due to infection are mediated by an antigen-antibody mechanism have failed. In the case of the positive Schick test it is the absence of antibodies that permits the toxin to act upon the tissues. To explain the short incubation period occasionally ob- served in serum sickness Zinsser, Enders and Fothergill suggest the possibility of intrauterine or intestinal sensitization but offer no statistical data to support such a correlation. Since the incubation period for serum sickness is frequently seven to ten days and since horse serum contains antigenic proteins one might exi)ect that frequently the correlation would be noted be- tween the appearance of antibodies and the development of symp- toms. This could easily be a coincidence. It is not likely that the union of antigen and antibody in the blood stream would cause symptoms as this phenomenon is offered as an explanation for the failure of symptoms to occur in sensitized animals. It has been called "masked anaphylaxis." Dean (1931) has suggested that perhaps tlie reason that antigen and antibody, at times, coexist in the blood without the formation of a precipitate is the ab- sence of optimal ratios of concentrations of each. Furthermore the symptoms in serum disease are not those of anaphylaxis. Con- trast the fever of serum disease with the subnormal temperature of anaphylaxis, the absence of pulmonary symptoms in serum disease and their presence in human anaphylaxis. In the rabbit, note that in serum disease there Ls an elevation of temperature, swelling and hyperemia of the ear but no cardiac symptoms while 314 IMMUNOLOGY in rabbit anaphylaxis there is lowering of the temperature, com- plete blanching of the ear and when death occurs it is due to right heart failure. The shortening of the incubation period in man following subse- quent injections is not proof of anaphylactic or antigen-antibody nature of serum sickness. As mentioned above a similar phenomenon occurs in drug allergy. The accelerated reaction is not an immediate one as in anaphylaxis but po.ssesses a delay of six hours or more. The incubation period may be short because of the toxic effect of the first injection upon the tissues permitting a more rapid entry of substances into the cells. While we are inclined to regard serum sickness as a manifesta- tion of some form of allergy, we agree with Coca that an antigen- antibody mechanism has not as yet been established. References Bradshaw, D. B. : A New Serum in the Passive Control of Scarlet Fever, Lancet 2: 6, 1939. Brodie, T. G.: The Immediate Action of an Intravenous Injection of Blood Serum, J. Physiol. 26: 48, 1900. Bullowa, J. G. M. : The Management of the Pneumonias, New York, 1937, Oxford University Press, p. 283. Coca, A. F.: Cited by Harten and Walzer, .T. Allergy 11: 69, 1939. Coca, A. F. : Essentials of Immunology, Baltimore, 1925, Williams & Wilkins. Coghill, Eobert D., Fell, Norbert, Creighton, Martha, and Brown, Gordon: The Elimination of Horse-Serum Specificity From Antitoxins, J. Im- munol. 39: 207, 1940. Dale, H. H. : The Anaphylactic Keaction of Plain Muscle in th« Guinea Pig, J. Pharmacol. & Exper. Therap. 4: 167, 1913. Davidsohn, I. : Isoagglutinin Titres in Serum Disease, in Leukemias, in Infectious Mononucleosis and After Blood Transfusion, Am. J. Clin. Path. 8: 179, 1938. Davidsohn, I.: (a) Heterophile Antibodies in Scrum Sickness, J. Immunol. 16: 259, 1929. (b) Further Studies on Heterophile Antibodies in Serum Sickness, Ibid. 18: 31, 1930. (c) Heterophile Antibodies in Serum Sickness, J. Infect. Dis. 53: 219, 1933. Dean, H. K.: The Precipitation Eeaction. A System of Bacteriologj-, His Majesty's Stationery Office, London (Vol. G, Immunity), 1931, p. 424. Fleisher, M. S., and Jones, L. R.: Serum Sickness in Rabbits With Manifesta- tions of Serum Sickness, J. Exper. Med. 54: 597, 1931. Influence of Various Sera Upon the Occurrence of Serum Sickness, J. Immunol. 24: 369, 1933. Immediate and Accelerated Reactions, Ibid. 24: 383, 1933. Serum Sickness in Rabbits. VI I. A Method for Removing or Destroy- ing tlie Factor Causing Serum Sickness, Ibid. 36: 511, 1939. Goodner, K., Horsfall, F. L., Jr., and Dubos, R. J.: Tj'pe Specific Anti- pneumococcic Rabbit Serum for Therapeutic Purposes: Production, Processing and Standardization, J. Immunol. 33: 279, 1937. Grabar, P.: Sur L 'action de la Pepsine sur Les Anticorps Antipneumo- cocciques, Compt. rend. Acad. d. sc. 207: 807, 1938. SKRUM REACTIONS 3l5 Hansen, A.: L 'action de La Pepnino e de L'acid Ghlorhydrique sur L'anti- toxine Dipliterique, Compt. rend. Soc. de biol. 129: 213, 1938. Harten, Max. and Walzer, Mattliew: S«riim Allergy, J. Allergy 11: 89, 1939. Harten, Max, and Walzer, Matthew: J. Allergy 11: 69, 1939. Hanzlick, P. J., and Karsner, H. T. : Anaphylactoid Phenomena From Thromboplastic Agents, J. Pharmacol. & Exper. Therap. 14: 229, 1920. Anaphylactoid Phenomena From the Intravenous Administra- tion of Various Colloids, Arsenicals and Other Agents, Ibid., p. 379. A Comparison of the Proph^vlactic Effects of Atropine and Epinephrine in Anaphylactic Shock and Anaphylactoid Phenomena From Various Colloids and Arsphenamine, Ibid., p. 425. Effects of Various Colloids and Other Agents Which Produce Anaphylactoid Phenomena on Bronchi or Perfused Lungs, Ibid., p. 449. Surviving Intestine and Uterus, Ibid., p. 463. Hemagglutination in Vitro by Agents "Wliich Produce Anaphy- lactoid Symptoms, Ibid., p. 479. Hooker, S. B. : Human Hypersensitiveness Induced by Very Small Amounts of Horse Serum, J. Immunol. 9: 7, 1924. Horsfall, F. L., Jr., Goodner, K., MacLeod, C. M., and Harris, A. H. : Anti- pneumococcus Rabbit Serum as a Therapeutic Agent in Lobar Pneu- monia, J. A. M. A. 108: 1483, 1937. Hutchinson, A.: Treatment of Diphtheria With Eefined Antitoxin, Brit. Med. J. 1: p. 381, 1039. Jones, L. R., and Flushner, M. S. : The Relation of Serum Fractions to Serum Sickness in Rabbits, Proc. Soc. Exper. Biol. & Afed. 30: llOf). 1933. See also J. Immunol. 26: 455, 1934. Kabler, Paul, and Sherwood, N. P.: Physiological Studies of the Hyper- sensitive Cat, TIniversity of Kansas Science Bull. 25: No. 5, June 1, 1938. Karsner, H. T.: Anaphylaxis and Anaphylactoid Reactions, Newer Knowl- edge of Bacteriology and Immunology, Chicago, 1928, ITniversity of Chicago Press, p. 966. Longcope, W. T., and Rackemann, F. M.r J. Exper. Med. 27: 341, 1918. Lord, F. T., and Heffron, R. : Pneumonia and Serum Therapy, New York, 1938, The Commonwealth Fund, p. 60 (cited by Harten, Max, and Walzer, Matthew, J. Allergy 11: 87, 1939). MacLeod, C. M. : Treatment of Pneumonia With Antipneumococcal Rabbit Serum, Bull. New York Acad. Med. 15: 116, 1939. rVfanwaring, W. H., Chilcote, R. C, and Hosepian, V. M. : Capillary Permea- bility in Anaphylaxis, J. A. M. A. 80: 303, 1923. Anaphylactic Reac- tions in Isolated Canine Organs, J. Immunol. 8: 233, 1923, Parfentjev, I. A.: (1936), U. S. Patent, 2,065,196. Park, W. H., and Williams, A. W. : Pathogenic Microorganisms, Philadelphia, Lea & Febiger, p. 403. Pope, C. G. : The Action of Proteolytic Enzymes on the Antitoxins and Proteins of Immune Sera, Brit. J. Exper. >ath. 20: pp. 132, 201, 1939. Powell, H. M., Jamieson, W. A., and Kempf, G. F. : On the Relation of Het«rophile Antigen to Serum Sickness, J. Immunol. 29: 267, 1935. Sherwood, N. P., and Stoland, O. O.r Bacterial Anaphylaxis, J. Immunol. 8: 141, 1923. Sherwood, N. P., and Stoland, O. O. : The Pulmonary Permeability in Normal and Sensitized Dogs and Its Relation to Anaphylactic Shock, J, Im- munol. 20: 101, 1930. Stoland, O. O., Sherwood, N. P., and Woodbury, E. A.: Chronaxie of the Cardiac Vagus Nerve in Dogs Sensitized to Horse Serum and in Anaphylactic Shock. J. Immunol. 21: 393, 1931. 316 IMMUNOLOGY Tuft, L.: Clinical Allergy, Philadelphia, 19ri7, W. B. Saunders Co.. pp. 6, 128. Tuft, L.: Serum Sensitivene.ss After Toxin-Antitoxin, a Clinical and Laboratory Study, J. Allergy 3: 235, 1932. Tuft, L,, and Ramsdell, S. G.: Antibody Studies in Serum Sickness, With Special Reference to the Prausnitz-Kiistner Reaction, J. Immunol. 16: 411, 1929, and Ibid. 17: 539, 1929. Vaughan, W. T., and Piper, D. M.: On the Probable Frequency of Allergic Shock, Am. J. Digest. Dis. 3: 558, 1937. Warner, W. P.: Antipneumococcus Rabbit Serum in the Treatment of Pneumonia, Canad. Pub. Health J. 30: 82, 1939. Weil, A. J., Parfentjev, I. A., and Bowman, K. L.: Antigenic Qualities of Antitoxins, J. "Immunol. 35; 399, 1938. Zinsser, Hans, Enders, John F., and Fothergill, LeRoy D.: Immunity, New York, 1939, The Macmillan Co., p. 391. CHAPTER XVII BIOLOGICAL AND ANTIGENIC SPECIFICITY Immunological Specificity, — In the previous pages attention has been directed to the phenomenon of immunological specificity wherein a specific antigen not only stimulates the physiological ]>roduction of specific antibodies but reacts only with the anti- bodies thus formed. Landsteiner (1936, p. 5) points out that specificity is not an entirely absolute quality but that there is always some overlapping. Loeb (1916), Wells (1929), Zinsser, Enders and Fothergill (1939) and others have called attention to the general biological significance of specificity and have reviewed the literature and offered experimental evidence pointing to a chemical basis for all specificity. Biological Specificity. — Experience in animal breeding and in cross fertilization has shown that success is attained only when eggs and spermatozoa of the same or very closely related species are used. Specificity and Incompatibility of Species.— In discussing the incompatibility of species not closely related, Loeb mentions the rigid specificity requirements for successful skin grafting or or- gan transplantation. It is the experience of every plastic surgeon that the best results are obtained when the skin from the patient's own body is used or from members of the family who belong to the same blood group. In any event skin from a different species cannot be used for a successful graft. The same applies to organ transplantation. Specificity^ and Fertilization. — Loeb also calls attention to the specificity requirements in the fertilization of an egg by a sperm. In this process many phenomena analogous to those observed in immunity are encountered. The first of these is positive chemo- taxis. Pfeiffer showed that fern spermatozoa are deviated in their course when they a]iproach an archegonium containing an egg, and that the attraction is sufficient to cause the sperm to enter the archegonium and permit contact and ultimate fertiliza- tion of the egg. Loeb says that it is a very common experience 317 318 IMMUNOLOGY ''that spermatozoa become very active when they reach the neigh- borhood of an egg. ' ' Lilly has shown this to be a specific effect or activation. When certain spermatozoa such as those of the Cali- fornia sea urchin, Strongylocentrotus purpuratus, or of certain annelids are put into sea water wliich has been in contact witli the corresponding eggs, specific clumping or agglutination of the spermatozoa occurs. Union of Egg and Sperm.— The fact that in fertilization, the sperm enters the egg and only the eggs of the same or closely related species, indicates not only specific invasive power on the part of the sperm but also specific susceptibility on the part of the egg for the same sperm. Toward the spermatozoa of other species it exlnbits a general resistance. After fertilization witli suitable spermatozoa has taken place the egg becomes immune to invasion even by spermatozoa of the same species. Tvoeb calls attention to the similarity of fertilization or union of sperm and egg to the phenomenon of phagocytosis. The sper- matozoan enters through a protoplasmic process (fertilization cone) wliich is comparable to the pseudopods of ameboid cells. As a result of the entrance of the spermatozoan, in the case of the sea urchins studied by him, the q^^ develops a membrane which acts as a barrier to the entrance of any more spermatozoa. This is obviously a specific defensive mechanism. Loeb has been able to modify these various mechanisms, and to a large extent control them by altering the salt content and pH of the sea water. He concludes that the biological specificity in fertilization is chemical in nature. Specificity and Genetics. — The exactness with which genet- icists can predict the appearance of certain characters in the off- spring of inbred strains of the grasshopper or white rat, plants, etc., indicates that specific reactions are at work and that the phenomenon of specificity is of broad biological significance. Production of Specific Hormones. — In the animal body specific chemical substances such as thyroxin, insulin, cortin, adrenalin, etc., are normal physiological products and each brings about certain specific physiological reactions. The constancy of pro- duction and the specificity of bacterial enzymes has long been BI0I.()(JI(;AI> AM) AXTKJKXIC SPKCIl'ICITY 319 known and used in l)H('lc'iial identification. That tiiis .si)ecific'ity is definitely due to chemical structure is now generally accepted. Thus it will be seen that immunological specificity is but another example of a phenomenon quite common in nature. Immunolog'ical Specificity. — CJrubei- and Durham ( 189(5) were really ])ioneers in the study of immunological specificity. They discovered, while working with bacterial antigens, specific and group agglutination. They noted that when they obtained an im- mune serum that agglutinated in high titer suspensions of the organisms used in the antigen, this same immune serum frequently agglutinated closely related bacteria at a lower titer. For example, an antityphoid immune serum that would agglutinate only suspen- sions of E. typJiosa when the serum was diluted 1 : 10,000 might agglutinate suspensions of closely related organisms when diluted to only 1 :100 or 1 :200. Fl Fig. 14. -Durham's conception of the multiplicity of cellular antigens. A, Major antigen. B, C, and D, Minor antigen.^;. Durham 's Explanation — Multiple Antigens. — Sometimes group agglutination at a higher dilution could be demonstrated. By using high titered serum they could demonstrate species speci- ficity, but the group reactions did not always indicate closely re- lated species. Durham attempted to explain the group reactions as due to the presence of identical antigens of different amounts in different bacteria. In his opinion, each species of bacteria con- tained a chief antigen that was species specific and a number of minor antigens that might be present in closely related organisms. This concept is indicated in Fig. 14. Here A represents the chief antigenic constituent of the cell while B, C, D, and E represent minor antigens. Any one of these might be found in some other closely related organism in different amounts. Varying amounts 320 IMMUNOLOGY of antibodies would be produced for each kind of cell antigen ; the amount of antibody response would correspond to the amounts of various antigens present. The major antigen would lead to the production of a major or chief agglutinin, while minor agglutinins would be produced for the corresponding minor antigens. Land- steiner (1936) in an excellent discussion of antigens considers cellular antigens as forming a mosaic pattern. This has been dis- cussed in the preceding chapter. Precipitins and Specificity. — Shortly after Gruber and Dur- ham's discovery of specific and group agglutination of bacteria, Kraus (1897) reported that filtered extracts of bacteria would give specific precipitates when mixed with their homologous im- mune serum. Two years later Tchistovitch (1899) found that blood serum (eel) when used as an antigen led to the production of precipitins in rabbits. There followed, according to Pick (1904), extensive investigations of antigens as to their nature, method of preparation, chemical and physical structure, and specificity. Nut- tall (1904) used quantitative precipitin tests in an extensive study of the zoological relationships in the animal kingdom. His results were in harmony with the accepted zoological relationships which had been based upon morphology. This indicated that the process of evolution included biochemical as well as anatomical and morphological changes. Precipitins and Species Relationship.— While the work of Nuttall (1904) and others indicated that the precipitin test could be used to identify the blood proteins of a species (species specificity parallels immunological specificity), a discovery had been made by Landsteiner (1902) which seemed to be at variance with this. He found that he could differentiate 3 or 4 groups within the human species by means of isohemagglutination. Serological Types Within a Species. — Thus while Nuttall and others had shown that the serum proteins of all members of a species were alike antigenically, Landsteiner had shown that the red cells of different groups of individuals within the species (human) were antigenically unlike and that the dissimilarity probably had an hereditary basis. Bacterial Types Within a Species. — In 1909, Neufeld de- scribed antigenic differences in a bacterial species, the pneumo- HTOLOdlCAL AND ANTIGKXK" SI'KCIFICITV 321 COCCUS, and since then Dochez, Aveiy, Blake and others have found, by means of aiLiglutination, antigenic types for the meningo- coccus, streptococcus and man}- other kinds of bacteria. Thus it will be seen that at least two kinds of specificity can be demon- strated by immune reactions, one that applies to species and the second to type variation within a species. Serum Proteins vs. Cellular Antigens. — Landsteiner (1928) has called attention to the fact that species relationships are indi- cated by a study of the serum proteins, while type differentiation within a species is determined by studies of the cellular antigens peculiar to the species. Wells (1929) considers that even the serum proteins show two kinds of specificity and this is borne out hy the work of Nuttall and others. Ehrlich (1910) says that his side chain theory of immunity implied that antigenic specificity was dependent upon the chemi- cal constitution of the antigen. Subsequent research has ap- parently confirmed this concept. Formerly it was thought that only complete proteins were antigenic but it is now agreed that a few bacterial polysaccharides (e.g., Type I pneumococcus) and perhaps a few lipid-carbohydrate complexes may stimulate specific antibody formation. Wells (1929) summarized the properties of antigenic protein substances somewhat as follows : Properties of an Antigen. — They are all complete proteins in a colloidal state, soluble in the fluids of the animal body and possessing a necessary number and kind of aromatic amino-acids. To elicit antibody production they must be introduced beneath the "epithelial coverings of the body," i.e., into the tissues of an animal not possessing similar antigens in its body proteins. (It is now generally believed that many of these properties are possessed by all antigens whether protein or carbohydrate.) To appreciate the basis for the above conclusion of Wells and others, it is necessary to compare the results of chemical and immunological investigations of both animal and plant proteins as well as experimentally modified proteins. Similar comparisons should also be made for the carbohydrates and lipoids. Animal Proteins. — Proteins of the animal body consist of the proteins ol' tlie l)lood, tissues, and normal secretions of the body. 322 IMMUNOLOGY Larjiely through tlie Avork of Lel)lane (1901), Hektoeii (1918), Dale and Hartley (1916), Doerr and Berger (1922), Lewis and Wells (1927) and others it has been shown that blood plasma contains at least five chemically different proteins that are also antigenically different. These plasma proteins are eus'lobnlin, psendo^lobnlin, albnmin, seromncoid and fibrino«'en. Of all these proteiiLs only one, fibrinogen, lacks species specificily. However, even with it there is a quantitative difference that is definite since the maximum precipitate occurs between an antifibrinogen immune serum and the species fibrinogen used to produce it. Goodner (1925) found that beef serum was antigenically different from beef chondromucoid or beef submaxillary mucin. Red Cell Proteins. — Hemoglobin present in the red cells is both cliemically and antigenically different from the proteins of the plasma. Even the globulin from the red cells shows antigenic differences from the serum glol>ulin. The red cells also contain lipoids and carbohydrates which form compounds with some of the red cell proteins, giving rise to chemical and immunological dif- ferences. This has been discu.ssed in the cliapter on Blood Group- ing. Tissue Proteins. — Downs (1925) has shown that tissue fibrino- gen is species specific antigenically, which is interesting in view of the fact that Hektoen and Welker (1925, 1927) showed that blood fibrinogen does not show species specificity. Goodner (1925) found marked antigenic differences between the mucins obtained from beef submaxillary glands and from the hog stomach. He calls attention to the fact that the conjugated proteins are relatively poor antigens and that their antigenic efficiency bears some relationship to the amount of protein pres- ent in the conjugate. This is in harmony with other experimental results which show that with a few exceptions tissue proteins do .show species specificity. Organ Specificity. — Pfeiffer (1910) even claimed that antigenic difference could l)e detected between the proteins of the liver, spleen, kidney, etc., but Pearse, Karsner, and Ei.senbrey (1911) failed to confirm Pfeiffer 's claims. Later Fleischer (1920, 1921) seem.s to have demonstrated some slight difference. Lens and Testicular Proteins Shonv Nonspecies Specificity. — Of the tissue proteins lacking species specificity, those of the BIOLOGICAL AND ANTIGENIC SPECIFICITY 323 crystalline lens of the eye and of the testis are possessed by all species, while Forssman's antigen and somewhat similar sub- stances recently described by Landsteiner and Levine (1932) are peculiar to only a limited number of species of animals as well as a few species of bacteria. Since Forssman's antigen is a lipoid- carbohydrate-protein complex, or at any rate a hapten-protein com- bination, it is possi])le that the hapten portion is the fraction com- mon to different species, etc. In regard to lens protein, Kraus, Doerr and Sohma (1908). Uhlenhuth (1910), Hektoen (1921, 1927) have found tliat ])rotein from the crystalline lens of the eye is antigenically and cliemically the same in all species. It even functions as an antigen when injected into the same species. Von Dungern and Hirschfeld (1910) claim to have demonstrated similar nonspecies specificity for testicular protein, although they did not find such marked organ specificity as exists in the case of lens protein. Keratin, Mucin, Etc., Lack Species Specificity. — AVells (1929) says that "it is altogether reasonable that lens proteins, keratin, mucin and other proteins whose function is not metabolism, should be nonspecific. Each of these proteins has quite the same function to perform in every species, and is set off from the active tissues to perform it." Thyroglobulin.— Hektoen and Scliulliof (1923) found that thyroglobulin is antigenically different from the other proteins of the same animal from which it is obtained but antigenically similar to thyroglobulin of other species. Milk Proteins. — Proteins of normal body secretions. Milk is a normal secretion of the mammary gland and contains proteins, carbohydrates, fats, and other ingredients. Casein, the principal protein, is chemically identical and antigenically similar for all species, but in each species its chemical and antigenic properties are different from other body proteins. On the other hand, the globulin present in milk is chemically and antigenically similar to the serum globulins of the s])ecies and shows the same .species specificity as the .serum globulins. Antigenic Components of the Egg.— Wells (1911) and Hek- toen and Cole (1928) have demonstrated five antigenic components 324 IMMUNOLOGY of hen's eggs corresponding to five chemically different proteins that have been isolated and studied. Wells has also called atten- tion to the chemical and antigenic similarity between the crystal- lized albumen of the egg from both the hen and the duck. Goodner (1925) found some antigenic relationship between two glucopro- Icins of the hen's egg. Biological and Immunological Relationships Among Ameba. — Heathman (1932) made extensive immunological studies of a num- ber of strains of free-living and pathogenic ameba. Her results corroborated the morphological classification of Schaeffer (1926). Her results indicated that there are both a highly specific and a common radical present in the antigen Avhich are responsible for the specific and nonspecific reactions. Slierwood and Heathman (1932) found two haptens present in Enfameha histolytica. The chief one was alcohol soluble and a second was solulile in saline, as were the haptens present in the free-living amebae. Vegetable Proteins. — Wells and Csborne (1916) and others working in Wells' laboratory have made extensive studies of the vegetable proteins and found them especially valuable for com- parative chemical and antigenic investigation. They isolated giiadin from both wheat and rye and found them chemically and immunologically identical. They could differentiate by immuno- logical methods proteins such as hordin of barley and glutenin of wheat not only from each other but also from giiadin. They also could detect by both chemical and immunological methods a protein legumin, common to the seeds of peas, vetch, lentil and horse bean. In their studies of the proteins of leguminous seeds, they isolated from each species two globulins, a and P, which are chemically and immunologically different. This was of especial interest since Wells had also shown that globulins from different beans may show chemical and immunological similarity. Algae. — Steinecke (1925) carried out extensive immunological and biological comparisons of algae, etc., with an idea of throw- ing light upon evolutionary development. He concluded that it is with the descendants of the euglenas that one is to look for the foundation of the animal kingdom. Elmore (1928) found that BIOf.OGI(L\L AND ANTKIRNIC Sl'KCIFICITY 325 Euglena (jmcilis Klehs is not a homogeneous group but consists of two distinct antigenic types. Tliese correlate with growth characteristics. Antigens in Fungi. — Hektoen (1901) noted antigenic properties for a strain of Blastomyces dermatitis. Recently Dnlaney (1930) confirmed this work, but could not demonstrate species specificity in the blastomyces. She says that "slightly positive results were obtained with closely related yeasts and specific results were ob- tained upon titration of the serums. ' ' In this connection it is interesting to note that IMueller and Tomesik (1924) obtained a soluble specific substance from yeast and found it to l)e identical With the "yeast gum" of Salkowski (1894). It yielded specific precipitates with homologous immune serum but was found to be nonantigenie. This work has been ex- tended recently by Tomesik (1930). Kesten, Cook, Mott, and Jobling (1930, 1931) liave isolated s])oeific i)olysaccharides from strains of monilia. Bacterial Antigens. — While this subject will ])e discussed in Chapter XIX, attention can be called here to the fact that they, like the algae, fungi, amebae and red blood cori:)uscles, represent cellular or protoplasmic antigens exceedingly complex in nature and in which haptens play an important role in the serological re- actions. By agglutination and absorption tests it has been possible to demonstrate species specificity for some species, e.g., E. typhosa while for others like Esch. coli only strain specificity has been demonstrated as yet. Only a small amount of albumin or globulin has been found in the bacterial extracts, the bacterial cell substance containing for the most part nucleoproteins. A continuation of the study of bacterial proteins such as the P^ and F^ fractions obtained by Furth and Landsteiner and of the cell haptens gives promise of a better understanding of bacterial antigenic specificity. Structure and Properties of Proteins. — When one looks at the lU'oteins from the standpoint of their chemical structure and biochemical pro])erties, one discovers a number of reasons why most antigens are probably protein in nature and how their struc- 326 IMMUNOLOGY ture may account for specificity. In the first place, they are colloids, chemically different yet possessing physical properties and chemical constituents similar to those of the tissues of the animal which give rise to antibodies. Second, the enormous number of possible combinations of the 20 or more amino acids might well account for the equally large number of species of living organisms; third, they possess the property of linkage with many different kinds of chemical elements that could account for further s])ecific diversity both in their character and also in their reactivity; and fourth, the different ])i'oteins are quite constant in their chemical composition. Importaxck of Physical and Chemical Changes in Antigen. — Zinsser calls attention to the fact that true antigens are composed of large molecular aggregates incapable of diffusion through a membrane and that this is probalily a necessary state to induce antibody response. When irreversible coagulation of an antigenic protein occurs, il becomes antigenically inert. Wells (1929) cites tlie irreversil)le coagulation of egg albumen by alcohol as an ex- ample of this. The same antigen coagulated by heat tends to redissolve and retains its antigenic property only so far as it goes back into solution. Wells (1909) further showed that when an antigen such as egg albumen is treated with hydrochloric acid, acid albuminate is formed without impairment of the antigenic properties. On the other hand, if the egg albumen is treated with alkalies with the formation of alkali albuminate, all antigenic properties are lost. This, according to Dakin (1912, 1913), is associated with a loss of optical activity and also the capacity to be acted upon by enzymes and is called ''racemization." It should be noted that racemized antigens, however, retain their solubility and coagulability and all of their amino acid constituents. The change, according to Wells (1929) and Dakin, is in those amino acids that have their amino groups linked to a carboxyl group Avithin the protein molecule. "The terminal amino acid groups containing a free carboxyl group remain un- changed." (Wells and Dakin.) They speak of this change as BIOLOGICAL AND ANTIGENIC SPECIFICITY 327 enolization and say that it consists of the loss of asymmetry by the a carbon atom. Wells* illustrates this as follows: OH OH II I I R— C— C— NH. » R— C = C— NH, I ■ I CH,, CH, Ketone form Enol form (Before racemization) (After racemizatioii) It -will l)e ol)served tliat in the ketone form the a carbon is linked to four different groups; this makes of it an as>mimetrie carbon atom. In the enol form it is linked to three, and hence is no longer asymmetric and can now form equal amounts of two isomers Avhich accounts for its loss of optical activity, etc. Enzyme Action Destroys Antigenic Propp:rty. — It has been quite well established that when proteins are acted upon by enzjmies their antigenic properties are diminished and entirely lost during the process of digestion. Relationship of Digestibility and Antigenic Property.— Since racemized proteins are neither antigenic nor digestible by enzymes and since complete ])roteins possess nondiffusible molec- ular aggregates and are both antigenic and digestible, it has been suggested that for antibody production the antigen must reach the surface of the tissue cells and call forth extracellular enzymes. It should be remembered that the molecular aggregates of an antigen may be phagocytized, so to speak, by the retieulo- endothelium and digestion occur within food vacuoles. Anti- bodies could be returned to the general circulation by a process llio reverse of that of phagocytosis. * Landstciner does not regard the lack of digestibility of racemized ])roteins as the reason for their lack of an antigenic property but thinks it Ls due to their lack of a chemical structure necessary to stimulate antibody formation. Possible Number of Compounds Formed by Amino Acids.— Abderhalden has calculated, according to Wells (1929), ''that 20 amino acids could form 2,432,902,008,176,640,000 different com- pounds, and this without including the enormously greater num- ber that might be made by varying the proportion of the different •Well.'', H. G. : The Chemical Aspects of Immunity, Reinhokl PublLshing Corporation. 328 IMMUNOLOGY amino acids in a single protein." Wells regards this as strong evidence that immunological and biological specificity depend upon the proteins. Subsequent work has shown, however, that a feiv apparently nonprotein polysaccharides or carbohydrate-lipoid complexes can stimulate specific antibody production. Importance of Aromatic Amino Acids in Protein Antigens. — Starin (1918) working in Wells's laboratory found that gelatin, which is a derived protein lacking tyrosine, is nonantigenic. From an extensive chemical examination of protein antigens Obermayer and Pick (1906) conclude that aromatic amino acids are essential to all antigens and conversely that compounds made up of ali- phatic amino and diamino acids are not antigenic. Acid and Basic Groups of Amino Acids. — It will be remembered that the aliphatic amino acids have an open chainlike structure while the aromatic amino acids have somewhere in their struc- ture a closed chain. The benzene ring is quite common. Each possesses one or more amino (NHo) groups and also a carboxyl (COOH) group. The former endows it with basic properties Avhile the latter, through its ionizable hydrogen, gives it acid properties. Normally these tend to balance each other. If the basic properties are removed, through linkage of some ethyl or methyl group to the nitrogen of the amino group by replacement of a hydrogen, the acid property is increased through this diminu- tion of the basic property. In a similar way the acid property may be reduced and the basic property increased. These basic and acid groups are shown in the following formula for alanine : ( Amino pr ] I basic grouj) ^ H NH, O I I II [Acid property due 1o ion- "j H-— C— C— C— OH-I izable hydrogen of the }- I I [ carboxyl ' (COOH) group J H H Alanine Aliphatic and Aromatic Amino Acids. — The student should re- member that the aliphatic or open chain amino acids can be acetylated or methylated but cannot be diazotized. This will be referred to again in the next chapter. The aromatic amino acids, on the other hand, can be methylated, acetylated, azotized or BIOLOGICAL AND ANTIGENIC SPECIFICITY 329 diazotized and nitroso compounds can also be prepared from them. This is largely because the diazo ( — N=N — ) compounds link readily to the nucleus, provided it possesses an OH or an NHg group to favor linkage. This will also be made more clear in the next chapter. A few of the aromatic amino acids are tyrosine, phenyl-alanine, tryptophane and liistidine. Their struc- tural formulae and importance in antigens will be apparent from a study of the chapter on modified antigens. Zozaya's Nonprotein Antigen. — Zozaya (1932) reports that a colloidal solution of collodion particles which had been made to absorb protein-free carbohydrate haptens of the pneumococcus functioned as an antigen. This has been referred to in a previous chapter. References Dale, H. H., and Hartley, P. : Anaphylaxis to the Separated Proteins of Horse-Serum, Biochem. J. 10: -108,"l916. Doerr, E., and Berger, W. : Ueber das Verhaltnis der Fraktionsspezifitat zur Artspezifittit bei den Eiweisskorpern der Blutsera, Ztsehr. f. Hvg. 96: 258, 1922. Downs, C. M. : The Antigenic Properties of Tissue Fibrinogen, J. Infect. Dis. 37: 49, 1925. Dulaney, A. D.: Immunologic Studies in Blastomycosis, J. Immunol. 19: 357, 1930. Von Dungern, E. 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Para- typhosus B. and B. dysenteriae Shiga, J. Immunol 22: 75, 1932. Landsteiner, K. : The Specificity of Serological Reactions, Springfield, 1936, Charles C Thomas. Loeb, J.: The Organism as a Whole, New York, 1916, G. P. Putnam's Sons. Mueller, J. H.. and Tomcsik, J.: The Chemical Nature of Residue Antigen Prepared From Yeast, .L Exper. Med. 40: 343, 1924. Neufeld, F., and Handel, L.: Cited by Avery, et. al. Acute Lobar Pneu- monia, ^Monographs of Rockefeller Institute No. 7, 1917, p. 55. Nuttall, G. : Blood Immunity and Blood Relationships, London, 1904, Cam- bridge L'niversity Press. Obermayer, F., and Pick, E. P.: Ueber die chemischen Grundlagen der Arteigenschaften der Eiweisskorper Bildung von Immunprazipitinen durch chemisch \ eranderte Eiweisskorper, Wien. klin. Wchnschr. 19; 327, 1906. Pearse, R. M., Karsner, H. T., and Eisenbrey, A. B. : Studies in Immunity and Anaphylaxis, The Proteins of the Liver and Kidney, J. Exper. Med. 14: 44, 1911. Pfeiffer, H.: Ztschr. f. Immunitat., p. 8, 1910. Cited by Zinsser, Resistance to Infectious Diseases, New York, 1931, The Macmillan Co., p. 360. Pick, E. P. : Handbuch der Technik und Methodik der Immunitatsf orschung, Kraus, R., and Levaditi, Gustav Fischer, Jena 1: 331, 1908. Salkowski, E.: Ueber die Kohlehydrate der Hefe, Ber. d. deutschen chem, Gesellschaft 27: 1, 197, 1894". Schaeflfer, Asa A.: (1926.) Investigations on Marine Amebas in Newfound- land and Labrador, Taxonomy of Amebas. Carnegie Inst., Washing- ton Yearbook 25: 312. Schmidt, C: The Chemistry of the Amino Acids and the Proteins, Ann. Rev. Biochem. 1: 151, 1932. Sherwood, N. P., and Heathman, L.: Further Studies on the Antigenic Properties of Pathogenic and Free Living Amebas. Complement Fix- ation in Amebic Dysentery, Am. .1. Hyg. 16: 124, 1932. BIOLOGICAL AND ANTIGENIC SPECIFICITY 331 Starin, W. A.: The Antigenic Properties of Gelatin, J. Infect. Dis. 23: 139, 1918. Steinecke, F. : Dcr Stannnbaum der Algen. nach sero-diagnostischen Unter- suchungen dargestellt, Botanisches. Arch. 10: 82, 1924. Tcliistovitch, T.: I5tudes Sur L 'Immuni.ssation centre Le Serum D'Anguilles, Ann. Inst. Pasteur 13: 406, 1899, Toracsik, J,: Ueber die Eolle des Hefegummis in der serologischen Dif- ferenzierung einzelner Hefearten, Ztschr. f. Iramunitatforsch. u. exper. Tlierap. 66: 8, 1930. Uhlenhuth, H.. and Haendel, D, : Unter.sucliungen ueber die praktische Ver- wertbarkeit der Anaphylaxie zur Erkennung und Unterscheidung verschiedener Eiweissarten, Ztschr. f, Immunitatsforsch. u. exper. Therap, 4: 761, 1910. Wells, H. G.: The Chemical Aspects of Immunity, New York, 1929, Eein- hold Publishing Corp., Formerly The Chemical Catalogue Co. Zinsser, H., Enders, J. F., and Fothergill, L. D. : Immunity, New York, 1939, The Macmillan Co., pp. 38-61. Zozaya, J. : Carbohydrates Adsorbed on Colloids as Antigens, J. Exper. Med. 55: 325, 1932. CHAPTER XVIII MODIFIED AND CONJUGATED ANTIGENS* Early Work on Modified Antigens. — The first work on modified antigens was that of Obermayer and Pick (1906). They made the important observation that chemical alteration of proteins leads to the appearance of new serological properties. They introduced iodine and NO 2 into proteins and thus formed iodoproteins and xanthoproteins, respectively. Antibodies produced by injecting these altered proteins gave visible (precipitin) reactions with any protein similarly altered but not with the original unaltered protein. It was the general belief at that time that the capacity of inciting antibody production as well as of reacting in vitro with antibodies was the property solely of ])roteins. This notion was retained long after the publication of Obermayer 's and Pick's papers and the idea that antibodies could react with simple chem- ical substances was not considered at all. It is now known that antibodies for xanthoprotein or iodoprotein will not react Avith NOo or lo (or KI), respectively, but are specific for chemically modified portions of the proteins. This should be kept in mind be- cause it really differentiates the discoveries of Landsteiner from those of Obermayer and Pick. Early Work on Conjugated Antigens. — Landsteiner began by confirming Pick's work and extending it to include acetylated and methylated proteins. He then started a new line of investigation, namely, an attempt to manufacture antigens by combining simple substances with proteins. From this work, he says, two unfore- seen facts emerged. First, that it is possible to produce antibodies which are specific for substances of known constitution. These results were pub- lished more than ten years after the studies of Obermayer and Pick. The second unforeseen result (1920) was that by means of a new method, namely, the "inhibition reaction" antibodies were *r>r. R. Q. Brewster of the Organic Chemi.stry Department collaborated with the author in the preparation of part of this chapter. 332 MODIFIED AND CONJUGATED ANTIGENS 333 shown to be capable of reacting witli very simple eiystalline sub- stances such as benzoic acid, etc. (Landsteiner, Specificity of Serological Reactions, 1936, p. IIS). Landsteiner has also dis- covered the importance of spatial relationships and of the relative position of chemical groups in determining specificity. For a better understanding of the early work of Obermayer and Pick and the distinction between their altered antigens and the new conjugated antigens of Landsteiner and others, structural formulae are employed in the following discussion : Altered or Modified Antigens. — Iodized Protein. — When iodine Ls added to a protein it very likely replaces the hydrogen adjacent to the hydroxyl (-0H) group of some aromatic nucleus; thus if tyrosine is in the ]-)rotein, tlie reaction could be illustrated as follows : H H O J H H O HO- ./' -C— C— C— OH + T.. = HO—/ \— C— C— C— OH + HI H NH, H NH, If an additional mole of iodine is added, the other hydrogen adjacent to the hydroxyl (-OH) group may be replaced to give a compound of the following formula : H H 0 HO— < >— C— C— C— OH \ / I I I H NH, In the case of phenylalanine it is hard to introduce iodine since there is no hydroxyl or other activating group on the ring, as will be seen from the following formula of phenylalanine : H H O <^ \— C— C— C— OH H NH, Iodotryptopiiane. — Tryptophane has not been extensively studied, but it is regarded as iodizable, probably through the re- placement of a hydrogen on the nucleus as follows : 334 IMMUNOLOGY y\ H H O I I I! N H -C— C— C— OH + I„ = I- I I H NH, H H O I I II -C— C— C— OH + HI I I H NH. N I H AcETYLATED Proteins. — The acetylated proteins have been ex- tensively studied by Landsteiner. In such compounds the acetyl group doubtless combines witli the liydroxyl (-0H) or amino (-NH2) groups of the protein molecule. Thus any amino acid might be acetylated in the following manner : H H I I R— C— N- I HO— 0=0 O H H O H'TcTi— C— CH, = R— C— N— 0— OH, ^■ HOI I HO— C=:0 In the case of tyi'osino the acetyl group might l)c attached to either the liydroxyl (-Oil) or amino (-NIL,) group, or porliaps to both. The hydroxvl linkase is ilhistrated as follows: 0 -0- H H 0 1 1 II CH3— C— i CI + H j- / \ 1 1 11 -^ ^— c— c— c— 01 Acetyl chloride N NH, Tyrosine 0 11 H H 0 1 1 II 1 1 / CH-C-0-/ N— C— C— C— OH + HCl -/ 1 1 H NH, In the case of tryptophane the reaction would be the replace- ment of either the single hydrogen on the nitrogen of the indol ring, or of the hydrogen in the amino (-NH,) group on the side chain. H H O /\ N -C— C— C— OH I I H NH, O i H + CI i— C— CH, MODIFUCD AND CONJUGATED ANTIGENS 335 Conjugated Antigens of Landsteiner. — Divzo Compounds. — By diazotization is meant the treatment of an aromatic amino com- pound -witli nitrous acid and hydrocliloric acid in the cold. As a rule Landsteiner diazotized com})ounds that he wished to add to liis proteins. It is very difficult to determine just where the diazonium chloride would couple to the protein molecule. From the stand- point of pure oi'^anic chemistry, however, the coupling of the diazonium chloride with the tyrosine w^ould be most logical, since this is in direct accord with the common procedure for making azo dyes. Students of organic chemistry will recall that plienols couple with phenyl diazonium chloride with the greatest of ease. For example, Landsteiner diazotized p-aminobenzenesulphonic acid by adding nitrous acid (NHOJ and hydrochloric acid (HCl) in the cold; water split off and the diazonium chloride resulted: O HO— S— Z' ^— N i"H;T0T=N— ! OH ■+ H' i CI I! ^ ^ -'- ' '■ ' o o HO— S— (f ^— N=X— CI + 2H,0 II ^ ^ O Coupling With a Protein. — This diazonium cldoride could be coupled to a i)rotein since it wnll unite Avith tyrosine and perhaps also other parts of the protein bj' replacing tlie hydrogen next to the hydroxyl (-0H) group on the benzene ring, as is illustrated below with tvrosine: O HO-^ H H O HO— s— / \— N=N— r'cr+ HH— . . II \ / '■ \/ -C— C— C— OH O H NH, The chloride of diazotized Tyrosine p-aminobenzenesulphonic acid. Arsonic Haptens. — In the same w^ay he diazotized p-aminoben- zenearsonic acid and coupled it with proteins forming a new anti- genic compound : 336 IMMUNOLOGY O HO— As— / N— r'NH7'+'0"'i=N— "OHTh"^— Cl= I ^ ^ ' ' OH O I' / \ HO— As— (^ y—N=N—Cl + 2H„0 OH This p-aminobenzenearsonic diazoniuin chloride could react with aromatic amino acids of the proteins replacing a hydrogen on the nucleus, as was the case with the sulphonic acid derivatives. Using tyrosine again as an illustration we have : O HO _/\ HO— As— / \ -N=N— r"ci"+ "h" OH H NH ^^-^^ H H O H H 0 I I II — C— C— C— OH = HO— As— <^ \ — N=N— C— C— C— OH OH H NH^ Arsonic Protein Compounds. — The compound of protein and the p-aminobenzenearsonic acid is a new antigen for which spe- cific antibodies are produced. Aliphatic Side Chains Not Diazotizable. — When an amino (-NHo) group is in the side chain of a compound, i.e., not at- tached to a carbon atom in a benezene nucleus or ring, as in benzyl amine ^ y — CH^NH,, that amino group is in general not diazotizable, the reaction with nitrous and hydrochloric acids under such conditions giving the corresponding alcohol, free nitrogen (N2) and water. This may be shown as below, using benzyl amine : H II ^-C— NH, + 0=rN— OH = H - / " \_ ^— C— OH + N, + H.O H MODIFIED AND CONJUGATED ANTIGENS 337 It is important that the diazotization of aromatic amino groups, i.e., amino groups attached directly to a carbon atom in a benzene nucleus or ring as in aniline TT N H NH, + H^ ; H + HO -N=:0 N=0 Azo Dyes. — Fur diazotization it is customary to use sodium nitrite and hydrochloric acid, the reaction between sodium nitrite and hydrocliloric acid that yields nitrous acid being as follows: NaNO., + HCl = HNO, +NaCl The preparation of azo dyes is accomplished by coupling dia- zonium chlorides in solution with phenols or aromatic amino com- pounds, \ ^— N==N— jCi'TH'"!— <^ N— OH = <(^ ^— N=N— <^ ^-OH + HCl ^— NH, -N=N— i CI + H <^ ^— N=N— / ^— NH„ + HCl In the protein a similar coupling can be accomplished whenever the aromatic nucleus has an -OH or an -NHo in it, as has been illustrated heretofore. Cysteine and Cystine. — In the case of cysteine, diazotization would not be possible, nor would it be in the case of cystine, since neither of these two compounds contains the aromatic nucleus. MODIFIED AND CONJUGATED ANTIGENS 339 However, acetylalion or niethylation of cysteine might be possible through replacement of the hydrogen on the sulphur as follows: H O H— C— S— rir+ ■ CTi— C— CH., I " " H— C— NH„ I c=o I OH Since there is no replaceable hydrogen on either of the sulphurs of cystine, it is not possible to acetylatc or methylale as shown by the structural formula : H H I I H— C— S fi— 0— H I I H~C— NH., H— C— NH., I ' I ' C=0 0=0 I I OH OH Cystine The chance for aeetylation or methylation of the amino grou]i of either of these two compounds or of any amino acid when combined in a protein is extremely remote, as explained under protein structure. Stereoisomers or Spatial Relationships. — Landsteiner calls at- tention to another interesting factor, spatial relationship, that may influence specificity, especially in the natural antigens con- taining carbohydrates. This type of specificity is due to the con- figuration around an asymmetric carbon atom. He finds when he couples stereoisomers to a protein such as horse serum, by diazo- tization, that each optical isomer-azo-protein complex constitutes a distinctly specific antigen for which corresponding antibodies can be produced. This is another example of hapten specificity. When phenyl glycine is treated with p-aminobenzoyl chloride there results phenyl-p-aminobenzoylamino acetic acid, of which Landsteiner prepared the two optical isomers as shown: 340 IMMUNOLOGY NH, NH, I c=o I N— H I H— C— COOH I I c=o N— H I HOOC— C— H Here the mere difference in relative positions of a hydrogen atom and a earboxyl group suffices to alter the serological reactivity. Ijandsteiner also succeeded in obtaining immune sera which differentiated sharply the three antigens obtained by coupling dextro-, levo- and meso-tartaric acids to proteins such as horse serum through diazotization of the corresponding p-aminotar- tranilic acids, formulae for which are shown below: OH OH 1 OH C=rO 1 C=0 1 C=0 1 H— C— OH 1 HO— C— H H- -C— OH 1 HO— C— H H— C— OH H- -C— OH C=0 -NH. C=0 1 -NH, 0=0 r< _>- |-< :>- f< 1 H Dextro H Levo H Meso -NH. Position Isomers. — Landsteiner also says that the relative posi- tions of groups are quite important in determining specificity. An example may be seen in a consideration of glycyl-alanine and alanyl-glycine. It would be difficult to differentiate them chemi- cally but they should be biologically unlike since their position relationship.s are different. The glycyl-alanine is formed as fol- lows : MODIFIED AND CONJUGATED ANTIGENS 341 HO HO I II I II 3 H— C— C— 0H+ PCl3= 3 H— C— C— CI + PCOH), I I NH, NH, H H O H CH3 H H O HO I I II II I I II I II H— N— C— C— i CI + H i— N— C— H = H— N— C— C— N— C— C— OH +HC1 I ■ -^ I III H C=0 H H CH3 Glycyl OH Glyeyl-alanine Chloride Alanine and in this compound the terminal carboxyl (-COOH) group is the one from the alanine molecule. Now if this process is re- versed, one may produce the alanyl-glycine with the terminal acid group that of the glycine molecule, thus, H H O H H O 3 H— C— C— C— OH + PCI3 = 3 H— C— C— C— CI + PCOH), II II H NH, H NH, HHO HHO HHO HO I I II I I II I I II I II H— C— C— C— iCl + Hi— N— C— C— OH = H— C— C— C— N— C— C— OH + HCl I I ' I I I I I H NH, H H NH3 H H Alanyl Glycine Alanyl-glycine Chloride Linkag'e With Salt-Forming- Groups. — While Pick and others regard the linking of the various haptens to the amino acids as replacement of hydrogen either on the ring or on an amino group attached to the ring, Landsteiner considers that linkage with salt-forming groups takes place in the formation of many of these new antigens. In some instances this might he explained as follows : Removal of Acid Properties of an Amino Acid. — If one con- siders a simple amino acid like amino acetic acid, the formula for which is H O H — C— C — OH ^- lonizable or acid hydrogen N— H I H \ Basic group 342 IMMUNOLOGY it will be observed to have both a basic (-NH^) and an acidic (-COOH) group. Thus, reacting as an alkyl amine it yields salts with acids and as an acid forms salts with bases. If the ionizable hydrogen is replaced by an ethyl group to form the ethyl ester, then the acid properties are destroyed and the basic properties are dominant, so that it might unite through the basic (-NHo) group with the acid (-COOH) groups of proteins. The formula for the ethyl ester of amino acetic acid is H O H— C— C— OCH^ I N— H I H l\EMo\"AL OF Basic I'roperties of ax Amino Acid. — On the other hand, if the same amino acid (amino acetic acid) is acetylated, a compound is formed in which one of the hydrogens of the basic (-NHo) group is replaced by the acetyl group, the formula for the resulting compound being H o H— C— C— OH I N— C— CH., H O In this ease the basic property is lost witli a simultaneous in- crease in acid property- so that the latter Ixx'omes dominant and union through replacement of the ionizable hydrogen to basic groups of other amino acids may occur and thus a new compound be formed through union with the salt-forming groups. Other Ways of Salt Formation. — Another way in which salts may be formed may be illustrated as follows : If one diazotizes p-aminocinnamic acid by means of nitrous and hydrochloric acids, there is obtained diazotized p-aminocin- namic acid which will couple Avith the nucleus of tyrosine as follows : MODIFIED AND CONJUGATED ANTIGENS 343 O H H ^^-| I H H O HO— C— C=C— / \— N=N— rcYTH'"!— I I— C— C— C— OH H NH, Now this prodiK't may uiiilo with the -NIL fjroiip of anotlier amino aeid either by replacement of a hydrogen throupjli loss of water to form an acid amide mroupins' as shown, H0-/\ H O H H R— N— r hT" HO "i— C— C=C— / \— N=N- H H O I I !! C— C— C— OH H NH, or by simple addition to form a substituted ammonium salt as shown by the fomiula H0-/\ H H O H H \y il I I / — \ R— N— O— C— C=C— <^ ^— N=N— H H O I I II — C— C— C— OH H H NH, Effect of Esterification and Methylation of Protein. — ^Land- steiner (1917, 1918) brought about esterification of protein by use of alcoholic acid solution, methylation by use of diazomethane and acetylation by means of acid anhydrides or acid chlorides. Cross Reactions. — Proteins thus altered showed marked anti- genic specificity depending upon the hapten introduced. Thus methylated horse protein reacted with immune sera for methyl- ated i")roteins from other species of animals as well as with meth- ylated plant proteins. This indicated that the hapten influenced the specificity of the protein-hapten complex. In the course of his studies he found that among the proteins coupled with di- azonium derivatives different phenomena Mere noted. Specific Reactions. — When he used them as antigens, those con- taining aniline, p-amino-azobenzenesulphonic acid, ortho-, meta-, and para-aminocinnamic acid and aminoazobenzenedisulphonic acid stinuilated the production of specific antibodies reacting only with the homologous antigens, while others showed a broader sphere of activity. 344 IMMUNOLOGY Formalized Rabbit Serum as an Antigen. — Lanclsteiner and Lampl (1917) also showed that formalized rabbit serum used as antigen would stimulate antibodies when injected into rabbits that reacted only with formalized rabbit serum and not with form- aldehyde-treated proteins of other species. This would indicate, as Wells (1929) has pointed out, that "the occupation of an amido group, e.g., lysine or alanine, by the methylene radical is almost devoid of any effect on specificity, and that a marked chemical change can take place in a protein without noticeable effect on the structural or species specificity." When formaldehyde is added to a protein, it couples with the nitrogen of the amino (-NHo) group with the splitting off of water, as may be seen in the follow- ing reaction of alanine and formaldehyde : H H H— C— H H H— C— H H— C— N i H, + O i = C = H— C— N=CH,, + H.,0 I '- ' I 1 " ' C=0 H C=0 OH OH This condensation removes the basic properties of the amino acid and permits the acid groups to become dominant. This is the basis of Sorenson's "formol" titration method for amino acids. Recent Work on Iodized Antigens. — Wormall (1930) and Jacobs (1932) have recently carried out extensive studies on iodinated sera. The latter prepared antigens, i.e., iodized serum proteins, in dilute ammonium hydroxide solution and also without acid or alkali and compared them immunologically. Jacobs also investigated the loss of original specificity as a result of iodine entering the protein as well as the specificity of the antibodies for iodized antigens and ascertained the amount of iodine necessary to alter the antigen. He also confirmed the observations of Wormall (1930) that diiodotyrosine suppressed the formation of specific precipitates that would ordinarily result when iodinated protein and its antiserum are mixed. This is another example of sup- pression phenomena frequently described by Landsteiner. Jacobs* summarizes the results of his work as follows : ♦Jacobs, J.: J. Immunol. 33: 3G1, 1932. MODIFIED AND CONJUGATED ANTIGENS 345 (1) "When iodine is added to animal sera (antigen) without the presence of acid or alkali, substances are formed which pre- cipitate witli antisera i)re])arcd from iodinated sera. (2) "This takes place in the presence of aqueous solutions as dilute as N/32. (3) "Wormall's observation that diiodotyrosinc, but not potas- sium iodide, inhibits precipitation specifically in sj^stems of iodinated proteins and their antisera was confirmed." Minimum Amount of Iodine to Change Antigenic Property. — To ascertain the minimum amount of iodine necessary to alter a protein antigenically, lie added 2.0 c.c. of iodine solutions corre- sponding to iodine concentrations of N/8, N/16, N/32, and N/64 to 1.0 c.c. of normal horse serum and the mixtures were allowed to stand at room temperature for fifteen minutes. Jacobs says that "a minimum amount of acetic acid was added and the pre- cipitates were centrifuged, taken up in 10 c.c. of saline with the aid of just enough dilute carlionate to adjust the pH to approxi- mately 7.5, and filtered through a Berkefeld V candle until clear." In the tests he used dilutions of this solution corresponding to 1:10, 1:50 and 1:250. "The supernatants were precipitated with three-fourths saturated ammonium sulphate, taken up in 10 c.c. of saline, pH adjusted to 7.5 and passed through a Berkefeld filter. Dilutions were made up comparable to those of the first precipi- tate. Both precipitates and supernatants M^ere examined im- munologically for the presence of normal horse serum and iodi- nated precii>itinogen." He found that it required at least N/32 iodine to alter the antigen immunologically. In these altered antigens there remained unaltered horse serum. He observed a diminution of unaltered antigen as more iodine was added until only a trace was left in the preparations containing the maxi- muni amount of iodine. Specificity for Iodized Proteins. — Like Obermayer and Pick (1906), Wormall (1932), Landsteiner and others, he found that the immune serum for iodized liorse serum reacted with other iodized antigens. Group Reactions Due to Different Haptens in the Same Mole- cule.— Hooker and Boyd (1933) have coupled diazotized arsanilic acid to egg white and gelatin, respectively, and found them both to be antigenic despite the fact that gelatin is not antigenic. The 346 IMMUNOLOGY antiserum for each produced the maxmium amount of precipi- tate with its homologous antigen, but the anti-egg-white-diazo- tized-arsanilie-acid protein gave weaker reactions with diazo- tized-arsanilic-acid-gelatin antigen and conversely the latter anti- serum gave weaker reactions with the former. Egg Albumen Contains Tyrosine and Histidine. Gelatin Con- tains HiSTiDiNE. — It occurred to them that egg white contained both tyrosine and histidine, while gelatin contained histidine but no tyrosine. Hence egg white would have at least two amino acids linked to the arsanilic acid while gelatin would have only one; i.e., histidine. These are the two amino acids which couple with the diazonium compounds. When these antigens are injected one would expect two antihaptens to be developed for the diazotized arsanilic acid egg white and one for the diazotized arsanilic acid gelatin antigen. To determine whether this assumption was true they made use of the phenomenon of suppression of a specific antigen-antibody reaction by compounds chemically similar to tlie specific hapten on the antigen. Use of Diazo Dyes for Suppression of Specific Reaction. — It will be remembered that Wormall showed that if he added diiodo- tyrosine to tubes containing antiserum for iodized protein (con- taining tyrosine) and its antigen, the specific precipitate was suppressed. In other words the addition of a hapten to a tube containing the hapten-protein antigen and its specific immune serum results in this case in the suppression of the precipitate. Hooker and Boyd prepared two dyes, one containing tyrosine- likc diazoarsanilic groups and a second containing histidine-like imidazoldiazoarsanilic groups. Tyrosinediazoarsanilic Groups in Protein Molecule. — In the protein molecule the tyrosinediazoarsanilic groups are tliought to be as follows : 0 1 0 HO— As— / \—N—^— 1 /\ 1 — N=N— / N— As— OH OH H— C— H — N— C— C=0 1 1 1 I H 1 H 1 MODIFIED AND CONJUGATED ANTIGENS 347 Tyrosine-Likk Diazoarsanilic Acid Dye. — In the dye compound wliieh they prepared to use as a synthetic hapten for suppression of the specific precipitate, the structure is not quite the same but it worked quilc Avell. They give its structural formula as follows: O HO— As— / \— N=N— <^ \— OH OH (Tyrosine-like) phenoldiiizoarsanilic acid. Histidindiazoarsanilic Acid Groups in Gelatin Molecule. — In the modified protein antigen the histidindiazoarsanilic acid groups are thought to be as follows : \— As— oil / I O /\ OH HO— As— / \— N=N- OH H=N— (^ N N ■i 1 1 H— C- -H 1 -N— C- -C=0 1 1 H H 1 Histidine-Like Diazoarsanilic Acid Dye. — The dye hapten which they prepared for use in suppressing the specific precipitate that normally results from the diazotized arsanilic acid protein (containing histidine) and its antiserum was not quite like the above, but it too worked satisfactorily in suppressing the specific reaction. The artificial dye hapten structural formula is given by Hooker and Boyd* as follows : O V HO— As— <^ S— N=rN OH N N (Histidine-like) imidazoldiazoarsanilic acid. •Hooker and Boyd: .1. Immunol. 25: 61, 1933. 348 IMMUNOLOGY They found that either dye will completely inhibit precipitation in either serum. The second, i.e., histidine-like, etc., "is relatively more efficient as an inhibitor for the antigelatin-diazo-arsanilic acid." They regard their results as indicating that "the injection of a single 'complete' protein coupled with diazotized arsanilic acid leads to the production of two different anti-haptens, one corresponding more closely to the tyrosinediazoarsanilic acid group, the other to the histidindiazoarsanilic acid group. In the case of the 'deficient' protein gelatin, the coupled protein seems to give rise to only one of these antihaptens. " These results indicate that more than one antibody may be produced by an antigen as suggested more recently by Landsteiner. All of the work on modified proteins supports Wells' (1929) con- tention that specificity may depend upon small groups in the mole- cule as well SLS other factors. For a brief summary of some of the more important points brought out in this cha]itoi' the student is referred to Chapter XX. Antibodies to Strychnine. — Using Landsteiner 's nu'tliods of producing new antigens Hooker and Boyd (1940) failed in an attempt to produce antibodies for morphine but were apparently successful in producing antibodies for strychnine. Precipitation with caseinazostrychnine was inhibited by the alkaloid and some of its derivatives but not by other substances, which like strychnine contain the indol nucleus. The sera they produced were too weak to neutralize the lethal effect of strychnine in mice. References Chamberlain, J. F.: Oiganic Cliemistrv, Pliiliulolpliia, 1928, The Blakiston Co. Heidelberger, M. : Immunochemistry. Annual Review of Biochemistry, Stanford University Press 1: 65.5, 1932. Hooker, S. B., and Boyd, W. C: The Existence of Antigenic Determinants of Diverse Specificitj" in a Single Protein I. Tyrosin and Histidin- diazoarsanilic Acids as Haptens, J. Immunol. 25: 61, 1933. Hooker, S. B., and Boyd, W. C: Antibodies to Strvchnine, J. Immunol. 38: 479, 1940. Jacobs, J.: Serological Studies on lodinated Sera. II. Anaphylaxis, J. Immunol. 23: 861, 1932. Matthews, A. P.: Physiological Chemistrv, New York, 1930, William Wood & Co. Landsteiner, K., and Jablons, B.: Ueber die Bildung von antikorpen gegen verandertes arteigenes Serumeiweiss, Ztschr. f. Tmmunitatsforsch. u. exper. Therap. 20: 618, 1914. MODIFIKD AND COXJUUATKD ANTIGENS 349 Landsteiner, K.: Ueber die Bedeutung der Proteinkomponente bei deii Pracipitinreaktionen der Azoproteine. XIII. Mitteilung ueber Antigene, Biocliem. Ztschr. 93: 106, 1919. Specific Serumreaktionen mit einfach zusammengesetzen Substanzen con bekannter Konsti- tution (Organischen Sauren). XIV. Mitteilung ueber Antigene und serologische Spezifitat, Ibid. 104: 280, 1920. Landsteiner, K., and Lampl, H.r Ueber die Einwirkung von Formaldehyd auf Eiwei Bantigen. VIII. Mitteihing ueber Antigene ausgefuhrt mit Unterstiitzung der fiirstlich Liechtensteinschen Spende, Ztschr. f. Immunitatsforsch. u. oxper. Therap. 26: 133, 1917. Landsteiner, K., and Van der Scheer, .James: On the Antigens of Eed Blood Corpuscles, J. Exper. Med. 41: 427, 1925. Landsteiner, K., and Van der Scheer, James: On the Specificit.y of Ag- glutinins and Precipitins, J. Exper. Med. 40: 91, 1934. On the In- fluence of Acid Groups on the Serological Specificity of Azoproteins, Ibid. 45: 1045, 1927. Serological Differentiation of Stereoisomers, Ibid. 48: 315, 1928. Landsteiner, K. : Cell Antigens and Individual Specificitv, J. Immunol. 15: 589, 1928. Landsteiner, K. : The Specificitv of Serological Kcactions, Springfield, 111.. 1036, Charles C Thomas. Obermayer, F., and Pick, E. P.: Ueber die cheraischen Grundiagen der anteigenschaften der Eiweisskorper. Bildung von TniTiiunprazipitinen durch chemiseh verandertc Eiweisskorper, Wien. klin. Wchnschr. 19: 327, 1906. Pick, E. P.: Handbuch d. Path. IVlikroorganismen, Kollc, W., and Wasser- mann, A. P. 1: 685, 1912. Wells, H. Gideon: Chemical Aspects of Immunitv, The Chemical Catalog Co., New York, 1929. Wormall, A.: The Immunological Specificitv of Chemicallv Altered Pro- teins, .1. Exper. Med. 51: 295, 1930. Zinsser, Hans, Enders, J. F., and Fothergill, L. D.: Immunitv, New York, 1939, The Macmillan Co. Supplementary References .Jacobs, J.: Serological Eeactions of Azoproteins Derived From Aromatic Hydrocarbons and Diaryl Compounds, J. Gen. Physiol. 20: 353, 1937. Landsteiner, K., and van der Scheer, J.: On Cross Eeactions of Immune Sera to Azoproteins, J. Exper. Med. 67: 709, 1938. Landsteiner, K., and van der Scheer, J.: On the Serological Specificitv of Peptids, J. Exper. Med. 69: 705, 1939. Landsteiner, K., and Parker, E. C: Serological Tests for Homologous Serum Proteins in Tissue Culture Maintained on a Foreign Medium, J. Exper. Med. 71: 231, 1940. van der Scheer, .J., and Landsteiner, K. : Serological Tests With Amino Acids, J. Immunol. 29: 371, 1935. CHAPTER XIX BACTERIAL ANTIGENS AND SPECIFICITY Bacterial Antigens. — Complexity. — Landsteiner (1936), Wells (1929), Zinsser (1928, 1939), and others have called attention to the complexity of cellular or protoplasmic antigens as contrasted with isolated and purified proteins. While species specificity can, as a rule, be demonstrated for intact red blood cells by means of high-titered immune serum, one frequently encounters difficulty in determining bacterial species by agglutination, absorption, and complement fixation. Living Attenuated Antigens. — Interest in bacterial antigens began Avith the immunization experiments of Pasteur. He found that attenuated, living cultures Avere efficient vaccines for protec- tive inoculation. Since that time the French scliool has continued to regard suspensions of living attenuated bacteria as the most desirable immunizing agents. Killed Suspensions as Antigens.- — Widal (1896), Bordet, Gruber and Durham and others who initiated the serological identification of bacteria by agglutination, noted that dead sus- pensions of E. tupliosa or Vibrio comma were quite satisfac- tory antigens. Later work by Felix and Pitt (1934) and others has shown the importance of labile antigenic factors in agglutina- tion reactions. Early Research on Antigens. — From Pick's (1908) review of the early literature it is evident that there were carried out exten- sive investigations of the effect of physical and chemical agents on the antigenic property of bacteria. He calls attention to tlie early use of heat-killed suspensions as well as to the use of phenol, formalin and other chemicals for the preservation of antigens or as germicidal agents. Species Specificity vs. Immunological Specificity. — Interest in the relationship between species specificity and immunological specificity developed early. In 1902 Castellani introduced the absorption technique. There soon developed among many bac- teriologists a belief that by the use of agglutination and absorption 350 BACTERIAL ANTIGENS AND SPECIFICITY 351 one could identify any bacterial .si)eeic.s. For a number of species such as E. iyphosa, Vibrio comma and several others, this is in a large measure true, although inagglutinable and atypically agglutinable strains of E. typliom have been described by many workers. Their inagglutinability is apparently due 1o a labile " Vi" antigen. (Felix and Pitt, 1934.) Limitation of Serological Methods. — While there is an in- cr-easing amount of evidence suggesting that each species possesses a s])ecies-specific antigenic fraction or component, yet this is not always demonstrable by agglutination or complement fixation using \ui altered bacterial suspensions. In fact, the result of agglutina- tion and absorption work would have one to believe that most species of bacteria are not represented by a species-specific antigen (in this case an agglutinogen) but on the contrary by two or more antigenic types. It is only by special methods of investiga- tion that one can frequently demonstrate species-specific antigenic substances. It may be that ultimately immunochemical studies will lead to a more accurate classification of bacteria. Stevens (1923) concludes that not all strains of a specific organ- ism can be recognized hy agglutination. Species Showing Antigenic Homogeneity. — The results of im- munological and biological investigations using agglutination and absorption and also complement fixation techniques indicate that bacterial antigens fall into three divisions. The first includes those species, each of w^iich consists of one dominant antigenic type. Practically all strains can be identified by means of an immune serum prepared against any single strain. Species Showing a Few Serological Types. — The second divi- sion is composed of species, each of which is represented by a small number of serological types and perhaps a heterogeneous one. With these it is possible to use a polyvalent immune serum for species identification and supplement this with other methods designed for the heterogeneous group of each. Species Showing Antigenic Heterogeneity. — The third divi- sion consists of those species, each of which shows so much antigenic heterogeneity that agglutination and absorption or even comple- ment fixation methods are not practical for species identification. 352 IMMITNOLOGY This will probably be best appreciated from a study of the fol- lowing list of species belonging to each of the divisions mentioned above, together with reference to specific immunological research relative to each species. Divisit)n T. Species wliieh can be identified serologically by means of a higli-titered monovalent, species-specific immune serum. E. typhosa (Downs, 1925) ; Mycohactermm tuber cidosis (Eice and Eeed, 1932, Wilson, 1925) ; Pfeifferella whitmorei (Stanton and Fletcher) ; Microsyira comma (Douglas, 1929) ; CoccohaciJ- lus foetidus osanea (Topley and Wilson 1:309); Pasteurella pestis (Topley and Wilson 1:486); H. pertussis (Topley and Wilson 1:505); Brucella abortus (Walton, 1930, and Topley and Wilson 1:515); CI. chauvei (Topley and Wilson 1:533); Treponema pallidum (Eice, 1932, also Topley and Wilson 1:570); Staphylococcus aureus (Julianelle, 1922, also Topley and Wilson 1:391). Division II. Species, each represented by a limited number of distinct antigenic types, some subtypes and perhaps a heterogeneous group. streptococcus hemolyticus (Topley and Wilson 1:373); Neis- seria gonorrhoea (Atkin, 1925, also Topley and Wilson 1:356); Neisseria meningitidis (Branham, 1932, also Topley and Wilson 1:341); B. dysenteria (Topley and Wilson 1:458); Friedlander bacillus (Julianelle, 1926, also Topley and Wilson 1:464, 465, 469); B. mallei (Topley and Wilson 1:307); CI. tetani (Topley and Wilson 1:557); B. paratyphosus B. (Jordan, 1923); B. enteritidis (Sherwood, Downs and McNaught, 1920). Division III. Species which show such extreme antigenic heterogeneity that immune serum is of no value in species identification. B. coli (Dulaney, 1928, Mackie, 1913, also Topley and Wilson 1:427); B. proteus (Topley and Wilson 1:408); Bhizobium leguminosarum (Topley and Wilson 1:315); C. diphtheriae (Topley and Wilson 1:287); Aerobic spore producers (Treece, 1920); CI. ivelchii (Topley and Wilson 1:540); Streptococcus viridans (Hooker and Anderson, 1929) ; Pseudomones pyocyan- eous (Sherwood, .Johnson and Radotinsky, 1926). Lack of Standard Procedure. — It should be remembered that these three divisions are based upon agglutination, absorption and complement fixation work by different individuals using various techniques and cellular antigens prepared in different ways. While type-specific polysaccharides have been found in a number of species, not many have been studied extensively to ascertain what haptens and antigenic fractions they contain and whether one or more are species specific. In some of the studies reported, methods of extraction have been used that have apparently racemized or denatured tlie bacterial antigens. One could deal more intelli- BACTERIAL ANTIGENS AND SPECIFICITY 353 gently with baetorial antigens if more were known about their chemistry. This sul),ieet lias been dealt with recently hy Baiim- gartel (1928), Branham (1928) and Zinsser, Enders and Fother- gill (1939). Some of the more recent literature has been reviewed by Heldelberger (1932, 1933) and Marraek (1938). Errors Due to Presence of Extraneous Material in Antigens. — In the i)reparation of bacterial antigens little attention has been paid to such factors although several have called attention to errors m immunological studies due to the fact that the bacterial antigens contained substances from the culture media that led to erroneous conclusions. This possibility of error in immunological research is indi- cated by the observation of Sordelli and Mayer (1931) reported by Heldelberger,* "that a 1:400,000 solution of agar (gelose) is enough to yield precipitates with antityphoid and antianthrax sera." Heidelberger suggests that this may account for cross- reactions reported by many w^orkers. Early Work on Antigenic Fractions. — Perhaps one of the earli- est studies on antigenic fractions of bacteria was that of Koch (1891, 1901) who found that extracts of the tubercle bacillus pro- duced specific reactions when injected into tuberculous animals. He regarded the extracts as containing an active principle of pro- tein nature which he called tuberculin. Subsequent Investigations. — This has led to extensive investi- gations by Long and Seibert (1926), Johnson and Coghill (1926, 1931, 1935), Anderson (1927, 1931, 1933), Zinsser and Rice and Reed (1932), Dienes (1929, 1930) and Dienes and Freund (1926), as well as many others, on the chemical and antigenic components of the acid-fast group, especially the various strains of the tubercle bacillus. Properties of Tuberculin. — Seibert (1926, 1933, 1934) suc- ceeded in crystallizing tuberculin and studying some of its prop- erties. The crystals, according to Long (1928), are water-soluble, take a methylene blue stain, give the biuret and Millon tests and are heat coagulable. The properties of a new Purified Protein Derivative of Tuberculin prepared by Seibert (1932, 1934) are discussed in Chapter XXVI. ♦Heidelberger: Ann. Rev. Biochem. 1: (j!j~>, 19.32. 354 IMMUNOLOGY In the past all attempts to employ tuberculin in antigen-anti- body reactions have failed. Seibert (1935) reports that the precipitin reaction between tubercle protein and its homologous antiserum is inhibited by her tuberculin preparation SOTT having a molecular weight of 3800. By means of tlie ultraeentrifuge and electrophoresis Seibert, Pedersen and Tiselius* (1938) have studied the constituents of tuberculin. They have isolated a protein with a molecular weight of 32,000 from culture filtrates. This protein, when injected into tuberculous guinea pigs, caused death. They isolated substances of lower molecular Aveight (9,000-16,000), from old tuberculin, that were active in producing skin reactions. Ac- cording to Chase and Landsteiner (1939), Maschmann (1937) has reported success in separating tuberculin into two fractions, one (resistant to proteolytic enzymes) that gives systemic and the other (destroyed by trypsin) that gives positive skin reactions. Specific Substances in Bacteria. — Early Discoveries.^ — In 1921 Zinsser obtained a specific substance from tubercle bacilli. It was obtained by alkaline extraction of ground tubercle bacilli, and was heat stable, alcohol-insoluble, and gave positive precipitin and complement fixation tests with specific immune serum. A gumlike substance that reacted with serum of animals im- munized with tubercle bacilli was isolated by Laidlaw and Dudley (1925). Tubercle bacillus polysaccharides were isolated by Heidel- berger and Menzel (1932) and Seibert, Pedersen and Tiselius (1938). Heidelberger and Menzel (1938) have continued tlieir studies of tubercle bacillus polysaccharides and have been able to isolate two whicli are immunologically active; one is low and the other is high in pentose. They "are apparently built up chiefly of d-arabinose and d-mannose units in varying proportions." Pick (1912) had isolated similar soluble specific substances from E. tjjphosa and Dochez and Avery (1917) had found in the urine and blood of pneumococcus pneumonia patients a substance that gave specific reactions with pneumococcus immune serum. In 1923, Zinsser and Parker found similar substances in pneumo- cocci, staphylococci, influenza and typhoid bacilli. Heidelberger and Avery have shown the soluble specific substances of pneumo- cocci to be polysaccharides and type-specific. These and other im- portant reacting carbohydrates and proteins isolated from a num- ber of other bacteria will be discussed briefly later in this chapter. •Seibert, F. B., Pedersen, K. 0., and Tiselius, A.: J. Exper. Med. 68: 413, 1938. BACTERIAL ANTIGENS AND SPECIFICITY 355 Antigenic Comparisons of Acid-Fast Bacteria. — Lewis and Seibert* (1931, 1933) have made extensive antigenic comparisons of different strains of the tubercle bacillus and the timothy bacillus. They found definite antigenic relationships and also differences between the human, bovine and avian strains but only slight relationship between any of these and the timothy bacillus. It has been shown that tubercle bacilli contain lipoids, pro- teins and carbohydrates. The question as to the immunological importance of each of these has led to much controversy. All three types of substances have been reported as giving serological reactions. They conclude that, "The sensitized animal body is able to make a quantitative distinction between the proteins from dif- ferent types of acid-fast bacilli, but is apparently unable to dif- ferentiate the proteins from different strains of the same type, such as between virulent and avirulent organisms." Lipoids. — In regard to the lipoids. Wells (1929) calls attention to the possibility that perhaps many of the lipoid preparations that have been studied contain a trace of protein. He considers that alcohol-soluble proteins may be present in bacterial extracts and that their presence might account for the results of Klop- stock and Witebsky (1927) who found that alcoholic extracts of bacteria were antigenic and Pinner (1927, 1928) who noted an increase in the antigenic property of alcoholic extracts of tubercle bacilli as the protein content was diminished by purification. Relative Importance of Lipoids, Proteins and Carbohy- drates.— There seems to be fairly good evidence indicating that the lipoid fraction of the tubercle bacilli is similar for all strains and that it does not determine serological specificity. On the other hand, the protein fraction, containing carbohydrate configurations, is quite specific. Long (1930) considers that the polysaccharides of the tubercle bacillus are immunologically specific as shown by Laidlaw and Dudley (1926), Mueller (1926) and Zinsser and Tamiya (1925), His conclusions" are apparently confirmed by the later work of Seibert, Pedersen and Tiselius (1938) and Heidel- berger and Menzel (1938). ♦Lewis, J. H., and Seibert, F. B. : J. Immunol. 20: 201, 1931. 356 IMMUNOLOGY Anderson (1932) and also Smedley-MacLean (1932) have re- viewed the work of Anderson (1930, 1931), Sabin and Doan (1927) and of Doan (1929) on the fatty acids and phosphatids isohited by Anderson from the members of the acid-fast group. Anderson (1940) has extended his work on lipids of the tubercle bacillus. He reports that two polysaccharide fractions isolated from two preparations of phosphatids gave different cleavage products. Tuberculo-Phosphatids and Fatty Acids.- — The tuberculo- phosphatids differ from phosphatids obtained from other sources in their phosphorus and nitrogen content. According to Anderson they also contain a large percentage of what appears to be a new type of polysaccharide. On hydrolysis the latter yields mainly mannose and inosite. Pangborn and Anderson (1933) and Anderson and Newman (1933) have also reported on the isola- tion of trehalose from the timothy and tubercle bacilli, respec- tively. Immuno-Chemical Studies of the Pneumococcus. — Another spe- cies of bacteria that has been both extensively and intensively stud- ied from the standpoint of immunochemistry is the pneumococcus. Attention has already been called to the early discovery of Dochez and Avery (1917), Zinsser (1921), Zinsser and Parker (1923), of soluble specific substances from pneumococci that are type specific in that they give specific precipitates or positive complement fixa- tion when mixed with their respective immune sera. These sub- stances have been carefully investigated by Heidelberger, Goebel, and Avery* since 1923. Their results may be briefly summarized as follows : Properties of Type Specific Polysaccharides. — In the begin- ning they found that all three types of specific soluble substances are polysaccharides quite dissimilar chemically as well as immuno- logically. Unfortunately they employed methods at first which deacetylated the natural soluble specific substance. In view of the observations of Schiemann and Casper (1927), Saito and Ulrich (1928), Enders (1930) and Wadsworth and Brown (1931) that pneumococci contain an antigenic polysaccharide, Avery and *Heidelberger, Goebel, and Avery: J. Exper. Med. 38: 73, 1923. BACTERIAL ANTIGENS AND SPECIFICITY 357 Goebel (1933) reinvestigated the type specific polysaccliaride of Type I and came to the following conclusions: 1. The soluble specific substance of Type I is an acetyl poly- saccharide. 2. The methods they employed in 1923 in isolating Type I SSS resulted in deacetylating the natural acetyl polysaccharide. 3. The acetyl polysaccharide is antigenic in that the injection of it into mice leads to the development of an immunity to Type I pneumococcus but not to the formation of precipitins. Zinsser and Bayne- Jones (1934) speak of this as a functional immunity, 4. The acetyl polysaccharide is quite soluble in water. 5. It shows a specific optical rotation in aqueous solution of +270. 6. It has a nitrogen content of 4.8 i^er cent. When treated witli nitrous acid in the cold, about 45 per cent of the nitrogen is li1)- erated in the amino form. 7. It yields reducing substances when h^^lrolized willi dilute mineral acids. When the reducing substance appears, the sero- logical specificity is lost. 8. The type-specific polysaccharide is also soluble in 80 per cent acetic acid. 9. Dilute aqueous solutions (0.5 per cent) are precipitated by silver nitrate, neutral and basic lead acetate and phosphotungstic acid. It is incompletely precipitated by barium hydroxide. It is precipitated by tannic acid but not by uranyl nitrate. 10. The ninhydrin, biuret, picric acid and sulphosalicylic acid tests are negative. 11. Neither phosphorus nor sulphur was detectable in highly purified preparations of the acetyl polysaccharide. Marrack (1938) describes Type I polysaccharide as containing a basic group and a trisaccharide with two uronic acid molecules and amino sugar. Brown (1939) has summarized in tabular form the chemical data on the soluble specific substances of Types I to XXXII pneu- mococci. Apparently they all contain dextrose although only Types V, XI, and XXXII will reduce Fehling's solution before hydrolysis. Types II, III and VIII are thought to be composed of dextrose and aldobionic acid in a carbohydrate chain. Accord- ing to Brown's data uronic acid was present in Types I, II, III, 358 IMMUNOLOGY VIII, IX, XII, XXII, XXV, and XXVI. She reports amino sugar (after hydrolysis) in Types I, IV, V, VII, IX, X, XII, XIII, XIV, XV, XVI, XIX, XX, XXI, XXIV, XXV, XXVII, XXIX, XXX, and XXXI. Types I, IV, V, XII and XXV contain the largest amount of nitrogen (about 5%). Type I alone has an ap- preciable amount of amino nitrogen (2%). Edwards, Hoagland and Thompson say that in the case of the specific polysaccharides of Types II, III and VIII the difference appears to be principally one of stereochemistry. These polysaccharides are haptens (Type I is definitely antigenic) coupled to protein common to all pneumo- cocci. The combination forms a complete pneumococcus antigen that is type specific. The type specificit.y is due to the polysac- charide hapten. Finland and Curnen (1938) report an interesting discovery concerning the specific carbohydrate of pneumococcus XIV. They observed tliat liorse immune sorum for this type of pneumococcus contained agglutinins for human red cells. According to Hoag- land, Beeson and Goebel (1938) this substance resembles to a great extent the group " A " substance from pig stomach. Difference Between Immune Serum from Horses and Rab- bits.— Heidelberger and Kendall (1933) have also found that partial hydrolytic products of Type III specific polysaccharide can be quantitatively freed from unhydrolyzed polysaccharide. They find that the fractions yield specific precipitates with Type III antipneumococcus serum obtained from horses but fail to give a precipitate with similar immune serum obtained from rabbits. In their opinion Felton's studies on antibodies for the pneumococ- cus may offer an explanation of this. Felton found that pneu- mococcus antibodies obtained from horse serum are precipitated with the water-insoluble fraction of the serum glol)ulin* whereas rabbit antipneumococcus sera yield no precipitate on dilution. Possible Explanation of Inhibition Phenomenon. — They say that ''The failure of the hapten fraction to form insoluble com- pounds with rabbit antibody may be connected with the greater tendency of rabbit globulin to remain in solution," They also suggest that this may explain the inhibition phenomenon of Tjand- steiner and van der Scheer. *J. Immunol. 21: 341, 1931. BACTERIAL ANTIGENS AND SPECIFICITY 359 Landsteinei' (1936, p. 119) suggests that tlie iiiliibition phe- nomenon may be due to an excess of antigen resulting in the forma- tion of soluble compounds containing a larger proportion of antigen, in comparison to antibody, than there is in precipitates. The "C" Species-specific But Not Type-specific Substance OF TiLLETT AND Fkancis (1930). — This substant'c has been studied by Tillett, Gocbel and Avery (1930). They find that it is a non- protein fraction distinct from the type-specific polysaccharide. The evidence seems to indicate that it is a ''nitrogenous i>oly- saccharide analogous in chemical behavior ])ut not in sei'ological reactivity to the Ty])e I soluble-specific substance." Since it passes through collodion membranes with ease and quite read- ily through parchment membranes, Tillett, Giroebel and Avery con- clude that the molecule is smaller than that of the SSS previously described. The "C" fraction is apparently common to all pneu- mococci. It is evidently not related to virulence since it is present in the avirulent "R" forms as well as the virulent "S" forms. Heidelberger and Kendall have found the "C" substance in Type IV pneumococci and have studied its chemical properties. Its rotation is given as +40° and its nitrogen content as 6.1 per cent. They also found approximately 0.9 per cent of amino nitrogen. It differs from the pneumococcus type-specific substance in con- taining phosphorus (4.0 per cent) (Heidelberger, 1932). The somatic substance is said to include skin irritating nucleic acids and nucleoproteins. Carbohydrate Fractions Adsorbed on Carbon Particles. — While all previous work has shown that the soluble-specific sub- stances (SSS) of Types II and III arc nonantigenic, yet Zozaya and Clark have recently (1933) reported active immunization of mice with the polysaccharides (SSS) of pneumococcus Types I, II, and III. They used polysaccharides adsorbed on carbon particles and also various dilutions of unadsorbed material. They claim that the dilution factor is important. Their work needs further con- firmation. It should also be noted that Wadsworth and Brown call attention to tlie importance of dosage in the demonstration of the antigenic nature of the carbohydrate fraction described by them. Synthetic Carbohydrate Haptens. — In Hie course of their in- vestigations of the role polysaccharides ])lay in determining im- munological specificity, Goebel and Avery (1929) synthesized two 360 IMMUNOLOGY optically isomeric polysaccharides, e.g., p-aminophenol-glucoside and the corresponding galaetoside. They diazotized these, using nitrous acid and hydrochloric acid in the cold as discussed in Chapter XVIII. These diazonium derivatives were readily coupled onto the proteins (horse serum globulin) in the presence of N/100 hydroxide. They then- studied and compared their antigenic properties. The structural formulae for the two isomeric glu- cosides used as haptens in these experiments are given by Avery and Goebel (1929) as follows: \. — 0- -C- -H / |\ H- -c- -OH \ HO- -c- -H\ \ H- -c- 1 I \ -OH / \/ H- -c NH, C >— O— C— H NH,<^ \— O— C— H ■ ^ -^ i\ H— C— OH i \ HO— C— H \ I HO— C— H \ \ / I / I / \/ H— G I I H,i=C— OH H„=C— OH p-aminophenol /3 glucoside p-aminophenol /3 galaetoside The specificity of the conjugated hapten proteins was checked further hj linking the same diazotized glucosides not only to horse serum globulins but also to egg albumen, Avery and Goebel* summarize their results as follows : 1. When two chemically different carl)ohydrate derivatives are bound to the same protein, the newly formed antigens exhibit dis- tinct immunological specificity. 2. When the same carbohydrate radical is conjugated with two chemically different and serologically distinct proteins both of the sugar-proteins thus formed acquire a common serological speci- ficity. 3. The newly acquired specificity of the artificially prepared sugar-proteins is determined by the chemical constitution of the carbohydrate radical attached to the protein molecules. Simple differences in the molecular configuration of the two isomers, glu- *Avery and Goebel: J. Exper. Med. 50: 533, 1929. BACTERIAL ANTIGENS AND SPECIFICITY 361 cose and galactose, suffice to orientate protein specificity when the corresponding giucosides of the two sugars are coupled to the same protein. 4. The unconjugated giucosides, although themselves not pre- cipitable in immune serum, inliibit the reaction between the homologous sugar-]U'()tein and ils specifie antil)ody. The inhil)i1ion test is specific. 5. The sugar derivatives unattached to protein exhibit the prop- erties of carl)ohydrate haptens ; they are nonantigenie but spe- cifically reactive, as shown l)y inliibition tests. Following this work, Avery and Goebel (1931) coupled the nitro- gen-free polysaccharide of Type III pneumococci onto horse serum globulin after preparing p-amino and p-nitromonol)enzyl ethers of the specifie polysaccharide. They produced active immunity and type-specific antibodies in rabbits for Type III pneumococ- cus by injecting the polj^saceharide horse serum globulin anti- genic complex. They were also able to protect mice specifically by immunization with the same. Hotchkiss and Goebel (1937) and Goebel (1938, 1939) have extended the work on synthetic conjugated antigens. The former synthesized azoproteins containing glucuronic acid and galac- turonic acid respectively. The galacturonic acid azoprotein gave positive precipitin reactions with Type I anti-pneumococcus serum. In 1939 Goebel produced an antigen containing cello- biuronic acid that produced active immunity in rabbits to virulent T3'pe III pneumococci. Serum from these immunized ra])bits conferred passive immunity to mice for Types II, III, and VIII pneumococci. The serum also gave positive Neufeld quellung re- actions. Friedlander's Bacillus. — In 1925 Heidelberger, Goebel and Avery became interested in the capsular substance of a strain of the Friedlander's bacillus. Earlier (1921) Toenniessen had iso- lated from the capsular substance of a strain of this organism, a snow-white nonreducing substance that was a polysaccharide sub- stantially free of nitrogen. Upon hydrolysis it yielded reducing sugars. Toenniessen found that these yielded an osazone which he regarded as that of galactose. His work was confirmed by Kramar (1922). The substance was not studied immunologically 362 IMMUNOLOGY until 1924-25 Avhen Mueller, Smith and Litarczek reported that the polysaccharide thus obtained caused specific precipitation of homologous immune serum. Heidelberger, Goebel and Avery (1925) found that the poly- saccharide from strain E (Type B of Julianelle) is chemically and imniunoloo'ically similar to but not identical with the soluble specific substance of Type II pneumococcus. They found tliat im- mune serum for strain E of Friedlander 's bacillus agglutinates both the homologous organism and also Type II pneumococcus and conversely immune serum for the latter agglutinates both organisms. Similar results were obtained using the respective polysaccharides and corresponding immune sera. When ab- sorption experiments were carried out, however, it was found that when Friedlander (strain E) immune serum was absorbed with Type II pneumococcus, the agglutinins for the pneumococcus were removed, but there remained agglutinins for the homologous organisms; and likewise when pneumococcus Type II serum was absorbed with Friedlander 's bacillus (strain E) the aggkitinins for the latter were removed, but there remained agglutinins for Type II pneumococcus. When either immune serum was absorbed with its homologous organism, agglutinins for both were removed. Similar results were obtained using the respective polysaccharides and immune sera. It is interesting to note that Julianelle (1926) found im- munological types to exist among forty strains of Friedlander 's bacillus studied. These strains fall into three specific types which he designates as A, B, and C and a heterogeneous one which he has named Type X. The strain whose capsular substance is similar to that of pneumococcus Type II corresponds to Type B of Julianelle. The soluble specific substances of Types A and C have been studied extensively by Goebel (1927) and Goebel and Avery (1927). The former investigated the hydrolytic products of Type A polysaccharide and found that it yielded on hydrolysis an aldobionic acid, glucose, and a second acid not identified. These three compounds were found to be present in the ratio of 1 :1 :1. Goebel found that the new aldobionic acid consists of a molecule of glucuronic acid linked to a molecule of glucose through its re- ducing group. It is iso7neric with an acid derived from the soluble BACTERUL ANTIGENS AND SPECIFICITY 363 specific substance of Type III pneiimococcus. The polysaccharide appears to be a condensate of 2 molecules of aldobionic acid and 1 molecule of glucose. The Type A substance yields specific immunological reactions with the homologous type immune serum, but does not give cross- reactions with any one of the three type specific pneumococcus immune sera. The failure to react with pneumococcus Type III serum, considering its chemical similarity to Type III polysac- charide, is apparently due to isomeric configuration. Goebel and Avery (1927) found that Friedlander 's Type C soluble specific substance is also a nitrogen-free polysaccharide and that it is chemically similar to tlie one obtained from Type B. Two distinct differences were noted; they show no cross-relation- ship immunologically; they differ in their solubility in water and in their behavior during purification. Pure Type B ia difficultly soluble, while the Type C substance is readily soluble in water. The former is readily precipitated by alcohol in tlie presence of hydrochloric acid, whereas the latter precipitates completely only after standing for one hour at 0° C. Avery and Goebel* (1927) think that the immunological dissimilarity observed in these two substances that are physically and chemically so much alike is probably due to "slight differences in the intramolecular link- age of sugar to sugar, or of sugar to sugar acid. ' ' Escherichia coli. — Stuart, Baker, Zimmerman, Brown and Stonet (1940) report the literature along with their own experi- mental studies of the relationship of colifonn bacteria. They say that Magheru (1937) found the entire "0" antigen of Escherichia coli is composed of a sugar-lipoid complex and that the complete antigen was not contained in all of the variants. Precipitin studies indicate many serological varieties for Escherichia coli. Torrey (1938) has found a virulence factor associated Avith the SSS ele- ment but not identical with it. E. typhosa. — According to Topley et al. (1937) two carbohy- drate-lipid complexes corresponding to the "0" and ''Vi" anti- gens of E. typhosa have been isolated. These two substances differ in their carbohydrate content and in a few other ways. ♦A.verv and Goebel: J. Exper. Med. 46: 601. 1927. tStuart. Baker, Zimmerman, Brown, and Stone: J. Bact. 40: 101. 1940. 364 IMMUNOLOGY Somewhat analogous findings have been reported, according to Chase and Landsteiner, for *S^. paratyphi G and certain Pas- tcurcllas. Shig-ella dysenteriae. — The antigenic complex of Shiga has been studied by Morgan (1936, 1937, 1938). He reports that it stimulates the production of both antibacterial and heterophile antibodies and is thouglit to be the endotoxin of Shiga. Upon fractionation he obtained in addition to the specific polysac- charide, lipoidal and other material. The polysaccharide con- tains 1.6 per cent nitrogen and yields 98 per cent reducing sugar on hydrolysis. The antigenic substance then appears to be a carbo- hydrate-lipid complex. V. cholerae. — Studies of cliolera vibrios has resulted in wliat appears to ])c the establishment of six groups according to Mitra. Two types were distinguished by differences in globulin fractions. Landsteiner and Chase (1939) say that when these results are considered together with three sorts of specific carbohydrates that have been isolated it ''permits of the establishment of six groups of vibrios." White (1937) studied the 0 receptor complex of strains of V. cholera found in India. He found the 0 receptors to be antigenic and located in the specific polysaccharide. He does not assume that the antigenic factors causing multivalence represent so many different substances. Instead he thinks of the multivalence as due to the presence of individual receptor substances in the polysaccharide molecules and to combinations of these functioning as complex receptors. He postulates the existence of three primary receptor groups in the cholera polysaccharide molecule. H. influenzae. — It was formerly thouglit that //. influenzae organisms constituted a heterogenous group. Zinsser and Bayne- Jones (1939) are of the opinion that the smooth H. influenzae represent a homogenous group. It is only after dissociation into "R" forms that they become serologically heterogeneous. Pitt- man (1931) found that the ''S" forms produced a soluble specific substance. Dingle and Fothergill have isolated this substance and identified it as a carbohydrate. Serological tests with this sub- stance gave positive precipitation in high titer with antisera ob- tained from both horses and rabbits. While numerous cross re- BACTERIAL ANTIGENS AND SPECIFICITY 365 actions were encountered with the immune scrum from horses, the rabbit immune serum was specific. H. pertussis. — While a number of types of //. pertussis have been reported in the literature it appears that the types reported represented serological differences between mutation forms de- scribed by Shibley and Hoelscher (1934). Leslie and Gardner (1931) described four dissociation phases of H. pertussis. They termed them Phases I, II, III and IV. It appears that the Phase I is the hemolytic smooth form that is found in whooping cough. It will be recalled that Sauer's vaccine is made from hemolytic, capsulated, virulent Phase I of //. pertussis. Brucella abortus. — There has been some controversy over the nature of the specific substance in Brucella abortus. Hershey, Huddleson and Pennell (1935) reported the specific reactions described by others as due to a noncarbohydrate substance. In later work they have found small amounts of carbohydrate in the antigenic material. Libby and Joyner (1941) report isolating an antigenic carbohydrate from all three strains of Brucella. Cutaneous tests with this carbohydrate in infected or sensitized subjects results in an immediate allergic reaction. There is no delayed reaction as in the tuberculin test. Hemolytic Streptococci. — By means of immune sera obtained from rabbits, Griffith has been able to divide hemolytic strepto- cocci from scarlet fever cases into four types and a heterologous group. Satisfactory typing of streptococci has not been as ade- quately solved by agglutination as it has by precipitation meth- ods using extracts of streptococci. Laneefield by means of the precipitin reaction has confirmed and extended Griffith's work on the division of hemolytic streptococci into groups and types. She has described groups ''A" to "G, " the group division de- pending on the occurrence of a specific carbohydrate (C) for each group. Tjq^e specificity in the case of group ''A" depends on the presence of an antigenic protein *'M. " In the case of the other groups, type specificity rests on the presence of polysac- charides designated as (S). These type specific substances are present in the mucoid and Matt type colonies of group "A" and in the smooth colonies of the other groups but are lacking in the glossy "A" colonies and in the rough colonies of other groups. 366 IMMUNOLOGY There is some correlation of type specific substance with viru- lence, but a strain of "A" group may lose its virulence and still retain the Matt type of colony. This work has assumed great importance because of the con- sistency with which group ''A" streptococci are associated with human infection. Recently another type specific antigen designated as "T" has been found in group A streptococci in both Matt and glossy strains. It has not been defined chemically but it is responsible for type specific agglutination. Anti "T" serum exerts no pro- tective effect in mice. The following antigens have been de- scribed by Lancefield : Group specific carbohydrates (C) Type specific protein for group ''A" (M) Type specific substances (T) Type specific carbohydrates for groups (S) other than "A" To obtain extracts for use in the precipitin work Lancefield extracts the streptococci with hot (boiling) N/20 IICl to de- stroy a Group factor "P" which overlaps with other organisms like pneumococei. She separates the type specific "M" sub- stance from the Group specific (C) carbohydrate by neutralizing the acid extract and precipitating out the "M" substance with alcohol. The Group specific polysaccharide "C" remains in the supernatant fluid. For detailed description of the technique involved the student is referred to Lancefield 's original paper. They might also be interested in a modification of Lancefield 's method reported by Brown* (1938) and in a new method sug- gested by Fuller (1938). Other Bacterial Specific Substances. — The specific substance of meningococcus Type I is reported by Scherp and Rake (1935) as sodium salt of a polysaccharide acid containing firmly bound phosphoric acid. Julianelle and Wieghard (1935) have described two type specific polysaccharides for staphylococci. Sievers and Zetterberg report that spontaneous agglutination interfered with their experimental study of aerobic spore forming bacteria. They obtained results with autolysates which suggests the existence of a *Brown. J. H. : J. A. M. A. Ill: 310, 193S. BACTERIAL ANTIGENS AND SPECIFICITY 367 specific antigenic structure in the different types. Ivanovies and Bruckner (1937) (1938) and Tomcsik and Ivanovies (1938) have described a new type of hapten which they have isolated from the capsular substance of two aerobic spore producers, B. anihracis and B. mesentcrkus. It is a polypeptide of liigli molecular weight containing only one amino acid. References Anderson, E. J.: The .Separation of Lipoid Fractions From Tubercle Bacilli, J, Biol. Chem. 74: 525, 1927. The Chemistry of the Lipoids of Tubercle Bacilli. XIV. The Occurrence of Linosite in the Phosphatide From Human Tubercle Bacilli, J. Am. Chem. Soc. 52: 1607, 1930. Anderson, E. J., and Eoberts, E. F. : The Chemistry of the Lipoids of Tubercle Bacilli. X. The Separation of Lipoid Fractions From Avian Tubercle, J. Biol. Chem. 85: 509, 1930. XL The Phosphatide Frac- tion of the Avian Tubercle Bacilli, Ibid. p. 519. XII. The Separation of the Lipoid Fractions From Bovine Tubercle Bacilli, Ibid. p. 529. XIX. Concerning the Composition of the Phosphatide Fraction Iso- lated From the Bovine Type of Tubercle Bacilli, Ibid. 89: 599. XX. The Occurrence of Mannose and Inosite in the Phosphatide Fractions From Human, Avian, and Bovine Tubercle Bacilli, Ibid. 89: 611. Anderson, E. J., and Eenf rew, A. B. : The Chemistry of the Lipoids of Tubercle Bacilli. XIII. The Occurrence of Mannose in the Phosphatide From Human Tubercle Bacilli, J. Am. Chem. Soc. 52: 1252, 1930. Anderson, E. J.: Chemistry of Lipids of Tubercle Bacilli; Studies on Phthioic Acid. Isolation of Levorotatory Acid From Phthioic Acid Fraction of Human Tubercle Bacillus, J. Biol. Chem. 97: 639, 1932. Anderson, E. J., and Newman, M. S. : The Chemistry of the Lipoids of Tubercle Bacilli. XXXIII. Isolation of Trehalo.'^e From the Acetone- Soluble Fat of the Human Tubercle Bacillus, ,L Biol. Chem. 101: 499, 1933. Anderson, E. J., Creighton, M. M.. and Peck, E. L.: The Chemistry of the Lipids of Tubercle Bacilli. LX. Concerning the Firmly Bound Lipids of the Avian Tubercle Bacillus, J. Biol. Chem. 133: 675, 1940. LXI. The Polysaccharide of the Phosphatide Obtained From Cell Ee.sidues in the Preparation of Tuberculin Ibid. 136: 211, 1940. Atkin, E. E.: Some Cultural Characteristics Exhibited by Serological Types of Meningococci, Brit. .1. E.xper. Path. 4: 325, 1923. Significance of Serological Types of Gonococci, Ibid. 6: 235, 1925. Avery, O. T., and Goebel, W. F. : Chemo-immunological Studies on Conjugated Carbohydrate-Proteins. Immunological Specificity of Synthetic Sugar Protein Antigens, J. Exper. Med. 50: 533, 1929. V. The Immunological Specificity of Antigen Prepared by Combining Capsular Polysaccharide of Type III Pneuraococeus With Foreign Protein, J. Exper. Med. 54: 437, 1931. Avery, O. T., and Goebel, W. F. : ChemoimnuuKilogical Studies on the Soluble Specific Substance of Pneumococcus I. The Isolation and Properties of the Acetyl Polysaccharide of Pneumococcus Tvpe I, J. Exper. Med. 58: 731, 1933. Avery, O. T., and Heidelberger, M. : Immunological Eelationships of Cell Constituents of Pneumococcus, J. Exper. Med. 38: 81, 1923. 368 IMMUNOLOGY Averj, O. T., Heidelberger, M., and Goebel, W. R: The Soluble Specific Substance of Friedlander 's Bacillus, J. Exper. Med. 42: 709, 1925. Branliam, S. E.: Serological Diversity Among Meningococci, J. Immunol. 23: 49, 1932. Branliam, S. E.: Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press. Brown, Rachel: Chemical and Immunological Studies of the Pneumocoecus. V. Tlie Soluble Specific Substances of Types I-XXXII, J. Immunol. 37: 445, 1939. ('astellani, A.: Die Agglutination bei gemischter Infektion und die Diagnose der Letzteren, Ztschr. f. Hyg. u. Infektionskr. 40: 1, 1902. Chase, M. W., and Landstein«r, K. : Immunochemistry, Ann. Rev. Biochem. 1939, 579-610. Coghill, R. D. : A Chemical Study of Bacteria. XII. The Albumin Globulin Fraction of the Tubercle Bacillus, J. Biol. Chem. 70: 439, 1927. XXIV. Approximate Chemical Analysis of the Timothy Bacillus, Ibid. 81: 115, 1929. The Nucleic Acid of the Timothy Bacillus, Ibid. 90: 57, 1931. Coghill, R. D., and Bird, O. D.: Chemical Study of Bacteria; Proximate Chemical Analysis of Timothy Bacillus, J. Biol. Chem. 81: 115, 1929. Coghill, R. D. : 1935. Personal communication. Dienes, L. : The Antigenic .Substances of the Tubercle Bacillus. II. The Examination of the Antigenic Properties, J. Immunol. 17: 85, 1929. III. The Chemical Examination of the Antigenic Substances of the Tubercle Bacillus Extracted With Lipoid Solvents, Ibid., p. 157. IV. Connection Between the Tuberculin Activity and Antigenic Properties, Ibid., p. 173. The Specificity of the Tuberculin Type of Sensitive- ness Produced With the Different Protein Substances of the Egg White, Ibid. 18: 279, 1930. Dienes, L., and Freund, J.: On the Antigenic Substances of the Tubercle Bacillus, J. Immunol. 12: 137, 1926. Dienes, L., and Schoenheit, E. W.: The Antigenic Substances of the Tubercle Bacillus. V. The Antigenic Substances of the Synthetic Culture Medium, J. Immunol. 18: 285, 19.30. Dingle, J. H., and Fothergill, L. D.: The Isolation and Properties of the Specific Polysaccharide of Type B Hemophilus Influenzae, J. Im- munol. 37: 53, 1939. Doan, C. A.: Diagnostic Significance of Precipitin Tests With Anderson Phosphatide Fractions From Human, Bovine, and Avian Tubercle Bacilli, Proc. Soc. Exper. Biol. & Med. 26: 672, 1929. Dochez, A. R., and Avery, O. T.: Tlie Elaboration of Specific Soluble Substance by Pneumocoecus During Growth, .J. Exper. Med. 26: 477, 1917. Douglas, S. R.: The Question of Serological Races of V. cholerae and the Relation of Some Other Vibrios to This Species, Brit. J. Exper. Path. 2: 49, 1921. Downs, C. M.: Antigenic and Metabolic Studies of Bacillus Typhosus, University of Kansas Science Bull. 6: 16, 1925. Dubos, R. J., and Avery, O. T. : Decomposition of the Capsular Polysaccharide of Pneumocoecus Type III by a Bacterial Enzyme, .1. Exper. Med. 54: 51, 1931. Dubos, R. J.: The Effect of Specific Agents Extracted From Soil Micro- organisms Upon Experimental Bacterial Infections, Ann. Int. Med. 13: 2025, 1940. Dulaney, A. D.: Microbic Dissociation of B. coli-communis, J. Infect. Dis. 42: 575, 1928. BACTKRIAL AXTIGKNS AND SPECIFICITY 369 Edwards, J. E., Hoagland, C. L., and Thompson, L. D.: Type Specific Polysaccharide Skin Test in Serum Therapy of Pneumonia, J. A. M. A. 113: 1876, 1939. Enders, J. F.: A Type Specific Substance Distinct J'rom the Specific Carbohydrate in Pneuniococi-us, J. Exper. IMed. 52: 235, ID.'iO. Finland, M., and Curnen, E. C. : Agglutinins for Human Erythrocytes in Type XIV Anti-Pneumococci Horse Serums, Science 87: 417, 1938. Francis, T., Jr.: Polysaccharide of Pncumococcus, Tvpe I in Man, Proc. Soc. Exper. Biol. & Med. 31: 493, 19.34. Fuller, A. T.: The Formamide Method for the Extraction of Polysac- charides From Hemolytic Streptococci, Brit. .T. Exper. Path. 19: 130, 1938. Goebel, W. F.: The Soluble Specific Substance of Friedlander 's Bacillus. IV. On the Nature of Hydrolytic Products of the Specific Carbo- hydrate From Type A Friedlander Bacillus, J. Biol. Chem. 74: 019, 1927. Goebel, W. F., and Avery, 0. T.: Chemo-immunological Studies on Con- jugated Carbohydrate-Proteins. I. The Synthesis of p-aminophenol b-glucoside, p-aminophenol, b-galactoside, and Their Coupling With Serum Globulin, J. Exper. Med. 50: .521, 1929. III. On the Isolation and Properties of the Specific Carbohydrates From Types A and C Friedlander 's Bacillus, J. Exper. Med. 46: 601, 1927. IV. The Synthesis of the p-aminobenzyl Ether of the Soluble Specific Sub- stance of Type III Pneumococcus and Its Coupling With Protein, J. Exper. Med. 54: 431, 1931. Heidelberger, M., Goebel, W. F., and Avery, O. T.: The Soluble Specific Substance of a Strain of Friedlander 's Bacillus, J. Exper. Med. 42: 701, 1925. The Soluble Specific Substance of Pneumococcus (third paper), Ibid., p. 727. Heidelberger, M., and Goebel, W. F.: The Soluble Specific Substance of Pneumococcus. IV. On the Nature of the Specific Carbohydrate of Type III Pneumococcus, J. Biol. Chem. 70: 613, 1926. The Soluble Specific Substance of Pneumococcus. V. On the Chemical Nature of the Aldobionic Acid From the Specific Polysaccharide of Type III Pneumococcus, Ibid. 74: 613, 1927. Heidelberger, M., and Avery, 0. T.: The Soluble Specific Substance (of Pneumococcus (first paper), ,J. Exper. Med. 38: 73, 1923. The Soluble Specific Substance of Pneumococcus (second paper), Ibid. 40: 301, 1924. Heidelberger, M.: Immunochemistry, Ann. Eev. Biochem. 1: 655, 1932. Heidelberger, M., and Kendall, F. E.: Some Physico-Chemical Properties of Specific Polysaccharides, J. Biol. Chem. 95: 127, 1932. The Molecular Weight of Specific Polysaccharides, Ibid. 96: 541, 1932. Heidelberger, M., and Kendall, F. E.: The Precipitin Reaction Between Type III Pneumococcus Polysaccharide and Homologous Antibody. II. Conditions for Quantitative Precipitation of Antibody in Horse Sera, J. Exper. Med. 61: 539, 1935. Heidelberger, M., and Kendall, F. E.: The Precipitin Eeaction Between Type III Pneumococcus Polysaccharide and Homologous Antibody. III. A Quantitative Study and a Theory of the Eeaction Mechanism, J. Exper. Med. 61: 563, 1935. Heidelberger, M., and Kendall, F. E.: Carbohydrate-Containing Proteins of the Hemolytic Streptococcus, J. Immunol. 30: 267, 1936. Heidelberger. M., and Seherp, H. W.: Protein Fractions of a Strain of Group' "A" Hemolytic Streptococci III, J. Immunol. 37: 563, 1939. Henderson, D. W., and Morgan, W. T. .1.: The Isolation of Antigenic Substances From Strains of Bact. Tvphosum, Brit. J. Exper. Path. 19: 82, 1938. 370 IMMUNOLOGY Henle, W., and Henle, G.: The Antigenic Structure of Hemolytic Strepto- cocci of Lancefield Group "A," J. Immunol. 37: 149, 1939. Hershey, A. D., Huddleson, I. F., and Pennell, R. B.: The Chemical Separa- tion and Biological Activity of the Polysaccharide Constituent in Brucella Cells, J. Infect. Dis. 57: 183, 1935. Hoagland, C. L., Beeson, P. B., and Goebel, W. F. : The Capsular Poly- saccharide of the Type XIV Pneumococcus and Its Relationship to the Specific Substances of Human Blood, Science 88: 261, 1938. Hooker, S. B., and Anderson, L. I\r.: Heterogeneity of Streptococci Isolated From Sputum, J. Immunol. 16: 291. 1929. Hotchkiss, R. D., and Goebel, W. F.: Chemo-Immunological Studies on the Soluble Specific Substance of Pneumococcus. III. The Structure of the Aldobionic Acid From the Type III Polysaccharide, J. Biol. Chem. 121: 195, 1937. Ivanovics, G., and Bruckner, V.: Chemische und immunologische Studien iiber den Mechanismus der Milzbrandinfektion und -Immunitiit; die chemische Struktur der Kapselsubstanz des Milzbrandbazillus und der serologisch identischen .spezifischen Substanz des Bacillus mesen- tericus, Ztschr. f. Immunitatsforsch. u. exper. Therap. 90: 304, 1937; addendum 91: 175, 1937. Ivanovics, G., and Bruckner, V.: Untersuchung der Spezifizitat der Milzbrandimmunsera mit verkuppelten Azoproteinen, Ztschr. f. Im- munitatsforsch. u. exper. Therap. 93: 119, 1938. Johnson, T. B., and Coghill, R, D. : The Chemical Analysis of tlie Tubercle Bacillus, Trans. 22nd Ann. Meet. Nat. Tuberc. Assii., 192(i, p. 277. Jordan, E. O.: The Differentiation of the Paratyphoid Enteritidis Group Strains From Various Mammalian Hosts, J. Infect. Dis. 36: 309, 1925. Julianelle, L. A.: Studies of Hemolytic Staphylococci. Hemolytic Activity — Biochemical Reactions, J. Infect. Dis. 31: 256, 1922. A Biological Classification of Encapsulatus Pneumoniae (Friedlander 's Bacillus), J. Exper. Med. 44: 113, 1926. Immunological Relationships of Encap- sulated and Capsule-Free Strains of Encapsulatus Pneumoniae (Fried- lander's Bacillus), Ibid., p. 683. Immunological Relationships of Cell Constituents of Encapsulatus Pneumoniae (Friedlander's Bacillus), Ibid., p, 735, Julianelle, L. A., and Wieghard, C. W. : The Immunological Specificity of Staphylococci. II. The Chemical Nature of the Soluble Specific Sub- stances, J. Exper. Med. 62: 23, 1935. Kramar, E.: Untersuchungen iiber die chemische Beschafifenheit der Kapsel- substanz einiger Kapselbakterien, Centralbl. f. Bakt. I abt. orig. 87: 401, 1922. Laidlaw, P. P., and Dudley, H. W.: A Specific Precipitating Substance From Tubercle Bacilli, Brit. J. Exper. Path. 6: 197, 1925. T>ancefield, R. C: The Immunological Relationships of Streptococcus A'iridans and Certain of Its Chemical Fractions. I. Serological Reac- tions Obtained With Antibacterial Sera, J. Exper. Med. 42: 377, 1925. II. Serological Reactions Obtained With Antinucleoprotefii Sera, Ibid., p. 397. Antigenic Complex of Streptococcus Hemolyticus. Demonstration of Type Specific Substance in Extracts of Strepto- coccus Hemolyticus, Ibid 47: 91, 1928. Chemical and Immunological Properties of Protein Fractions, Ibid. p. 469. Chemical and Im- munological Properties of Species Specific Substances, Ibid., p. 481. Lancefield, R. 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Pittman, M. J. : Variation and Type Specificity in the Bacterial Species Haemophilus Influenzae, J. Exper. Med. 53: 471, 1931. Przesmycki, F. : Specific ' ' Residue Antigens ' ' of Different Types of Meningococci, J. Infect. Dis. 35: 537, 1924. Rice, C. E.: A Study of the Antigenic Activity of Preparations IMade From A'arious Strains of Treponema Pallidum, J. Immunol. 22: 67, 1932. Rice, C. E., and Reed, G. B.: Studies in the A^iriability of Tubercle Bacilli. VII. Antigenic Activity of "S" and "R" Cultures as Measured by Complement Fixation, J. Immunol. 23: 385, 1932. 372 I.MMT-XOLOOY Sabin, F. R.. and Doan, C. A.: The Biological Reactions in Rabbits to the Protein and Phosphatide Fractions From the Chemical Analysis of Human Tubercle Bacilli, J. Exper. IMed. 46: 645, 1927. Saito, T., and Ulrich, W. : Ueber die Gewinnung des spezifischen Kohle- hvdrates der II Pneumokokken und seine Chemischen. Eigenschaften. Ztsehr. f. Hyg. u. Infektionskr. 109: 103, 1928. Scherp, H. W., and Rake, G. : Studies on Meningococcus Infection, Type I Specific Substance. J. Exper. Med. 61: 753, 1935. Schiemann, O., and Casper, O. W. : Sind der spezifisch pracipitablen Sub- stanzen der 3 pneumokokkentvpen Haptene? Ztsehr. f. Hvg. u. In- fektionskr. 108: 220. 1927. Seibert, Florence, and Munday, B.: The Chemical Composition of the Active Principle of Tuberculin. XVII. A Comparison of the Nitrogen Partition Analysis of the Proteins From Different Acid-Fast Bacilli and the Relationship to Biologic Activity, J. Biol. Chem. 101: 763, 1933. Seibert, F. B.: The Purification and Properties of the Purified Protein Derivative of Tuberculin, Am. Rev. Tuberc. 30: 713, 1934. Seibert, F. B.: Cited b}- Long (1928) in Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1929, I^niversity of Chicago Press, p. 1021. Sievers, O., and Zetterberg, B. : A Preliminary Investigation Into Antigenic Characters of Spore-Forming, Aerobic Bacteria, J. Bact. 40: 45, 1940. Sherwood, N. P., Johnson, T. L., and Radotinsky, I. I.: Studies on Bacillus Pyocyaneus, University of Kansas Sc. Bull. 16: 91, 1926. Sherwood, N. P., Downs, C. M., and McNaught, J. B.: Nonlactose Ferment- ing Organisms From the Feces of Influenza Patients. .T. Infect. Dis. 26: 16, 1920. Shibley, G. S., and Hoelscher, H. : Studies on Whooping Cough. I. Type- Specific (S) and Dis.sociation (R) Forms of Haemophilus Pertu.'isis, J. Exper. Med. 60: 403, 1934. Smedley-MacLean, I.: The Chemistry of the Lipins, Ann. Rev. Biochem. i: 135, 1932. Sordelli, A., and Mayer, E. : Les Precipitines de la Gelose, Compt. rend. See. de biol. 107: 736, 1931. (Cited by Heidelberger.) Spielman, M. A. : The Chemistry of the Lipids of Tubercle Bacilli. XXXIV. The Constitution of Tubereulostearic Acid, J. Biol. Chem. 106: 87, 1934. Stanton, A. T., and Fletcher, W. : Melioidosis and Its Relation to Glanders, J. Hyg. 23: 347, 1925. Stevens, J. W. : Can All Strains of a Specific Organism Be Recognized bv Agglutination? J. Infect. Dis. 33: 557, 1923. Tillett, W. S., and Francis, T., Jr.: Serological Reactions in Pneumonia With a Nonprotein Somatic Fraction of Pneumococcus, J. Exper. Med. 52: 561, 1930. Tillett, W. S., Goebel, W. F., and Avery, 0. T.: Chemical and Im- munological Properties of a Species Specific Carbohydrate of Pneumo- eocci, J. Exper. Med. 52: 895, 1930. Toenniessen, E.: Untersuchungen iiber die Kapsel (Gummihulle) der pathogenen Bakterien. II. Die chemisehe Beschaffenheit der Kapsel und ihr dadurch bedingtes Verhalten gegeniiber der Fixierung und Farbung, Centralbl. f. Bakt., I abt. orig. 85: 225, 1921. Tomcsik, J., and Ivauovics, G.: Ueber die Herstellung des Antikap.sel- Immunkorpers des Milzbrandbazillus, Ztsclir. f. Immunitatsforsch. u. exper. Therap. 93: 196, 1938. i;ArTi:KiAL antickxs and srHciiicirv 373 Toplej-, W. W. C, Raistrick, H., Wilson, J., Stacey, J. W. M., Challinor, S. W., and Clark, R. O. J. : The Immunizing Potency of Antigenic Components Isolated From Different Strains of Bact. Tvphosum, Lancet 232: 252, 1937. Topley, W. W. C, and Wilson, G. 8.: The Principles of Bacteriology and Immunity, Xew York, 1929, William Wood & Co. Treece, E. L. : Personal Communication. University of Kansas, Lawrence (1920). Unpublished work. Wadsworth, A., and Brown, R. : Chemical and Immunological Studies of the Pneumococcus. I. A Specific Antigenic Carbohydrate of Type I pneu- mococcus, .J. Immunol. 21: 245, 193L III. Cellular Carbohydrate Fractions, Ibid. 24: 349, 1933. Walton, S. T. : Complement Binding Properties of Brucella Abortus of Bovine and Porcine Origin, J. Immunol. 22: 19, 1932. Wells, H. G.: The Chemical Aspects of Immunity, Xew York. 1929, The Chemical Catalog Co. White, P. B.: The 0 Receptor Complex of V. Cholerae and Its Antibodies, J. Path. & Bact. 44: 706, 1937. Whitmore, A. : An Account of a Glanderslike Disease Occurring in Rangoon, J. Hyg. 13: 1, 1913. Wilson, G. S. : The Serological Classification of the Tubercle Bacilli by Agglutination and Absorption of Agglutinins, J. Path. &: Bact. 28: 69, 1925. Zinsser, H.: Newer Knowledge of Bacteriology and Immunity, .Jordan and Falk, University of Chicago Press, 1928. Resistance to Infectious Diseases. Xew York, 1931, The Macmillan Co. Zinsser, H., and Bayne-.Jones, S.: Textbook of Bacteriologv, New York, 1939, D. Appleton-Century Co., p. 359. Zinsser, Hans, and Mueller, J. H. : Antigenic Properties of the Bacterial Cell. Xewer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press, p. 721. Zinsser, H., and Parker, J. T. : Further Studies on Bacterial H\-persuscepti- bility. II. J. Exper. Med. 37: 275, 1923. Zinsser, H.: Studies on Tuberculin Reaction and on Specific Hypersensitive- ness in Bacterial Infection, J. Exper. Med. 34: 49.5, 1921. Zozaya, J., and Clark, J.: Active Immunization of Mice With Polysac- charides of Pneumococcus T^-pes I, II. and III, J. Exper. Med'. 57: 21, 1933. CHAPTER XX RECAPITULATION OF CHAPTERS ON SPECIFICITY Examples of Specificity in Nature. — In the three preceding chapters the subject of specificity is discussed rather extensively. It is illustrated by the observations of Loeb and others made dur- ing fertilization experiments, by the physician in skin grafting and blood typing, by the immunologist who is interested in the acute infectious diseases and in biological relationships, by the physiolo- gist in his studies of hormones, enzymes, and various physiological phenomena, by the pharmacologist in liis experiments dealing with the nature and pharmacological action of drugs and by the geneti- cist in his investigation of inheritance. In fact, it is evident that the phenomenon of specificity is a common one in Nature. It is important in medicine because all of our serological tests and many therapeutic procedures must be reasonably specific to be of value. Antigens. — In earlier chapters attention is called to the fact that the injection of some substances into the animal body may lead to the production of specific antibodies, while the injection of other substances never leads to the production of antibodies. Spe- cific antibody stimulators are called antigens. Their properties are reviewed in these chapters. They are all colloids and aside from a few apparent exceptions, belong to the group of organic com- pounds called proteins. The antigenic properties of various pro- teins are compared with their chemical structure and a number of important facts recorded: Properties of Protein Antigens. — (a) Proteins are made up of amino acids linked together through their amino and carboxyl groups. They differ from each other according to the number, kind, and arrangement of the amino acid components. Since all proteins give the IMolisch reaction, it is possible that each contains a carbohydrate radical. There is some evidence that globulins contain a lipoid. If carbohydrate or lipoid is present, it will increase the number of possible combinations of 374 SPECIFICITY o/'> protein elements and thus add to the nuni])er of different proteins. It has been estimated that with the twenty amino acids it is pos- sible to build 2,432,902,008,176,640,000 different kinds of proteins. It appears from the work of Seibert and others that the size of the molecular aggregate forming the particles of colloid is an- other important factor determining the antigenicity of a substance. (b) Only those proteins containing certain of the aromatic amino acids are antigenic. (c) For a protein to retain its antigenic property, the optical activity of its amino acids must be vmimpaired. (d) AntigeiLS must be soluble in the body tiuids. (e) It has been determined by comparing the chemical structure and antigenic property of a large number of plant and animal pro- teins that immunological specificity is dependent upon the chemical constitution of the antigen. Species-Specificity and Type-Specificity. — Certain proteins such as those in the crystalline lens of the eye are common to all mammals and are therefore not species-specific. The same can be said of casein, thyroglobulin, testicular protein and blood fibrin- ogen. Some proteins are shared by a few species only, as, for ex- ample, gliadin is present in wheat and rye, while other proteins such as the serum globulins, albumins and tissue fibrinogens are species-specific. Thus it will be seen that the body contains many chemically and therefore antigenically different proteins, some of which are species-specific and others are not. Differences Within a Species. — Attention is called also to bio- chemical and antigenic differences within a species. There are four antigenic types of human red blood cells, over fifty types of pneumococci, several types of tetanus bacillus, etc. When one in- spects these cellular antigens in which each species is represented by several antigenic types, he discovers that all types of any one species have a species-specific antigenic protein in common, and that each type is composed of a different hapten associated with the species-specific protein. Thus, all pneumococci have a species- specific pneumococcus protein in common, but Type I has, in ad- dition, a hapten (polysaccharide) that distinguishes it from other pneumococci ; likewise Tj-pes II, III, etc., each pos.sesses distin- guishing haptens (polysaccharides), all chemically different from oach other. In the case of the four types of human red cells, all 376 IMMUNOLOGY possess a species-specific human protein in common, but there are two haptens, ''A" and "B," which make possible these four types. One type is represented by the species-specific protein not associ- ated with either hapten, this is type "0." A second type exists be- cause of the presence of hapten "A," a third because of hapten "B," and a fourth has both "A" and "B" associated with the species-specific protein. It is interesting to note that haptens may be carbohydrate-lipoid complexes, as, for example, the ''A" and "B" factors of red cells or the hapten fraction of Forsmann's antigen or perhaps just lipoids as suggested by Eagle for the antigen used in the Wassermann reaction. They may be pure carbohydrates such as one finds as partial antigens representing many bacterial types within a species as well as characterizing some species; or the hapten may be some chemical group such as tartaric acid, etc., attached to a protein. Hapten May Dominate a Serological Reaction. — Most haptens are incapable of stimulating antibody formation when separated from the protein fraction and injected into the animals. Anti- bodies formed as a result of injecting the hapten-protein complex can react with either the hapten alone or the hapten-protein com- bination. It seems that the hapten dominates the picture to the extent that one may not observe the presence of antibodies for the species-specific protein part of the antigen in immune serum which theoretically should contain them. Suppression Phenomenon of Landsteiner. — In connection with haptens the "suppression phenomenon" of Landsteiner is of in- terest. This is illustrated by the work of Wormall. He iodized a protein that contained tyrosine and then prepared a good pre- cipitating antiserum for the iodized protein. He found that when he added diiodotyrosine to tubes containing the iodized antigen and its antiserum, the specific precipitate did not form, it was sup- pressed. Arsanilic Acid Coupled to Histidine and Tyrosine by Hooker AND Boyd. — By means of this plienomenon Hooker and Boyd were able to prove that the arsanilic acid which they linked to gelatin couples to histidine, and when linked to egg white it couples with both tyrosine and histidine. They prepared good precipitating antisera for the arsanilic acid gelatin and egg white antigens re- spectivel}'. They then sj'uthesized tyrosine-like ar.sanilic acid and SPECIFICITY 377 liistidinc-like arsanilic acid haptens and found tliat only the latter would suppress the precipitate when the arsanilic-acid-gelatin anti- gen was mixed with its antisernin. When either of the synthesized haptens was added to the tubes containing arsanilic-acid-egg white and its antiserum, the amount of specific precipitate formed was reduced, and when l)oth synthesized luiptens were added, the pre- cipitate was completely suppressed. This showed that arsanilic acid was coupled to histidine in gelatin and both tyrosine and histidine in the egg white. This agrees with the known respective chemical structures, since gelatin contains histidine but not tyrosine, while Qgg white contains both of these amino acids. In this connection it should be remembered that the formation of a visible precipitate it not essential to the union of antigen and antibody, in fact it is a secondary reaction. It is evident that Landsteiner's suppres- sion phenomenon is due to some form of interference on the part of the unattached hapten added to the antigen-antibody mixture. Apparently it is due to the fact that the formation of immune precipitates is diminished or prevented when the antigen is present in excess (Landsteiner, 1936, p. 119). Its value in determining the nature of an unknown hapten is well established. Haptens Responsible for Cross-Reactions. — While some haptens are species-specific, many are not. The latter is indicated by the presence of the "A" and "B" factors of human cells, in the red cells of the anthropoid apes; of a hapten in one strain of Fried- lander's bacillus sufficiently similar to the polysaccharide of Type II pneumococcus, as to be responsible for cross serological reac- tions ; of a hapten in Proteus X19 so similar to one in the Rickettsia of typhus fever, as to make the Felix-Weil reaction possible. Such results enable one to interpret correctly numerous cross-reactions. Pneumococcus Type-Specific Polysaccharide Serum Globulin Antigen. — Many immunological facts have been discovered as a result of extensive investigations of haptens carried on for many years. It has been found that when a pneumococcus type-specific polysaccharide is combined with suitable serum globulin there re- sults an antigen which when injected into mice stimulates anti- bodies that protect the mouse against the pneumococcus correspond- ing to the liapten employed. Spatial Relationships and Specificity Stereoisomers as Hap- tens.— The possibility that spatial relationship may be one way of 378 IMMUNOLOGY determining specificity is illustrated when one employs antigens having a common protein fraction and differing only in that their haptens are stereoisomers of each other. Thus when dextro-, Icvo-, and meso-tartaric acids are attached respectively to pig serum, three different antigens are formed. An antibody can be pro- duced for each of these antigens lliat Avill not react with either of the other two. Landsteiner's Method of Producing- New Conjugated Anti- gens.— In order that the student may appreciate the significant work of Landsteiner on conjugated antigens, a brief discussion is given of the method he employs in introducing chemical groui)s (haptens) into an antigen. The discussion calls attention to the importance of these aromatic amino acids that have an OH group attached to the benzene ring in preparing a conjugated antigen. By first diazotizing the compound, the hapten can be linked to the nucleus replachig a hydrogen adjacent to the OH group. It is suggested also that linkage of the hapten with the salt-forming groups may occur. Specific antibodies representing each hapten- protein antigen can be produced. Suggested Explanation of Drug Allergy. — Since it has been shown tliat an animal's own protein when modified by the addition of a hapten will stimulate antibodies if injected into the same animal, it has been suggested that some of the drug allergies oc- cur when the drug, or some portion of it, acts like a hapten and unites with the patient's proteins and forms an antigen capable of stimulating antibody formation and sensitization of the patient. Mosaic Structure of Antigens. — As one contemplates the pos- sible significance of all these facts about antigens, one can appre- ciate ])erliaps tlie significance of Landsteiner's suggestion that antigens may have a mosaic structure. It is recommended tlmt the student read Landsteiner's monogry])h on "The Specificity of Serological Reactions" (1936). CHAPTER XXI THE IMPORTANCE OF ANTIBODIES IN DIAGNOSIS In the previous chapters antibodies have been defined and dis- cussed and methods of measuring them have been suggested. While there is some controversy over their importance in the body's defense against infectious agents, there is general agreement that they are frequently of great value in diagnosis. Discovery of Role of Ag-glutinins in Diagnosis. — The Widal Test. — Gruber and Durham (1896) investigated the phenomenon of bacterial clumping by immune serum and named it agglutina- tion. Almost simultaneously Widal (1896) studied the blood of typhoid fever patients and found that such bloods specifically clumped suspensions of living or dead typhoid bacteria. Both Widal and CI ruber recommended that the reaction be iLsed as an aid in the diagnosis of typhoid fever. In honor of its discoverer, the test has been known as the Widal or the Gruber-Widal reaction. Scope of Subsequent Investigations. — Since then the phenom- enon of agglutination has been investigated to ascertain the prev- alence of normal agglutinins, the effect of vaccination on ag- glutinin titer, the time of their appearance and the titers at- tained in both experimental infections and clinical cases due to a variety of infectious agents. The agglutinin reaction has also found wide application in the identification of bacteria isolated from pathological lesions and other sources. When the test was first introduced it was not realized that serological types exist among the pneumococci, meningococci, streptococci and other bac- teria. Perhaps this was because B. typhosus (Eherthella typhosa) was until recently regarded as being represented by one uniform type. While types are not as yet described for this organism, Downis (1925) and White (1926) have reviewed the literature and shown antigenic variation of practical importance. In preceding chapters a more comprehensive discussion of antigenic factors and variants now known to exist among bacteria have been discussed. Antigens Used in the Widal.- — At this point it is sufficient to .say that Widal discovered that suspensions of living organisms 379 380 IMMUNOLOGY (twenty-foiir-liour Ijrotli cultures or saline suspensions washed from a twenty-four-hour agar slant) or dead suspensions pre- served with formalin were quite satisfactory for use in the test. The suspensions should be' diluted to match a satisfactory turbidity standard and tested for agglutinability by serums of known potency. The importance of flagellar and somatic antigenic factors is discussed later in this chapter. It is quite important that a smooth motile strain be used in antigen preparation. Two Techniques Employed. — There are two methods employed to determine whether the patient's serum will agglutinate at a di- 71 Twbe Result V ViJ s^ ^^ v2/ '0 Mom,al R„.b« Pot,enti ^al.ne, PoS.k.ve M ^ I 1. ^ 1 ,'^°"i,'o' Widal Reaction Modtjq. Complete Cor.t>eriis SaVine donirol Negative Wiial Rea.c-t.on Fig-. 16.— Negative W^iclal test. clumps. Tlie patient's serum is in tube three and this tube is to be compared with tubes one and two. If it is negative, it will be like the former, while if it is positive it will be like the latter. The saline control, tube four, should show uniform turbidity. Figs. 15 and 16 show positive and negative Widals, respectively. Microscopic Method. — The microscopic method has inherent in it several sources of error to which exceptions are taken, but it does give some information not discoverable Ij.y the macroscopic test. In carrying out the microscopic technique, one prepares reagents as for the macroscopic test and studies them in hanging drops under the microscope. If living cultures are used, it will be ob- 382 IMMUNOLOGY served that in preparations containing normal serum or saline, the organisms remain motile and no clumps appear, while in prepara- tions containing agglutinins the organisms lose their motility and come together in definite clumps. It is also customary to test the patient's serum, in the same way, to see whether it agglutinates suspensions of aS^. 'paratyplii A or B* since these organisms produce clinical pictures that cannot be differentiated by physical examination and histor;/ of onset, al- though the course of each is usually milder and the duration shorter than in typhoid fever. Controls are very necessary in carrying out the test, since it is not impossible for the laboratory technician to get his cultures contaminated and use a suspension of the contaminant for the typhoid or paratyphoid suspension. Where the suspension is made from a pure culture of E. fijphom or of the aS'. puratuphi A and B* the tubes containing known positive serum will sliow defi- nite agglutination. The normal serum and saline controls should be negative to rule out spontaneous agglutination. When even pure cultures are used, if they are made from rough colony types, espe- cially of 8. pamtupM B* they quite often show spontaneous agglutination. When this occurs, the normal scrum and saline con- trol tubes should show perceptible agglutination. Aside from errors due to antigenic variation and technical pro- cedure, there are others due to mistakes in the interpretation of the results. The Widal is essentially a test to ascertain whether specific agglutinins for E. typJiosa or one of the paratyphoid organisms is present in the blood stream in sufficient amounts to give positive agglutination at the diagnostic titer used. If infection is present, the efificiency of the body tissues in the production of antibodies will determine the time of appearance and amount in the blood stream. Variation in Time of Appearance of Agglutinins. — Marked variation is to be observed in different individuals; some respond readily with early and abundant antibody production while others are poor antibody producers, the antibodies appearing later or in meager amounts. This accounts for the fact that the Widal may be positive in some cases of typhoid as early as the fifth day, while in others not until the third or fourth week. Gay (1918) feels ♦S'. schottmiilleri. IMPORTANCE OF ANTIBODIES IN DIAGNOSIS 383 that in approximately 100 per cent of tlie cases of typhoid fever, it will be pasitivc at some time during the course of the disease. Agcg-lutinins in Carriers. — It should also be borne in mind that clironic carriers or persons who have been recently vaccinated against typhoid may have a sufficient amount of agglutinins in their blood to give a positive Widal. If they should develop influenza or a streptococcus endocarditis or miliary tuberculosis, or any other infection that gives a clinical picture similar to typhoid fever, the Widal might be positive and therefore lead to a mistaken diagnosis. Thus it will be seen that there are many l)0ssibilities of error not only in performing the test but also in the interpretation of the results. To understand the significance of diagnostic tests based upon the presence of specific agglutinins in the blood of patients, one needs to know something of normal agglutinins and of the effect of vaccination and of infection on agglutinin titer. Normal Agg-lutinins and Diagnostic Titers. — The lilood of normal individuals not infrequently contains a small amount of agglutinins for various organisms. The normal agglutination titer for E. typhosa varied from 0 to 1 :20 with an occasional finding somewhat higher, but with the average being about 1 :5. Titers of 1 :80 are considered of diagnostic value. In the case of P. tular&nsis or Br. abortus the normal agglutinating titer may be somewhat higher than for E. tjiphosa. Titers should be about 1:80 to sug- gest active infection with either of the organisms. In active infec- tions one commonly finds a titer of at least 1 :320 or higher. The occurrence of previous unrecognized infection is commonly men- tioned as the explanation of the presence of normal agglutinins, but there are a few individuals who believe that in some cases these agglutinins may be normal physiological products just as the iso- and hetero-hemagglutinins are. Perhaps both explanations are valid. Flagellar, Somatic and Labile Agglutinins. — In 1903, Smith and Reagh in their studies on a motile organism concluded that ag- glutinins for the flagella as well as for the bacterial cell (soma) were produced. This indicated that motile organisms possess flagellar antigens as well as somatic antigens. Felix and Weil (1917) although disagreeing with Smith and Reagh found two antigens which they named "11" and "0" respectively. The 384 IMMUNOLOGY former letter is now used to designate flagellar antigens, while the latter is applied to the somatic ones. The extensive studies of Andrewes (1922, 1925) and of White (1926) have thrown a great deal of light upon the complex anti- genic pattern of the Salmonella group. According to White, E. t]/p]iosa is related to ;S^. eiiteritidis tlirough a common somatic or () antigen. Apparently the H antigens of E. typhosa, S. ixiratjjphi A, and S. e7iteyritidis are type specific, while the H antigen of S. paratyphi B, S. aertrycke and a few others are diphasic and may occur in one of two forms. They may change from one H form to another H form. The somatic antigens remain unaltered. The latter, however, as in the case of >S'. cnteritidis, E. typhosa, S. pullorum and *S'. sanguinarium, may be shared by a number of organisms. This somatic antigenic relationship between E. tjijjhosa and S. enteritidis must be kept in mind in interpreting a Widal designed to test for fine-flocculating agglutinins. The H antigen is contained in cultures showing motility. To prepare 0 antigens, advantage is taken of the solubility of the H antigens in alcohol. Bien and Gardner suggest adding an equal volume of alcohol to thick suspensions of the motile organisms and incubating the suspension at 37° C. for 24 to 36 hours. The alcohol destroys the H antigen. They are not injured, however, by formaldehyde. In Chapter VI a description of ''Vi" antigens discovered by Felix and Pitt is given. This is a labile surface antigen that is present in varying amounts in living virulent typhoid bacteria and tends to interfere with agglutination. It is thought to be respon- sible for inagglutinable strains of virulent E. typhosa. Variations in Ag"g"lutination Titer Following Vaccination. — In- formation relative to the variation of agglutinin titer following vaccination has been obtained by studying the antibody response in the lower animals and in man following vaccination. Jorgensen and Madsen (1902), Levin (1902), Staubli (1903) and others very early in this century made notable contributions along these lines. More recently Felix (1924), Stuart and Krikorian (1928-29), and Dulaney, Wikle, Stewart, Rayfield, et al. (1933), have in- vestigated the relative agglutinin response following vaccination IMPORTANCE OF ANTIBODIES IN DIAGNOSIS 385 with typhoid vaccines. The last named authors find both agglu- tinins produced contrary to the conclusions of Felix who held that only "H" agglutinins were formed as a result of vaccination, while "0" agglutinins were produced by infection. In a series of in- dividuals who were immunized against typhoid, the "H" ag- glutinin titer was much higher than the " 0, " the former showing a pronounced steady drop from an average titer of 1 :95 for 20 weeks after the last injection followed by a more gradual decrease in titer until a titer of about 1 :80 was reached in 140 weeks. The " 0 " agglutinations reached, on an average, a titer of only 1 :80 and fell during the same period of time to approximately 1:40. Effect of Allergy on Ag-g-lutinin Titer. — Lamson and Kessel (1932) noted low titers in a group of allergic individuals given multiple injections of typhoid vaccine. They assumed that im- munity was produced in spite of the low antibody response. In- quiry should always be made of patients who are suspected of having typhoid fever to ascertain whether they have been vac- cinated with a typhoid vaccine, since an increase in titer would be expected in such individuals. Experimental Results on Agglutinin Variation. — The result of vaccinating a large series of rabbits, some being given 'one in- jection while others are given two, three or more injections, is quite surprising in that a wide variation in response is found. Some animals are very poor antibody producers, while others yield exceptionally high titers. If only one injection is given to an animal which produces antibodies readily the titer viaj/ change from zero on the day of vaccination to 1 :500 or 1 :700 on the fifth to seventh day, although often it is less. The con- centration of antibody rarely continues to rise longer than the tenth day and frequently is dropping by that time to reach a low level in from three to six weeks. Where several injections of a vaccine are given, it is customary to administer them at from three- to seven-day intervals. This is done to keep the antibody titer rising. As a rule there is a slight drop in titer after each injection, followed by a fairly sharp rise during the next few days. Time of Appearance of Maximum Titer.- — The maximum titer Is usually attained after four to six injections and is reached usually five to seven days after the last injection. 386 IMMUNOLOGY With typhoid fever in an unvaeeinated individvial the agghitinin titer does not increase perceptibly nntil the fifth or seventh day of the disease. Occasionally it is the third week before an increase can be noted. The titer occasionally may not go above 1 :100, bnt as a rule it will go mnch higher. Hence early in the disease when the ))acteria arc in tlie blood stream, a blood culture is the lal)07*atoi'y procediii-c of choice since the Widal is likely to be negative. During hiter weeks the opposite will hold true. The Usk of the \Vn)AL ix Vaccinated Ixdividi^vls. — Where an examination is being made of tlie blood of an individual who has previously been vaccinated against E. tjfphosa the question arises as to whetlier the abnormally high titer is due to the vaccine or to an actual existing infection. It is not impossible for a vaccinated person to acquire an infection from massive doses such as may be present in contaminated milk. When such a question arises, it is answered by titrating the patient's blood daily using serial dilutions of his serum, and noting whether there is an increase in the titer, as one would expect if infection with E. typhosa is present, or whether the titer remains constant as it might after it had reached a Igav level several months following vaccination. Since iiifections due to *S'. paratyphi A and b.t: 2»2 Pig. 17. — Graph showing- fluctuation in asglutinins in partially immune and im- mune animals during- infection -with P. tularensis. (After Downs.) partially immune and immune rabbits following and during infec- tion. Downs ( 1932 ) has extensively investigated these phenomena. Agglutinin Response to Infection in Partially Immune Animals. — Fig. 17 shows the fluctuations in agglutinin titer in a partially immune rabbit, Number 282, and a highly immune rabbit, Number 274, following the intradermal injection of a dose of living organisms that would kill a normal i-abbit within a few days. It is interesting to note the drop of agglutinin titer simultaneously with the appearance of the bacteria in the blood stream. IMPORTANCE OF ANTIBODIES IN DIAGNOSIS 389 It will be observed from a study of the graph that at the time ])oth animals were inoculated with virulent organisms the partially immune rabbit's blood show^ed an agglutinin titer of 1:80 while blood from the immune rabbit (No. 274) had a titer of 1 :160. The agglutinin titer of neither animal was constant during the suc- ceeding days, but varied as indicated by the graph. Blood cultures were positive for the partially immune rabl)it on the ninth da>' and for the immune animal on the twelfth day. In both aninuils there was a drop in titer associated with blood stream invasion and til is was followed by an increase in titer. It is interesting to note that the partially inununc animal died on the eighteenth day wlien the titer had reached 1 :160, wliicli was twice as much as at tlie beginning of the infection, while the titcc for tlie immune animal stayed low (1:80) between the sixteenth and the twenty-fourth day, when it began to increase. This lasted several days, then dro])ped and finally Avent up to 1 :640 on the fortieth day after the initial infection. Infection in Normal Animals.^ — The picture is quite different in the normal animal. It shows that blood stream invasion occurs much earlier and that no agglutinins can be demonstrated either initially or during the infection. The failure to demonstrate agglutinins is probably due to the fact that death usually occurs on the fifth or sixth day which is too short a time for agglutinins to appear. It is also interesting to note that the immune animal did not show a high titer (1:160) when infection was produced, but nevertheless, it survived blood stream invasion and completely recovered. These results show lack of accurate correlation be- tween antibody titer and immunity. The Effect of Specific Infection on the Titer of Ag'g-lutinin for Other Organisms. — There are many specific infectious diseases in which there occurs an increase in agglutinins for bacteria other than the causal agent. This phenomenon is of importance in typhus fever, tularemia, and also undulant fever. Felix- Weil Phenomenon. — Typhus fever is caused by certain exceedingly minute organisms called Rickettsiae. They have been successfully cultured by Nigg and Landsteiner (1932). Within the ])lood stream of typhus patients there appear agglutinins for a strain of B. proteus vulgaris called Proteus X19. The phenom- 390 IMMUNOLOGY eiioii of their appearance was first observed by Felix and Weil and is regarded as being- sufficiently constant to be of value in the diag- nosis of typhus fever. The agglutination of Proteus X19 by the serum of a typhus fever patient is called the Felix-Weil reaction. Castaneda and Zia (1933) have demonstrated a common antigenic factor in Typhus Rickettsia and Proteus X19. This accounts, ap- parently, for the presence of agglutinins for the latter. Tularemia — Specific and Cross Reactions With Br. Abortus Antigens. — In clinical cases of tularemia, it is not uncommon to observe an increase in agglutinins for Br. ahortus. Conversely, in undulant fever, there may be an increase of agglutinins for P. tuhrensis. Francis (1926) has investigated the relative occurrence of agglutinins for Br. ahortus in the blood stream of patients suffering from tulai'omia and has tabulated his results as shown in Table IX. Table IX U. S. PiTBLic Health Reports, .TrxE, ]02fi TIME AFTER TULA- MELI- CASE ABORTUS TREAT^^E^rT ov SERiiivr OX SET RENSE TENSIS R. R. S. IS days (340 40 40 Unheated glycerin 2<) days 1,280 1,280 640 Unheated glycerin 7 months 640 320 320 55° C, no preservative 9 montlis CAO 320 320 55° C, no preservative 1 year 320 320 320 55° C, trikresol 1 year, 4 nio. (540 320 320 55° C, trikresol B. F. T. ?, days 0 0 0 Unheated glycerin 9 days 80 0 0 Unheated glycerin 16 days 1,280 160 320 Unheated glycerin 2?« days 320 160 320 Unheated paracresol 42 days 320 160 160 Unheated trikresol E. W. M. 5 days 0 0 0 Unheated glycerin 11 days 160 0 0 Unheated glycerin IS days 320 160 160 Unheated glycerin 25 days 1,280 320 160 Unheated paracresol 71 days 320 80 160 Unheated trikresol S7 days 320 80 80 Unheated glycerin J. W. G. 40 days 640 160 160 Unheated glycerin 53 days 640 160 160 Unlieated trikresol A.M. 11 days 160 0 Unheated, tive no preserva- 24 days 2,560 160 Unlieated, tive no preserva- oo days 1,280 80 80 55° C, no preservative 79 days 640 80 40 55° C, no preservative Permission of author and Surgeon General, U. S. Public Health Service. IMPORTANCE OF ANTIBODIES IN DIAGNOSIS 391 From nil inspection of this table, it will be observed that patient R. R. S. developed aj^glutinin titers for Br. abortus quite com- parable with those for P. tularensis and that the titer for Br. mcUtcnais was noticeably increased. In the remaining four cases of tularemia, Francis records a definite increase in titer for both strains of Brucella organisms, but in no case did he observe such a striking res])onse as in that of patient R. R. S. These results may l)e due to similar antigenic su])stances common to the Brucella group and to P. fularens{.s and also to the i)ossibility that when tularemia develops in a patient who has a latent infection with some member of the Brucella group agglutinins for both result. Since the diagnosis of either of these diseases is usually confirmed by the agglutination test, an appreciation of such phenomena is of importance in interpreting laboratory reports. Antigenic Relationships Between S. (Iallinarum and E. Typhosa. — The commercial laboratory is not infrequently called upon to test the blood of chickens for the presence of agglutinins for S. gallinarum or *S'. pullorum. Bushnell (1926) has recommended an agglutination ])rocedure designed to determine whether active infection exists in the fowl. Tliese organisms are of interest since they possess remarkable antigenic similarity to E. tuphosa. Sher- wood and Hoffman (1929) observed that the blood sera of several typhoid patients agglutinated a suspension of S. gallinarum (sanguinarum) at a higher titer (1:800) than the suspension of E. typhosa used in the Widal. IMulsow (1919) working in Jordan's laboratory concluded that the organisms could be dif- ferentiated only by careful absorption experiments. Theobald Smith (1915) has suggested that E. ti/phosa may be a variant of .S'. (jalUnarum. If this should eventually i)rove to be true, it might ex])lain many things at present difficult to understand in regard to the epidemiology of typhoid fever. As mentioned earlier in this chapter. White (1926, 1932) reports the existence of a somatic antigen ("0" antigen) common to E. typhosa, S. gaUinarum, S. pulloruui and S. enteritidis. Attention has already been called to tiie discovery of carbohydrate-lipid complexes in E. typhosa by Topley, Raistrick and their associates (1933, 1938). These sub- stances are highly antigenic and incite the production of the ' ' granular ' ' agglutinins. 392 IMMUNOLOGY . The Ag-g-lutination Reaction in Other Specific Infectious and Toxemic Diseases. — S. Dysenteriae. — There are several strains of >S^. dysenteriae which differ antigenically. Infection with the Shiga strain induces agglutinins in the patient for that strain but not for the one described by Flexner. The converse also holds. Shiga discovered the dysentery baciUus by isolating various organisms from the stools of clinical cases and ascertaining which were agglu- tinated by the patient's serum. Glanders. — In glanders, specific agglutinins develop as a rule for P. mallei. Tlie agglutination test has found definite applica- tion in the diagnosis of this disease. Human infection witli P. mallei is relatively rare. Whitmore (1913) described a glanders- like disease in man due to a new organism that has since been called B. whitmori by the English bacteriologists but named Flavobacterium pseudomallei by Bergey. Stanton and Fletcher (1925), Thompson (1933) and also Sherwood and Lan (1933) have observed antigenic relationships between B. whitmori and certain strains of P. mallei. The last named authors also noted some slight antigenic relationship between B. whitmori and an organism iso- hited by Sherwood from a case of meningitis in the central part of the United States. This latter organism has been named Flavohaderium orchitidis by Bergey. It is culturally and other- wise quite similar to B. whitmori except for the weak antigenic relationship. This is of interest since no strain of the latter organ- ism has been isolated from any source outside of Asia. Asiatic Cholera, and a Few Other Diseases. — The agglutina- tion reaction is of little or no value in the diagnosis of Asiatic cholera, cholera carriers, diphtheria and scarlet fever because of the great irregularity in the appearance of agglutinins in tliese diseases. Test for Heterophile Antibodies in Acute Infectious Mononu- cleosis.— The titer of normal hetcrohemaggliitinins in liuman blood for sheep cells is usually less than 1 :32. Paul and Bunnell (1932) discovered that this titer is increased quite extensively in most cases of acute infectious mononucleosis. Their discovery is now considered of diagnostic importance. Identification by Ag-g^lutination or Precipitation. — In the study of bacteria isolated from various sources such as typhoid fever. IMPORTANCE OF ANTIBODIES IN DIAGNOSIS 393 bacillary dysentery, Asiatic cholera, and the study of gram- negative diplococei from suspected meningococcus carriers and other conditions, it is often necessary to ascertain whether the organism isolated and culturally identified is agglutinated by known immune serum. If it is agglutinated by high dilutions of the known antiserum, its identity is considered as established. Thus if we isolate a motile gram-negative bacillus that is culturally similar to E. typhosa, the next step will be to see whether a sus- pension of the organism isolated is agglutinated by antityphoid serum and at a titer at which the antiserum will agglutinate a suspension of E. typhosa. In addition to this it has usually been decreed that for final identification an immune serum must be prepared against the new organism and this immune serum must react with a suspension of E. typhosa by agglutination and ab- sorption tests in exactly the same way that it does with its homologous organism. This is the "mirror reaction." Bacterial Types. — Since cellular antigens and specificity are discussed extensively in earlier chapters, it is sufficient to state at this time that each species of bacteria is represented usually by a number of types which can be differentiated from each other by agglutination. There are four or more types of meningococci, fifty-five specific types of pneumococci and an heterogeneous group not yet subdivided, four or more types of dysentery bacilli that also differ in some of their cultural reactions. The colon group is antigenically so diverse that the agglutination test is of no value in identification, hence one depends entirely upon morphology, staining, and cultural reactions. In regard to the staphylococci, only morphology, cell grouping and i)igment produc- tion are used as a rule in identification. In the more recent work on streptococci (Bliss, Gordon, Tunnicliff, Lancefield, and others) it has been reported that hemolytic streptococci can be subdivided into a number of groups and types by agglutination or precipita- tion. Virulent organisms from human sources fall into Lancefield 's group A. The green producing streptococci are more hetero- geneous, i.e., show greater antigenic diversity. C. DiPHTHERiAE. — In Identifying C. diphtheriae, agglutination Is not only of little value, but it is unnecessary since toxin produc- tion and specific neutralization of toxin by diphtheria antitoxin are excellent criteria for the final identification of the organism. All 394 IMMUNOLOGY strains produce the same kind of toxin, although they may show marked variation in agglutination. Cl. Tetani. — CI. tetnni has been divided into at least six types by the agglutination test but tliese, too, all produce one kind of toxin that is neutralizable by tetanus antitoxin. Cl. Botulinum. — An interesting exception to tlie rule that a single toxin is produced by all strains of an organism is to l)e found in Cl. hotulinum. The three types of this anaerobe produce antigenically different toxins althougli all three toxins produce botulism in appropriate test animals. Toxin from Type A is neutralized only by antitoxin for Type A and not by antitoxin pro- duced against the toxin of Type B or C. This holds true for each toxin. Neutralization and protection tests, rather than agglutina- tion, are the final criteria in identifying these organisms. E. Typhosa (B. typhosus). — E. typho.-pes 1 to 32 (Cooper). With Recommendations for Terminology of All Tvpes Reported Through 1940, J. Immunol. 41: 279, 1941. Weil, E.: (1905.) Cited by E. Volk in Kraus and Levaditi 's Handbuch fiir Immunitatsforschung 2: 629, 1909. White, P. B.: Cited by Zinsser and Bayne- Jones, Textbook of Bacteriology, ed. 8, New Y'"ork, 1939, D. Appleton-Century Co., pp. 505, 50(5. CHAPTER XXII THE BASIS OF BACTERIAL COMPLEMENT FIXATION TECHNIQUE Introduction. -Ill the pi-ecediiig' chapters the subjects of aiilijieus, antibodies and coiuplenicut liavc been discussed along witli our i>resent concepts of spccificit.y, cellular sensitization and the binding of complement by sensitized cells. It remained for Bordet and Gengou (1901) to discover that one could ascertain Avhether a given sample of serum contained antibodies for a specific bacterial antigen by mixing the two together with complement and later testing with sensitized red cells to see whether the comple- ment had been bound. If the serum under investigation contained antibodies for the bacterial antigen, one would expect sensitization of the bacterial cells and a subsequent binding of complement. One could determine whether complement was bound or free by adding sensitized red cells, incubating and examining for hemol- ysis. Bordet and Gengou saw at once that such a procedure might be helpful in tlie diagnosis of certain specific infectious dis- eases to ascertain the kind of antibodies in the blood stream. They also realized that by this means an unknown organism might be identified as accurately as by the agglutination test. In the present chapter it is proposed to discuss the methods of perform- ing the test and call attention to the reasons for adopting certain standard procedures. The Original Bordet-Gengou Technique. — The original comple- ment fixation technique was introduced by Bordet and Gengou in 1901 and can best be understood by a study of the accompanying protocol from their paper (Table X). These tubes were incubated for five hours at 15-20° C, and to each tube was added 0.2 c.c. of sensitized rabbit blood cells, and the tubes were shaken and in- cubated at 37° C, for one hour, when the results were read and recorded. The tubes appear as in Plate V. It was found that in Tube A no hemolysis occurred after the sensitized red cells were added. The reason is presumably that the anti-pestis serum sensitized the bacteria in the pestis emulsion 401 402 IMMUNOLOGY Table X* TUBE COMPLEMENT j^ NTIGEN SERUM USED A 0.2 C.C. guinea pig com- 0.4 c.c. pestis emul- 1.2 C.C. anti-pestis plement sion serum (heated) B 0.2 C.C. guinea pig com- 0.4 c.c pestis emul- 1.2 c.c. normal horse plement sion serum (heated) C 0.2 c.c. guinea pig com- plement None 1.2 c.c. anti-pe.' 1-. > 1, ^ W \-J ABC F Plate V. — OuuiixAL Bordet-Gengou Bacterial Complement Fixation Test A B C D E F Complement 0.2 p.e. 0.2 c.c. 0.2 c.c. 0.2 c.c. Antigen 0.4 c.c. 0.4 ex. 0.4 e.e. 0.4 c.c. Antipestis serum (heated) 1.2 e.e. 1.2 cc. 1.2 c.e. Normal hor.se serum ( heated) 1.2 e.e. 1.2 c.c. 1.2 c.c. In<'ubate for fifteen to twenty hours at room temperature and add 0.2 c.c. sensi- tized led cells and incubate at 37° C. for one liour. BACTERIAL COMPLEMENT FIXATION TECHNIQUE 403 horse. It is used to some extent as an aid in the diagnosis of tuber- culosis, but in general the tuberculin reaction has been found much more satisfactory. The agglutination reaction rather than comple- ment fixation is used almost exclusively in the identification of unknown organisms and in the diagnosis of typhoid fever, un- dulant fever and tularemia. The complement fixation technique has found its greatest application as an aid in the diagnosis of syphilis. This will be discussed in Chapter XXIII. More recently Craig (1927, 1928, 1929, 1930) and Sherwood and Heathman (1932) have studied its use in amebic dysentery cases and carriers. Dulaney's* (1940, 1941) work suggests that the test may be of value in malaria; Lennette and Horsfall (1941) have employed complement fixation in their influenza work and Witebsky, Wels and Heide (1941) report that the complement fixation test is sui)erior to the precipitin test in trichino.sis. Reagents and Factors Involved. — A study of the protocol illus- trating the technique employed by Bordet and Gengou shows that the following seven reagents were used ; namely — complement, bac- terial antigen, bacterial antibody, normal serum, red blood cells, hemolytic amboceptor and physiological saline. It also involves certain arbitrary expressions of total volume in each tube, time, temperature and conditions of incubation as well as the use of test tubes and pipettes. Development of Modern Technique. — Modern complement fixa- tion is a result of intensive and extensive studies of each of these factors with a view toward obtaining greater accuracy, simplicity, and standardization of procedure. Its evolutionary development may be traced through the following progressive steps : 1. The introduction of a complement fixation technique by Bordet and Gengou in 1901. Their technique defined a unit of red cells (rabbit), specified physiological saline as a diluent, a primary incubation of 15 to 18 hours at 20° C, and a secondary incubation of one hour at 37° C. They apparently did not em- ploy a uniform total volume nor did they recommend a method of titrating amboceptor, complement, or antigen. 2. Ehrlieh, V. Dungern, Wassermann, Bruck and others intro- duced a standard total volume (Von Dungern preferred 2.0 c.c, Wassermann and Bruck 5.0 c.c.) aiid substituted sheep for rabbit *Dulaney, A. D.. and Stratman-Thomas, W. K. : Complempnt-Fixation in Malaria, J. Immunol. 39: 247, 257. 1940. 404 IMMUNOLOGY red cells, and introduced methods for titrating amboceptor and antigen. Wassermann and Bruck employed 1.0 c.c. of a 1 :10 dilu- tion of complement in their test. They also specified that both primary and secondary incubation be carried out at 37° C. The definition for the unit of amboceptor suggested by the Ehrlich school is essentially that employed today. While their final con- centration of red cells was approximately the same as now em- ployed, they did not prepare their suspension from packed cells. 3. Numerous modifications of the early technique have been used. Noguchi employed an antihuman hemolytic system and a total volume of 1.0 c.c. while the New York City and State Boards of Health have employed a total volume of 0.5 c.c. Others have employed various hemolytic systems and total volumes of 2.5, 3.0, 4.0, and 5.0 c.c. The Kolmer technique which is becoming more or less of a standard one in the United States specifies a total volume of 3.0 c.c. 4. The amount of complement used in the test has varied from 0.2 c.c. of undiluted complement employed by Bordet and Gengou, and a titrated amount of a 1 :10 dilution employed by Noguchi to a titrated amount of a 1 :30 employed by Kolmer. 5. While the time and temperatures employed in the secondary incubation have remained fairly constant since the beginning, the time interval and temperature of the primary incubation have varied. Bordet and Gengou used 15 to 24 hours at 20° C; the Ehrlich school, Noguchi, Craig and others, one hour at 37° C. ; and Kolmer, although not the first to suggest it, has employed 15 to 18 hours at 6°-8° C. 6. The amount of patient's serum employed in the test has varied from 1.2 c.c. suggested by Bordet and Gengou to 0.1 c.c. recommended by Noguchi and also by Kolmer until recently. Kolmer now employs 0.2 c.c. The time of inactivation of serum has varied from 15 minutes to 30 minutes at 56° C. The former time is recommended by Kolmer for the quantitative test and the latter time for the qualitative although he states that 30 minutes may be employed in preparing serum for either test. 7. Neither Bordet and Gengou nor the Ehrlich school attempted to standardize the glassware employed in the te,st as is done by BACTERIAL COMPLEMENT FIXATION TECHNIQUE 405 Koliner. There are, however, good reasons for adopting standard equipment and standardized procedures. The pipettes he recom- mends enable the technician to combine accuracy with reasonable speed. The test tubes are of dimensions to insure a satisfactory height to the column of content.s and permit adequate mixing of reagents by shaking the rack. There are equally good reasons for most of his other requirements. 8. In regard to the diluent used (0.85 per cent saline) in com- plement fixation work, it has remained essentially the same since its introduction by Bordet and Gengou with the exception that Kolmer adds 0.10 gm. of magnesium sulphate per liter. Since Kolmer has increased the sensitivity of his modification of the Bordet- Wassermann test by reducing the concentration of com- plement and by increasing the lipoid content of his antigens and by interpreting a one-plus reaction as positive, there has been a growing insistence upon the use of purer saline solutions. 9. As regards bacterial antigens to be employed in complement fixation considerable progress has been made but there is room for improvement. Bordet and Gengou used untreated bacterial sus- l)ensions Avhile Wassermann and Bruck used bacterial extracts. Since then the New York City Board of Health has employed polyvalent antigens defatted with alcohol and ether; Wadsworth (1929) describes a "Dialyzed Distilled Water-Extract" method for the preparation of an antigen from tubercle bacilli ; Price (1932) dissolves a suspension of the gonococcus in NaOH and later adds sufficient HCl to precipitate out an antigenic substance and Kolmer and Boerner (1941) describe other methods of bac- terial antigen preparation. Torrey (1940) recommends Price's antigen for gonococcal complement fixation. Theoretically every antigen should l)e tested to see if it is hemolytic, anticomplementary or antigenic, but Kolmer (1941) says that when the bacterial antigens are prepared according to his method they are rarely hemolytic and therefore the hemolytic titration may be safely omitted. For the sake of completeness we will include protocols of the hemolytic, anticomplementary and antigenic titrations as recommended by Kolmer. They are as follows ; 406 IMMUNOLOGY Hemolytic Titration of Bacterial Antigen HEATED 00 o „• HUMAN .SA- i-i cc :2 2% C .SERUM C.C. LINE o 5 RED -^ HYPOTHETICAL TUBE ANTIGEN CELL rH OF 1:10 SOLN. READING DIL. OR C.C. SUSP. O SALINE rt Xi 1 umlil. (*usp. 0.5 1.5 0.5 25% hemolysis 2 1:2 dil. 0.5 1.5 o . no hemolysis 4 1:4 dil. 0.5 1.5 fi-§ 0.5 no hemolysis 0 1:6 dil. 0.5 1.5 0.5 no hemolysis () 1:(5 dil. 0.5 1.5 0.5 no liemolysis In the above protocol it will be noted that 0.5 c.c. of a 1 :2 dilu- tion of antigen was the least amount of antigen producing some hemolysis. Kolmer would call this the hemolytic unit. The next protocol is given to illustrate one method Kolmer and others have used to determine the smallest amount of antigen that produces a slight inhibition of hemolysis. Tliis amount is called the anticomplementary unit. The results are illustrated in Plate YI, Fig. 1, except that Tubes 11 and 12 are not shown on the plate. The anticomplementary unit in the above protocol would be 0.5 C.C. of a 1 :2 dilution. Kolmer regards the anticomplementary titration as very important and suggests that it be done at frequent intervals with most bacterial antigens. The next protocol is an example of antigenic titration according to a method suggested by Kolmer. The results are illustrated in Plate VI, Fig. 2, except that the serum control Tube 11 and the control on the hemolytic system Tube 12 are omitted from Plate VI, Fig. 2. In the accompanying j)rotocol the antigenic unit, i.e., the least amount of antigen giving a + + + + fixation of complement (in- dicated l)y complete inhibition of hemolysis), is contained in Tube 8. This unit then is 0.5 c.c. of a 1:200 dilution of antigen. (See Plate VI, Fig. 2.) If positive serum is not available, Kolmer suggests employing a dose of antigen in the test, equivalent to one-third the anti- complementary dose. Referring to the anticomplementary titer given earlier, it is found to be 0.5 c.c. of a 1 .-2 dilution of antigen. Fig. 1. — Antic()iri|il('niontiii y litiatiou of antigen in tlie presence of normal serum. ^cz:^ fcr r j> <-.. " 11 \\ rwvJ n Fig. 2. — Antigenic titration in tlie presence of positi\e serum. Plate VI. — AxTico.MPi.K.NtKXTAKY and Axtige.vic Titratiox ok Axtigex. BACTERIAL COMPLEMENT FIXATION TECHNIQUE 407 EXAMPLE OF READING AS PER PLATE VII, FIG. 1 tc 7i 'co 'co 'aj 'K 'x '33 '» o o o o o'o'o'o'o tnwaassasssa « j^j^.^" — — --'— ~ — — ~ [^ -■-raaaaaaaaa -^ OOoOOOOOOOO o •.moij I joj q}Bq .io)tj_\\^ h3 . LO iq iO iq ic in iq iq iq iq ira to d d d d d d d d d d d d HEMOLYSIN 2 UNITS IN 0.5 c.c. iq iq LO iq iq iq lo in in o m a A •ai[ z JOjf A'luo i()Bq J8;BAi ut pa^Bqnaui ao i Hieq ja^B.u dm m •uini OS -^q pa.ttO|ioj '•.n\ 81 o; sx JO J '0 o8 o; „9 ;b aiTjqnoui COJIPLEMENT 2 FULL UNITS IN 1.0 C.C, OOOOOOOOOOO fl ^' ^" _j _J _: _: _; _; _; _; _: o HEATED NORMAL SERUM C.C. 1:10 DIL. iq iq iq iq iq iq iq iq iq in in g o < undil. susp. 1:2 dil. 1:3 dil. 1:4 dil. 1:6 dil. 1:8 dil. 1:10 dil. 1:12 dil. 1:16 dil. 1:20 dil. None ; 0.5 c.c. saline None ; 2.5 c.c. saline -1 ci cc -f m "xi t^- 00 oi o i-H ci i-l r-l r-l 408 IMMUNOLOGY O s ^ + + + + + + + + + 1 1 1 + + + + + + + + + + + + + + + + •jq X joj q;Bq ja^BAV >n in iq iq iq iq iq iq iq iq iq iq o o, o o o o o o c o o o HEMOLYSIN 2 UNITS IN 0.5 C.C. uq iq iq iq iq iq iq iq in in uq iq o o o o o o* o o o o o o Xq paMonoj 'uq gl o; gi joj "0 o8 0* o9 *'B uox;Tjqnoni XiBraiJj COMPLEMENT 2 FULL UNITS IN 1.0 C.C, oooooooooooo HEATED POSITIVE SERUM C.C. OF 1:40 DIL. 0) iq iq iq iq iq in iq iq iq iq iq C o o d d d d d d d d d ^ DILUTION OF ANTIGEN SUSP. C.C. .9 .9 o o o o o ^ ^ ri(M-^«aOi-(i-l(MCOTt( .. r-lr-lrHrHrHi-lr-lr-li-lr-l o O im O d r-i TUBE r-lC-lrO-^inXit^OOClOr-ICvl I— 1 t— 1 I— 1 BACTERIAL COMPLEMENT FIXATION TECHNIQUE 409 The test dose based upon using one-third of this would be 0.5 c.c. of a 1 :6 dilution of antigen. If, instead of the above, antigenic titration was done and it was desired to use 10 antigenic units in the test, it would only be necessary to use 0.5 c.c. of a 1 :20 dilution of this hypothetical antigen since one antigenic unit is 0.5 c.c. of 1 :200 dilution. After the antigenic titration has been completed and a test dose of antigen is decided upon, one is then ready to set up a com- plement fixation test to determine whether antibodies correspond- ing to the antigen are present in the patient's serum. In sucli a test it is necessary to establish by experiment (adequate controls) the validity of the results obtained. Three methods are available for complement fixation. One is the quantitative, a second is the qualitative, and third is the simplified test. A protocol for the simplified test is given in Table XI and is illustrated in Plate VII. In order for the student better to understand the significance of the results indicated in Table XI and Plate VII the following discussion is presented : From a careful inspection of tlie above table it will be evident that Tube 5, which contains patient's serum, antigen, complement and sensitized red cells, constitutes the only test of the patient's serum for the presence of complement fixing antibodies. The other nine tubes are obviously controls. These controls are not only necessary from the standpoint of accuracy of results, but also fre- quently enable the laboratory worker to ascertain the cause of his failure. No Hemolysis in Tube 5. — If either no hemolysis or partial hemolysis occurs in Tube 5 containing patient's serum, antigen, complement, red cells and hemolysin, the following questions arise : 1. Is it because the patient's serum contains sufficient antibodies to sensitize the antigen and thus give specific fixation? 2. Is the patient's serum anticomplementary? 3. Is the antigen anticomplementary? 4. Is it because of insufficient amount of complement due to a drop in titer or to errors in making up the standard dilution or to pipetting into the tubes? 5. Is it due to hemolysin deficiency for similar reasons ? 410 IMMUNOLOGY X H 3 m H 0) _ ^^ O > CS CS «H '•*= S fl g ^ »- 4i >s'S >s o Sc P^. o ^.^ ^ 2 >^ a -^ ^ fl O c; o< =e ts rt tH -tj fl CQ "S (3 ei 1° a =H M "S S g 0) 'J-l ^ O 9 =H 0) fl g ■— ' ca a "^ri! a^ 'p^^ S-2 a £ a a £ a a 2 a o ti 3 O -M 3 ^ cs e; -w S « fl ?: w a t. •r3 o 4) P, ^ X, 0 O OJ V2 o a, PI O X CU -«! *^ -< _ 03 O TiJTjq ja:^!?^. «I '0 o2S ^^ .inotj 1 YSIN ITS C.C. o •6^ «' o J :^ ic w 6 « «j OPo o o iq iq d d d d W - O '-I Z • w ^" ^ o c3 o w o^co^ o « « o cJ (M S P '=^ m i-O iq iq d d d d ' INCUBA- N PERIOD FIXATION COMPLE- NT OVER- IGHT IN CE BOX 01 01 A'q peMoiioj -0 „8 o\ •0 ot) ;b uor;Bxy ^ s.moq gX"!! s! O S3 El, W :z " ►i ' "^ . !2: • a g ^ :^ " ^. 1:30 : comp: MENT 2 FU UNITS 1.0 c q q q q r-5 i-i rH '4. m X O s§ si ^ lO i.O y^ . > r< ^_, ;r ^ ''< lO . o w p • ^• W; tr< OS \\ ►- gss-° ;= g < S LO . « w o i g ^" e. ^ O i r^ « «3 iq 3 g M d d + '^ '^ »n . o c3 ^ g"^ s o o o S ;: fl d lO d w w ca 1—1 ?q f-j ■^ P H BACTERIAL COMPLEMENT FIXATION TECHNIQUE 411 0^ tH 5 es a 4) *- QJ 3 -ti p< tX 'T rt •— ■ ■S 2 - a o «^." O of antibodies by tliis tube). (■). Anticomplementary control on patient 's seiuin. 7. Control on hemolytic system. S. Control to determine if amboceptor is hemolytic. 9. Antigen control (to determine if antigen is hciiiolytic). .10. Control to determine if saline is hemolytic to 1)0 dcteiniincd BACTERIAL COMPLEMENT FIXATION TECHNIQUE 413 occur if tlie antigen is not anticomplementary. It is suggested that the student follow Kolmer's suggestions. Quantitative and Qualitative Tests. — The technique of the quantitative test is similar to that of the simplified test except that in the former the serum is used in amounts of 0.2, 0.1, 0.05, 0.025, and 0.005 c.c. with 0.2 c.e. in the control tube. In the qualitative test Kolmer uses 0.2 and 0.1 c.c. and 0.2 c.c. in the control. Positive, negative and other controls should be included in both tests. Reading the Results. — Kolmer states that the readings should be made immediately after the secondary incubation of one hour or they may be made 10 minutes after complete hemolysis occurs in the serum, antigen and hemolytic system controls. Reporting- Results. — Kolmer states that results may be reported as ''positive," ''doubtful" or "negative." He considers fixation of one plus or more as ' ' positive ' ' ; the + reactions are called "doubtful" and when complete hemolysis occurs the report is "negative." Not infrequently the physician desires to know the degree of fixation observed. In such an event the report is given as four, three, two or one plus positive or + as doubtful and complete hemolysis as negative fixation. Correlation With Clinical Findings. — In the case of gonococcal complement fixation, Price obtained 85 per cent positive results in known cases of infection. Park, AVilliams and Krumwiede re])ort 75 per cent to 95 per cent positive results for complement fixation in clinically active pulmonary tuberculosis. In glandular tuberculosis and tuberculosis of the l)ones and joints, only 58 per cent and 22 per cent, respectively, of the sera gave positive results using complement fixation. Discussion. — It should be remembered that the complement fixation test is just one of several techniques employed to detect antibody in blood serum and it is perhaps one of the most sensi- tive tests employed for that purpose. The reason for lack of cor- relation with clinical findings may be due to : 1. Defective reagents such as antigen, complement, hemolysin and saline. 2. Defective technique in preparing glassware or reagents, or errors in inactivation, incubation, or in setting up the test, «tc. 3. The serum being tested. 414 EVIMUNOLOGY Prom previous discussions bearing upon the complexity of cellular antigens and the effect of cultural environment, variation, heat and chemicals upon certain important substances associated with virulence it is obvious that obtaining a perfect antigen for complement fixation is not as yet passible. It is desirable to learn as much as possible about antigens so that better methods of prep- arations may be devised. The diflSculties that arise from defective technique are to a great extent within man's power to prevent. In this category are placed errors due to utter carelessness or ignorance of available knowl- edge concerning these tests. It is quite possible that as our under- standing of the underlying physical and chemical mechanisms be- comes more perfect our technique will improve. The difficulties that are due to tlie serum under investigation are to a large extent beyond one's power of control. If the patient's tissue cells do not respond to antigenic stimulation with the produc- tion of antibody, then the negative results of complement fixation may be poor from the clinician's point of view but actually the results would be accurate from the standpoint of what the test is designed to accomplish, i.e., to test for specific antibodies. On the other hand the test might be positive in the case of a high normal antibody titer or when the antibody titer is increased due to vaccination, e.g., B. C. G. vaccine, and the positive results would not correlate with the true clinical condition of the patient. A few specifie examples taken from actual laboratory records should cause the physician to realize that the lack of correlation between laboratory and clinical findings is not always due to faulty tech- nique but that biological variation may be a factor. Variation in Titer of Hemolysin. — In the study of hemolysin production, 23 rabbits of about the same weight were used. Each received a daily intravenous injection of 0.4 c.c. of a 50 per cent sheep cell suspension over a period of 10 days. Tliey were bled and the serum titrated for antibodies on the seventh to the ninth day after the last injection. The antibody titers observed are interesting. Seven gave a titer of 1 :10,000, two of 1 :5,000, five of 1 :2,500, three of 1 :2,000, three of 1 :1,500, one of 1 :1,000, and two yielded a zero titer. Variation in Agglutinin and Precipitin Titer. — Similar re- sults were obtained with two additional series used for precipitins BACTERIAL COMPLEMENT FIXATION TECHNIQUE 415 and bacterial agglutinins, respectively. It is realized that some of the variation may be due to variations in the time interval for maximum antibody production, but it is certain that some animals are entirely refractory as judged by our present methods of in- vestigation. In the commercial j^roduction of diphtheria antiloxiji, hoi-scs are given repeated injections oC toxin for the most pai-t in the form of toxin-antitoxin mixtures or of anatoxin, which is toxin detoxified with formaldehyde. It has been the general experience that some horses yield little or no antitoxin while others produce high titered antitoxic sera with all degrees of variation between these two extremes. Antibody Variation During Infection. — Likewise, the physio- logical production of antibodies resulting from infection is variable even when the infectious agent is a good antigen. This is quite definiteh' illustrated by a study of the agglutinin response in nor- mal rabbits infected with P. tularensis producing a disease com- mon to rabbits. In a series of 11 normal rabbits, showing zero agglutinins before inoculation, and all surviving for almost the same length of time. Downs observed that one developed a titer of 1 :1,200, three of 1 :320, one of 1 :160, two of 1 :40, two of 1 :20 and two continued with a zero titer until death. In the case of typhoid fever, the literature indicates that the Widal reaction is positive at some time during the disease in 80 to 92 per cent of the cases. Gay reported 100 per cent positive Ijy the fifth week in a series that he studied. It should be remembered that E. typhosa is an unusually good antigen represented by practically one type in contrast to the numeroiLS groups or types of pneumococci, gonocoeci, and meningococci found associated with their respective infections. Summary of Recommendations. — In this chapter some of the factors influencing the results of bacterial complement fixation and the underlying philosophy of hemolysin, complement and antigen preparation and titration have been presented by discussion and protocols. In conclusion it would seem advisable to summarize most of the specific recommendations that have been presented rela- tive to a standard procedure for complement fixation. It will be observed that these conform very closely to Kolmer's recommendations : 416 IMMUNOLOGY 1. The glassware, which includes pipettes, test tubes, volumetric flasks and graduates, should be chemically clean since minute traces of acids, alkalies, dyes, and many other substances may interfere with the successful performance of the tests. 2. The size of the test tubes should follow ]U'oferably Kolmer's recommendations, i.e., 15 mm. by 85 mm. 3. Sterile, cold, properly prepared physiological saline contain- ing 0.1 gm. of magnesium sulphate per liter should be used uniformly as a diluent. 4. The final volume in each tube is to be 3.0 c.c. f). One unit of cell suspension is contained in 0.5 c.c. of a 2 per cent suspension of sheep cells prepared from waslicd packed cells. 6. A unit of hemolysin (antisheep) is contained in 0.5 c.c. of the highest dilution that brings about complete hemolysis of a unit of red cells in the presence of 0.3 c.c. of a 1 :3() dilution of complement when incubated for one hour at 37° C. 7. An exact iniit of complement is the lea.st amount oL" a stand- ard dilution 1:30 of complement that will comi)letely hemo- lyze a unit of red cells in the presence of 2 units of hemolysin when incubated for one hour at 37° C. in a water bath. A full luiit is 0.05 c.c. more than a unit, (complement should be titrated in the presence of a test dose of antigen contained in 0.5 c.c. of saline. 5. Pooled complement from 3 or more guinea pigs is ])referable to complement from one pig. }). (^om])lement nuiy be preserved either hy adding 10 limes the normal concentration of dry NaCl or by freezing. A modi- fied Sonnenscheine method as suggested by Boerner and Ijukens (1940) has been successfully used in this laboratory. Ijyophil or cryochem complement is preferred. 10. High titered inactivated antisheep hemolysin is preferably preserved by adding an equal amount of C.P. glycerin. 11. For antigen titration 2 full units of complement and two units of hemolysin are recomnu^ided. 12. It is recommended that six to fifteen hours at 6° to 8° C. be used as the time and temperature for primary incubation. 13. For the second incubation it is recommended thai one hour in a 37° C. water bath be employed. BACTERIAL COMPLEMENT KLXATION" TECHNIQUE 417 14. The method of antigen preparation depends to some extent upon the organism used. It is suggested that the Kolmer or New York City or New York State Board of Health methods be used. For purposes of instruction defatted antigens are recommended. 15. An antigenic unit is the least amount of antigen or 0.5 e.c. of the highest dilution of antigen that gives complete (+ + + +) fixation of 2 full units of complement in the pres- ence of 0.1 c.c. of known positive human serum. 16. A satisfactory antigen must not be hemolytic or anticom- plementary when 10 antigen units are present in the tubes. 17. All immune, positive, negative, and patient's sera are to be inactivated at 56° C. in a water bath for fifteen or twenty minutes for the quantitative test and thirty minutes for the simplified complement fixation test. References Bonlet, J., and Gengou, O. : Sur 1 'existence des substance.s sen.sibilisatrices dans la plupart des serums antimicrobiens, Ann. de I'Inst. Pasteur 15: 289, 1901. Craig, Charles F. : Observations Upon the Hemolytic, Cytolytic and Com- plement-Binding Properties of Extract.* of Endamebic Histolytica, Am. J. Trop. Med. 7: 225, 1927. Complement Fixation in the Diagnosis of Infections With Endameba Histolytica, Ibid. 8: 29, 1928, The Technique and Kesults of a Complement Fixation Test for the Diagnosis of Infection With Endameba Histolytica, Ibid, 9: 277, 1929. The Diagnostic Value of the Complement Fixation Test in Amebic Infection, .1, A, M. A. 95: 10, 19.'?0. Downs, C. At.: Personal communication. Khrlich, Paul: Studies in Immunity, translated by Bolduan, Xew York, 1910, .lohn Wiley & Sons. Gay, F. P.: Typhoid Fever, New York, 1918, The Macmillan Co. Gengou, O. : Sur les sensibilistrices des .serums aetifs contre les substances albuminoides, Ann. de I'lnst. Pasteur 16: 734, 1902. Kolmer, J. A., and Brown, C. P. : Studies in the Standardization of the Wassermann Reaction. III. The Red Corpuscle Suspension for the Wassermann Reaction. The Preservation of Red Blood Cells, Am. J. Syph. 3: 169, 1919. Kolmer, J. A. : Infection, Immunity, and Biologic Therapy, ed. 3, Phila- delphia, 1925, W. B. Saunders Co. Kolmer, J. A., and Boerner, F. : Approved Laboratory Technic, New York, 1941, D, Appleton-Century Co., p. 637. Kolmer, J, A.: Technics of Serodiagno.'^tic Te.sts for Syphilis, U. S. P. H. Service, Supplement 11, p. 56, 1940. Mhile antigen. Since occasionally one encounters a normal serum with a higli heterophile antibody titer, it is conceivable that false poxitives may, on rare occasions, be obtained. They further recom- mended the preparation of an alcoholic extract of beef heart using methods of grinding and drying, preliminary and final extrac- tions of the tissue and later cholesterinizing part of the extract, that are quite similar to the more recent nu'thods recommended by Kolmer. They further suggested that a cholesterinized and an acetone-insoluble lipoid antigen be prepared from portions 428 IMMUNOLOGY of the plain alcoholic extract of beef heart, thus insuring a certain uniformity in lipoid content of the three antigens. Fifth Stage: Standardization of Wassermann Reaction by KoLMER. — The fifth stage began with the extensive investigations of every aspect of the complement fixation test for syphilis by Kolmer and his colleagues in 1919 and ended with his recommen- dations for a standard qualitative and quantitative Wassermann technique in 1922 and 1925. These are generally spoken of as the Kolmer or Kolmer- Wassermann qualitative or quantitative com- plement fixation reactions in syphilis. A sixth stage exists at the present time and is one of revalua- tion, reconciliation, reinvestigation, and coordination. The com- plement fixation test for syphilis is assuming greater value in all laboratories where an unmodified Kolmer technique is employed. Clinical pathologists who have either published or adopted various modifications of the Wassermann technique have been slow to aban- don these methods and adopt Kolmer 's recommendations in their entirety, but a perusal of the literature seems to indicate that there is a slow but definite trend in that direction. The American Public Health Association is actively interested in the standard- ization of the Wassermann reaction as evidenced by the fact that it has a committee of which Ruth Gilbert is chairman, working to- ward this end. An extensive literature that has been augmented recently shows rencAved interest in the physicochemical aspects of complement fixation and may lead to further improvement in technique. There is also a growing conviction among serologists that a flocculation test such as the Kahn or Kline should be done simultaneously with the Kolmer complement fixation test on each serum. In order that the student may appreciate the value of the ex- tensive investigations of the Wassermann reaction carried out by Kolmer, a list of his publications leading to the first Kolmer com- plement fixation test is included in references at the end of the chapter. Kolmer Test. — In the preceding chapter (XXII) the technique is given for Kolmer 's bacterial complement fixation test. Except for the differences in antigens the technique for the Kolmer modification of tlie Wassermann used in the diagnosis of syphilis is the same. It is described in detail in United States Public COMPLEMENT FIXATION IN SYPHILIS 429 Health Supplement No. 11 to Venereal Disease information. This can be obtained from the Superintendent of Documents at a nominal cost. Since the Public Health service plans to issue re- visions of Supplement No. 11 from time to time it would seem advisable for medical students, and others interested, to be on the alert for such revisions. The antigen used in the Kolmer test is a cholesterolized and lecithinized alcoholic extract of heart muscle. It is prepared by extracting powdered beef-heart with acetone for 5 days to remove all the acetone soluble substances since they are thought to be un- desirable. The acetone extract is filtered off and discarded. The dry residue is next exti*acted with chemically pui-e absolute elhyl alcohol for 5 days and filtered. This filtrate is saved since it con- tains tlie acetone insoluble, alcohol .soluble lipoids used in the Kolmer antigen. To improve the antigen, i.e., to make it more sensitive, Kolmer adds 0.2 gm. of cholesterol to each 100 c.c. of the alcoholic solution of lipoids. After shaking and heating for one hour in a water bath at 55° C. to aid in the solution of the cholesterol it is allowed to stand at room temperature for 2 or 3 days and finally filtered to remove any precipitates that may have formed. This cholesterolized alcoholic extract is standard stock solution of antigen. Kolmer also prepares a neiv antigeyi which is more sensitive than the above. The only difference is that the new antigen has not only all of the lipoids of the first antigen but there has been added to it, just before he added the cholesterol, all of the ether soluble but acetone insoluble lipoids obtained from the first 4 ether ex- tracts of heart muscle that are usually discarded in the preparation of either Kahn or Eagle antigen. These added lipoids make the new Kolmer antigen more sensitive. Titration of Antigen. — Both of the antigens mentioned are titrated in the same way. Kolmer says that it is unnecessary to titrate these antigens for hemolytic or anticomplementary units. It is, however, necessary to titrate for antigenic activity. This he does in accordance with the method of Boerner and Lukens. A brief summary and protocol is as follows : He first prepares a 1 :80 dilution of antigen by adding, drop by drop, 0.1 c.c. of the cholesterinized alcoholic solution of lipoids to 7.9 c.c. of saline, shaking between each drop. Beginning with this 430 IMMUNOLOGY he prepares the following higher dilutions, 1 :160, 1 :320, 1 :640, 1 :1,280 and 1 :2,560. He then prepares 5 dilutions of inactivated positive serum in such a way that by pipetting 0.5 c.c. of each he can arrange six sets of 5 test tubes in a rack with the amounts of serum shown in the following protocol. Each tube also has 0,5 c.c. of the antigen dilution indicated. Antigen Titration antigen in 0.5 c.c. amounts SERUM IN 0.5 c.c. 1:80 1:100 1:320 1:640 1:1280 1:256( 0.005 - - + + - - - 0.0125 - + + + + + + + + + + + 0.025 + + + + + + + + + + + + + + + + + + 0.05 + + + + + + + + + + + + + + + + + + + + + 0.10 + + + + + + + + ■i + -f- f ^ 4 + f + + + + + + + Kolmer defines the dose of antigen to be used in the Kolnier complement fixation test as the lai-gcst amount of antigen giving a 4r>t- reaction with the smallest amount of serum. He says that, if three dilutions all give 4 plus reactions with the smallest amount of serum, one should choose a dose midway between the highest and lowest. In the above protocol the test dose would be 0.5 c.c. of a 1 :320 dilution. As mentioned previously in this chapter the technique of the Kolmer complement fixation test used to detect syphilitic reagin is the same as that described in the previous chapter for bacterial complement fixation except for the antigens used. The lipoid antigen is substituted for the bacterial antigen in the Kolmer modification of the Wassermann. The tests are also reported the same. It will be recalled that the committee on the Evaluation of Serodiagnostic Tests for Syphilis of the United States Public Health Service cooperating with the American Society of Clinical Pathologists recommend that four plus, three plus, two plus or one plus reactions in the first tube be reported positive while plus or minus be called doubtful and com- plete hemolysis negative. Kolmer, however, prefers to call four plus and three reactions in the first tube as strongly positive, two plus reactions or one plus reactions as weakly positive and plus- minus as doubtful reactions and complete hemolysis in first tube as negative. COMPLEMENT FIXATION IN SYPHILIS 431 For a more complete description of the technique the student should read Supplement No. 11 issued in June, 1940. Kilduffe's Ten Basic Principles in Serolog-ical Diagnosis. — In regard to tlie present status of the serological diagnosis, Kilduffe* (1933) discusses ten basic principles which he regards as impor- tant. His observations are Avorthy of serious consideration and are summarized as follows : "1. The diagnosis of syphilis, however achieved, should l)e surrounded by every ])ossible safeguard, regardless of the time, the labor, the ex])ense of the minutiae involved. "2. The diagnosis of syphilis should be based upon a careful, well-balanced consideration of all the data, however obtained, rather than predicated solely upon one or two isolated particu- larized facts or findings. "3. A careful and intelligent stud}^ of syphilis is hnpossible without eonstant recourse to laboratory avenues of investigation, especially serological studies. "4. Laboratory procedures nuist be regarded solely as con- stituting a single phase in the examination of the patient, ''5. A joint and interlocking responsibility rests upon both serologist and clinician entitling each to demand somewhat of the other and obligating both to a joint obligation of their com- bined resources in the interest of the patient. ' ' 6. He states that in his opinion the Kolmer complement fixation test (quantitative) is one of the most valuable laboratory pro- cedures available, ' ' but it must be said that this applies only when the method is used as described without distortion of its principles or evasion or omission of its essential minutiae. "7. So great is the practical specificity of precipitation tests under })roperly controlled conditions, that they may be accepted as valuable additions to the serological study of syphilis, and as useful, if not essential, adjuncts to the complement fixation test. "8. While both the complement fixation and the precipitation reaction are biologically nonspecific, they possess, nevertheless, an extraordinary degree of practical specificity when properly per- formed under carefully controlled technical conditions, so much so, that positive reactions are consistently encountered in only one disease other than syphilis, namely yaws. ♦Kikluffe: Am. J. Clin. Path. 3: 61, 1933. 432 IMMUNOLOGY "9. There is no serological procedure at present available, nor is it probable that one will ever be devised, with which a false negative reaction may not be obtained. "10. The simultaneous use of both complement fixation and precipitation tests on every serum should be practiced routinely." This paper by Kilduffe (1933) as well as the reports of Denison and McDonald (1933), Heathman and Higginbotham (1932) and Nigg and Larsen (1928), previously mentioned, as well as many others indicate that there is a growing appreciation of the value of the Kolmer-Wassennann complement fixation technique and of tlie need for a standardized procedure that will undoubtedly in- clude some form of flocculation test a.s a check on the former. The Provocative Wassermann Reaction. — The Effect of Treatment on the Reagin Content of the Blood. An excellent discussion of the provocative Wassermann reaction is given in a paper by Belding (1929). He says that Gennerich (1910) and Milian (1910) reported, almost simultaneously, that in a certain percentage of syphilitics with negative blood Wassermanns, the complement fixation becomes positive following a single injection of arsphenamine. Others have reported various drugs as well as foreign protein as apparently producing provocative reactions. The average dose of arsphenamine recommended is apparently 0.3 Gm. Belding says that a few investigators believe that the phenom- enon called a ''provocative Wassermann reaction" is a myth. They explain the positive results as due either to errors in laboratory technique or to a coincidence in the occurrence of reagin follow- ing the injection of a drug or foreign protein. While Belding con- cludes that the majority of provocative reactions reported in the literature may be "ascribed to technical error" he reports that 3.3 per cent of 338 patients under treatment for syphilis .showed an increase in reagin content of the blood following the first treatment. He regards the test as of doubtful value because of the many possi- bilities of error. Wassermann-Fast Cases. — An excellent discussion of Wasser- mann-fast syphilis is given in a paper by Tobias (1928). He says that the term includes both those cases that are apparently refrac- tory to treatment as indicated by the persistence of symptoms and a positive Wassermann, and second, cases that become, after adequate treatment, free of all symptoms except a positive blood Wasser- COMPLEMENT FIXATION IN SYPHILIS 433 inann. His paper is devoted to a discussion of the latter group. He reports that 48 or 13 per cent of 364 consecutive cases of syphilis of all types appeared to be Wassermann-fast. Of this series 11 per cent were of tertiary or late syphilis, 15 per cent latent, 30 per cent neurosyphilis, and 40 per cent congenital. He regards it as inadvisable to consider a case Wassermann-fa.st unless the complement fixation test is positive after four or more courses of treatment. In his opinion, these are all cases of incompletely cured syphilis. Whether such a conclusion is correct is a disputed point among clinicians. Effect of Malarial and Diathermy Treatment. — Nicole and Fitz- gerald (1931) report on a series of malarially treated cases of general paralysis. Before treatment was instituted, 100 per cent liad a positive spinal fluid and 89 per cent a positive blood. After treatment there were 66.7 per cent whose spinal fluids and 70 per cent whose blood sera showed the presence of syphilitic reagin as determined by complement fixation. Epstein and Paul (1933) report upon a series of cases of neuro- syphilis treated by means of diathermy. They state that the sero- logic changes were not striking considering the group as a whole. An excellent short review of the use of diathermy in the treat- ment of syphilis is given by Schamberg and Butterworth (1932) and O'Leary and others (1940). They report some cases showing definite serological improvement, although clinical improvement occurred in others without demonstrable blood changes. Immunity to Syphilis. — ^The subject of immunity in syphilis is well reviewed by Chesney (1927), Zinsser, Enders and Fother- gill (1939) and others to whom they refer. The following brief summary of present concepts may be of interest to the student : 1. Syphilis seems to be a purely human disease that may be transmitted artificially to monkeys and rabbits. Zinsser (1939) says that the reported transmission of syphilis to the Ihuna is not borne out by subsequent investigation. 2. There is no evidence of natural, individual or racial im- munit^^ 3. In acquired syphilis, reinfection is possible during the in- cubation period and most of the primary stage of the disease. A refractory stage develops which, all agree, persists during the secondary stage and in many cases much longer. 434 IMMUNOLOGY 4. Neisser maintains that the refractory stage or immunity to reinoculation is evidence of an existing infection, but Chesney (1927) concludes that a definite immunity persists after the body is free of infectious agents. 5. Clinical evidence indicates that reinfection is rarely ob- served. This question is also discussed by Cannon (1933) in a report of a case of reinfection. 6. A number of syphilologists hold that antibodies do not seem to play a role in acquired immunity to Treponema pallidum. Two mechanisms are suggested by Neisser. One of these is a condition of cellular indifference to the presence of the spirochetes. This phenomenon he has named ''anergy." The second is a state of allergy or hypersensitivity that may function in the body 's defense. 7. There is some controversy over the role of phagocytosis in immunity to Treponema pallidum. Chesney (1927) cites the in- vestigations of both Ehrmann and Levaditi who report having observed phagocytosis of Treponema pallidum in stained sections. Both Zinsser (1939) and Chesney (1927) suggest that perhaps the reticulo-endothelial system may play a role in acquired im- munity in syphilis. Nonspecific Wassermann Reactions. — It is generally admitted that false positive Wassermann reactions may occur in such con- ditions as acute infectious mononucleosis, malaria, leprosy and perhaps a few other diseases. Landsteiner and Van der Scheer (1927) say that it is known that rabbits infected with trypanosomes develop positive Wassermann reactions. They report that similar positive results were obtained by injecting dead trypanosomes into rabbits. Kemp, Fitzgerald and Shepard (1940) iiave summarized the literature bearing upon the occurrence of positive serological tests for syphilis in animals other than man. It is evident that positive serological tests, especially flocculation tests, are of frequent occur- rence in many species of animals. Their blood contains a normal biologic reagin. Whether genuine nonspecific reactions occur in man has been the object of a recent survey by the United States Public Health Service. Eagle (1941) summarizes the results of this study in- volving 40,545 initial specimens taken from as many college stu- dents representing twenty-five schools. All specimens giving posi- COMPLEMENi' FiXATION fX SYPHILIS 435 tive reactions were re-checked. The incidence of positive tests in these students, all of whom were nonsyphilitie, as judged by history and physical examination, was approximately 1:1,125. As Eagle says, at first sight, this incidence appears disturbingly high. However, when the results are analyzed in the light of the incidence of known .syphilis in the respective campuses he found a rather liigh correlation between the number of known cases of syphilis and the number of apparent false positive reactors. When these facts are taken into consideration a revised estimate would suggest that the number of biologic false positives was not over 1 in 4,000 students. It should be remembered that these data have been obtained only for the age group of college students. Our results (Sherwood, Bond and Canuteson, 1941) on 1,018 stu- dents conformed quite closely to the results obtained for the whole group. Kahn (1941) i-oports tiiat the noi-mal biologic reagin can be differentiated from .syphilitic reagin by a new flocculation tech- nique which he calls the V erification test. He finds that animal and human sera that give false positive reaction will sliow .stronger reac- tions at 1° C. than at 37° C. whereas syphilitic sera give weaker or negative reaction.s at 1° C. and stronger reactions at 37° C. This test will be described briefly in the next chapter. Mechanism of the Wassermann Reaction, — The mechanism of the Wassermann reaction is discussed extensively by WelLs (1929), Zinsser (1931), lOagle (1929, 1930, 1931) and others. The exact origin and nature of syphilitic reagin is at present unknown. While it has the properties of an antibody, it has been generally regarded as not specific for Treponema pallidum as Wa.s.sermann originally thought, but consistently sensitizes certain lipoids when they are properly dispersed in saline. Eagle and Hogan (1940), however, regard the reagin as specific for a lipoid common to both animal tissue and Treponema pallidum. According to Wells (1929) Sachs suggests that perhaps infection with Treponema pallidum cau.ses the liberation or formation in the l)ody of li})o-proteins for which the tissues produce an antibody which we call reagin. He conceives of the lipoid or liapten portion as present in the tissues of many animals and that reagin will sensitize such a hapten anti- gen in vitro just as the haptens previously discussed will react in vitro with homologous immune serum. 436 IMMUNOLOGY The mechanism of complement fixation may be discussed with reference to plain alcoholic and cholesterolized antigens, respec- tively. When one adds a plain alcoholic extract of heart muscle to saline the lipoids are dispersed in the saline and to some extent peptized by it. Those finely dispersed lipoid particles can adsorb reagin globulin and the resulting complex will bind complement. When a cholesterolized alcoholic extract is added to saline, the cholesterol is dispersed as fine particles more rapidly than the lipoids present. The latter are, however, dispersed and adsorbed by the cholesterol particles. The lipoids form more or less of a sur- face film over the latter. Because of the presence of cliolesterol, tliere is little or no peptization of lipoids by the saline. For this reason the sensitivity of the antigen is increased. The lipoid-coated particles of cholesterol adsorb syphilitic reagin from positive serum and the resulting antigen-reagin complex adsorbs complement in the same way that antigen-antibody complexes are known to adsorb or fix complement. Summary. — In this chapter a large amount of material has been presented in relatively few pages. It is obviously impossible to summarize condensed material of this type adequately. To facilitate a rapid review of the chapter the following conclusions and observations may be suggestive : 1. Syphilis was probably introduced into Europe from the West Indies about 1493. It acquired its name from a Latin poem written by Fracastoro in 1530. 2. The etiological factor is Treponema pallidum discovered by Schaudinn and Hoffmann in 1905. 3. In the primary stage of syphilis, only laboratory methods designed to detect the parasite are of value in diagnosis. 4. In the secondary and later stages of the disease, the labora- tory methods used as an aid in the diagnosis of syphilis are those designed to detect syphilitic reagin in the blood or spinal fluid of the patient. The frequency of occurrence of syphilitic reagin in the various stages of syphilis is discussed. The data given are based upon complement fixation and fiocculation tests. 5. In 1905, Wassermann and Bruck became interested in the bacterial complement fixation test previously discussed. They modified the Bordet-GengoVi technique and undoubtedly improved its efficiency. As soon as it was established that Treponema pal- COMPLEMENT FIXATION IN SYPHILIS 437 Kduni is tlie cause of syphilis, it oeciu-red to Wasserinann that this disease might be diagnosed by bacterial complement fixation. He and his colleagues prepared an antigen by extracting a syph- ilitic fetal liver, rich in spirochetes, with saline. They did this five years before Noguchi taught the scientific world how to cul- tivate Treponema pallidum. They found that the saline extract possessed some antigenic property as measured by complement fixation. In their opinion this complement-fixing property de- pended upon the spirochetes present. Within the next year, Marie and Levaditi (1907) and also Landsteiner, Miiller and Potzl (1907) showed that spirochetes are not the antigenic factors in the Was- sermann antigen but that alcohol-soluble lipoids from normal heart muscle and other tissues are the substances that adsorb syphilitic reagin and thus bind complement. 6. Six stages in the development of our present serological metliods used in the diagnosis of syphilis are next discussed. These include the original Wassermann technique and concepts; the discovery of lipoid antigens ; the discovery that cholesterol increases the sensitivity of lipoid antigens ; the modification of Neymann and Gager ; the modifications of Kolmer and the present stage in whicli a general standardization of the Wassermann based upon extensive experimental, clinical and statistical studies is nearly completed. The flocculation test is becoming an accepted adjunct to the Was- sermann test. 7. The various experimental studies of Kolmer and his col- leagues are summarized and discussed. Likewise the recommen- dations of the Committee on Adherence to Conventional Technique in the Performance of Reliable Serologic Tests for Syphilis. Kilduffe's ten basic principles governing the serological diagnosis of syphilis are also presented. 8. The student is made acquainted with the importance of um- bilical cord Wassermanns and of the meaning of the terms "Was- sermann-f ast " and ''provocative Wassermann." The effect of diathermy and malarial treatment on the blood Wassermann is also mentioned. 9. Reference is made to Chesney's (1927) excellent monograph on Immunity in Syphilis and to a recent paper on reinfection in syphilis. Chesney seems to approve of Neisser's views that "anergy" and "allergy" are important factors in the body's k 438 IMMUNOLOGY defense against reinfection. Both Chesney (1927) and Zinsser, Enders and Fothergill (1939) state that the retieulo-endothelial system may also be an important defensive mechanism, 10. A discussion of nonspecific Wassermann reactions is given. References Belding, D. L.: The Wassermann Test, Am. J. Syph. 4: 541, 1929. Cannon, A. B.: Keinfection in S}T)hilis, Am. J. Syph. 17: 459, 1933. Chesney, A. M. : Immunity in Syphilis, Baltimore, 1927, Williams & Wilkins Co. Citron, J. B.: Immunity — Methods of Diagnosis and Therapy and Their Practical Application, Philadelphia, 1912, The Blakiston Co. Citron, J. B.: Die Technik der Bordet-Gengouschen Komplement bindungs — Methode in ihrer Verwendung zur Serodiagnostik der Infektions- krankheiten speziell der Syphilis, sowie zur Eiweissdifferenzierung. Handbuch der Technik und Methodik der Immunitat., Kraus and Levaditi 2: 1076, 1909. Craig, C. F.: A Comparison of the Practical Value of the Wassermann and Kahn Tests in the Diagnosis of Syphilis in the Military Service, Am. J. Syph. 13: 206, 1929. Denison, G. A., and McDonald, Evelvn G.: Serodiagnosis of Syphilis, Am. J. Syph. 17: 90, 1933. Eagle, H.: The Mechanism of Complement Fixation, .T. G«n. Physiol. 12: 825, 1929. Study 1. Mechanism of Flocculation Reaction, J. Exper. Med. 52: 717, 1930. Study 2. Physical Basis of the Wassermann Reaction, J. Exper. Med. 52: 739, 1930. Study 4. A More Sensitive Antigen for Use in the Wassermann Reac- tion, J. Exper. Med. 53: 605, 1931. Study 5. The Cause of Greater Sensitivity of the Ice Box Wassermann; Zone Phenomenon in Complement Fixation, J. Exper. Med. 53: 615, 1931. An Explanation of the Mechanism of the Wassermann and Precipitation Tests for Syphilis, Bull. Johns Hopkins Hosp. 47: 292, 1930. Eagle, H.: Specific Agglutination and Precipitation. 1. The Mechanism of the Reactions, J. Immunol. 18: 393, 1930. Eagle, H.: A Method for the Titration of Complement, J. Gen. Physiol. 12: 821, 1929. Eagle, H., and Brewer, G.: Mechanism of Hemolysis by Complement, J. Gen. Physiol. 12: 845, 1929. Eagle, H.: Studies in the Serology of Syphilis, VI. J. Exper. Med. 55: 667, 1932. Eagle, H. : On the Specificity of Serologic Tests for Syphilis as Determined bv 40,545 Tests in a College-Student Population, Am. J. Syph., Gonor. & Ven. Dis. 25: 7, 1941. Eagle, H., and Hogan, R. B.r On the Presence in Syphilitic Serum of Antibodies to Spirochetes, Their Relation to So-Called Wassermann Reagin and Their Significance for the Serodiagnosis of Syphilis, J. Exper. Med. 71: 215, 1940. Epstein, N. N., and Paul, S. B.r The Treatment of S^-philis With Hyper- pyrexia Produced by Diathermy, Am. J. Syph. 17: 72, 1933. Gilbert, Ruth: Standardization of the Complement Fixation Test in Syphi- lis, Am. J. Syph. 17: 238, 1933. COMl'LKMEXT FIXATION" IN SYl'IIILLS 439 Heathnian, Lucy S., and Higf^iubotliam, MarfjartU: A Study of the Kolmer, Kahn and Kline Tests Witli Spinal Fluids, J. Lab. & Clin. Med. 18: 1287, 1933. Jones, H. W., Ratlimell, T. K., and Wagner, C. W. : The Transmission of Syphilis by Blood Transfusion, Am. J. Svi)h. & Neurol. 19: 30-39, 1935. Kahn, R. L., McDermott, E. B., and Marcus, S.: Effect of Temperature on Kahn Reactions. I. With Serologically Positive Sera of Lower Animals, Am. J. Syph., Gonor. & Ven. Dis.'25: 1.51, 1941. II. With Serologically Positiye Sera of Human Syphilis, Ibid. 25: 1.57, 1941. III. With Serologically Positive Sera in the Absence of Syphilis, Ibid. 25: 162, 1941. IV. With Serologically Negative Sera in the Absence of Syphilis, Ibid. 25: 173, 1941. Kemp, J. E., Fitzgerald, E. M., and Shepard, M.: The Occurrence of Positive Serologic Tests for Syphilis in Animals Other Than Man, Am. J. Syph., Gonor. & Ven. Dis. 24: 537, 1940. Kilduffe, R. A.: Present Status of Serological Diagnosis of Syphilis, with Special Reference to Basic Principles, Am. J. Clin. Path. 3: 61, 1933. Kolmer, J. A.: The Specificity, Sensitiveness and Practical Value of the Kolmer- Wassermann Reaction, Am. J. Syph. 13: 248, 1929. Kolmer, J. A., and Schamberg, J. F.r The Influence of Reticuloendothelial "Blockade" and Splenectomy Upon Experimental Trypanosomiasis and Syphilis and the Chemotherapeutic Properties of Arsphenamine and Neoarsphenaniine, Am. J. S^^jh. 17: 176, 1933. Kolmer, J. A.: Studies in the Standardization of the Wassermann Reac- tion. I. Introduction. The Demand for and Requirements of a Standardized Wassermann Reaction and Method of Investigation, Am. J. Syph. 3: 1, 1919. Kolmer, J. A., and Brown, C. P.: Studies in the Standardization of the Wassermann Reaction. II. The Preparation of Glassware and Saline Solution for the Wassermann Reaction. The Influence of Acids, Alkali, and Other Factors, Am. J. Syph. 3: 8, 1919. Kolmer, J. A., and Brown, C. P.: Studies in the Standardization of the Wassermann Reaction. III. The Red Corpuscle Suspension for the Wassermann Reaction. The Preservation of Red Blood Cells, Am. J. Syph. 3: 169, 1919. Kolmer, J. A., Matsunami, T., and Trist, j\f. E.: Studies in the Standard- ization of the Wassermann Reaction. IV. A General Study of the Complements of Various Animals with Special Reference to Human and Guinea Pig Complements and Methods of Collection, Am. J. Syph. 3: 407, 1919. Kolmer, J. A., Matsunami, T., and Trist, M. E. ; Studies in the Standardi- zation of the Wassermann Reaction. A'. The Preservation of Comple- ment Serum, Am. J. Syph. 3: 513, 1919. Kolmer, J. A., and Flick, A. M. : Studies in the Standardization of the Wassermann Reaction VI. Human Versus Guinea Pig Complement in the Serum Diagnosis of Syphilis with Special Reference to Meth- ods Employing Human Complement, Am. J. S_yph. 3: 541, 1919. Kolmer, J. A., Trist, M. E., and Flick, A. M.: Studies in the Standardi- zation of the Wassermann Reaction. VII. A Study of the Natural Thermolabile and Thermostable Hemolysins and Hemagglutinins in Human Serum in Relation to the Wassermann Reaction, Am. J. Syph. 4: 111, 1920. 440 IMMUNOLOGY Kolmer, J, A., and Eule, A.: Studies in the Standardization of the Was- sermann Eeaction, VIII. The Influence of Natural Antisheep Hemolysin in Human Sera Upon the Wassermann Eeaction, Am. J. Syph. 4: 135, 1920. Kolmer, J. A., Matsunami, T., and Eule, A.: Studies in the Standardiza- tion of the Wassermann Eeaction. IX. A Comparative Study of Complement Fixation in Syphilis with Antihuman, Antichicken, and Antisheep Hemolytic Systems, Am. J. Syph. 4: 278, 1920. Kolmer, J. A., and Eule, A.: Studies in the Standardization of the Wasser- mann Eeaction. X. A Study of Methods for the Preparation and Preservation of Hemolysins, Am. J. Syph. 4: 484, 1920. Kolmer, J. A., Matsunami, T., and Eule, A.: Studies in the Standardiza- tion of the Wassermann Eeaction. XL A Study of Methods for Ad- justing the Hemolytic System with Special Eeference to the Titra- tion of Complement, Am. J. Syph. 4:' 518, 1920. Kolmer, J. A., Eule, A., and Trist, M.: Studies in the Standardization of the Wassermann Eeaction. XII. The Titration of Hemolysin and Sensitized Versus Plain Eed Blood Corpuscles in Complement-fixa- tion Tests, Am. J. Sj^pt. 4: 616, 1920. Kolmer, J. A., Eule, A., and Trist, M.: Studies in the Standardization of the Wassermann Eeaction. XIII. The Influence of Heating Serum Upon Complement-fixation in Syphilis, Am. J. Syph. 4: 641, 1920. Kolmer, J. A., and Eule, A.: Studies in the Standardization of the Wasser- mann Eeaction. XIV. The Influence of Temperature and Duration of Primary Incubation Upon the Hemolytic Activity of Complement, Am. J. Syph. 4: 675, 1920. Kolmer, J. A., and Trist, M.: Studies in the Standardization of the Was- sermann Eeaction. XV. The Influence of Temperature and Duration of Primary Incubation Upon the Anticomplementary Activity of Organ Extracts (Antigens) and Sera, Am. J. Syph. 5: 30, 1921. Kolmer, J. A., Eule, A., and Yagle, E.: Studies in the Standardization of the Wassermann Eeaction, XVI. The Influence of Temperature and Duration of Primary Incubation Upon the Velocity and Amount of Complement Fixation in Syphilis with Different Organ Extracts (Antigens), Am. J. Syph. 5: 44, 1921. Kolmer, J. A., Matsunami, T., and Trist, M.: Studies in the Standardiza- tion of the Wassermann Eeaction. XVII. A Comparative Study of Methods for Conducting the Primary Incubation for Complement Fixation in Syphilis with the Technic Eecommended for a Standard- ized Test, Am. J. Syph. 5: 63, 1921. Kolmer, J. A,: Studies in the Standardization of the Wassermann Eeac- tion. XVIII. The Influence of the Order of Mixing Serum, Antigen and Complement and Total Volume Upon Complement-fixation Eeac- tions in Syphilis, Am. J. Syph. 5: 290, 1921. Kolmer, J. A.: Studies in the Standardization of the Wassermann Eeac- tion. XIX. A Study of Factors Eelating to the Serum and Serum Control Tube, Am. J. Syph. 5: 439, 1921. Kolmer, J. A., Yagle, E., and Eule, A.: Studies in the Standardization of the Wassermann Eeaction. XX. A Study of Factors Influencing the Amount of Hemolysin Employed in Complement-fixation Tests, Am. J. Syph. 5: 451, 1921. Kolmer, J. A.: Studies in the Standardization of the Wassermann Eeac- tion. XXI. A Study of Methods for Conducting the Secondary In- cul)ation and Time of Eeading of Complement Fixation Eeactions in Syphilis, Am. J. Syph. 5: 614, 1921. COMPLEMENT FIXATION IN SYPHILIS 441 Kolmer, J. A.: Studies in the Standardization of the Wasscrmann Ke- action. XXII. A Method for Preventing the Influence of Natural Antisheep Hemolysin Upon Complement-fixation Reactions After the Secondary Incubation, Am. J. Syph. 5; 628, 1921. Kolmer, J. A.: Studies in the Standardization of the Wassermann Reaction. XXIII. A Study of Methods for Conducting Quantitative Comple- ment Fixation Tests and of Reading Scales for Recording Reactions, Am. J. Syph. 6: 6-i, 1922. Kolmer, J. A., and Trist, M. E.: Studies in the Standardization of the Wassermann Reaction. XXIV. A Comparative Study of Tissue Ex- tracts (Antigens) and Methods of Preparation, Am. J. Sj'ph. 6: 289, 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXV. A Superior Antigen for Complement-Fixation Tests in Svphilis. (A Cholesterolized and Lecithinized Alcoholic Extract of Heart Muscle.) Am. J. Syph. 6: 74, 1922. Kolmer, J. A., and Yagle, E. M.: Studies in the Standardization of the Wassermann Reaction. XXVI. A Study of Methods for the Preserva- tion of Tissue Extracts (Antigens), Am. J. Syph. 6: 319, 1922. Kolmer, J. A., and Trist, M. E.: Studies in the Standardization of the Wassermann Reaction. XXVII. A Study of Factors Influencing the Titration of Antigen, Am. J. Syph. 6: 461, 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXVIII. A Study of Factors Influencing the Amount of Antigen to Employ in Complement-Fixation Tests in Syphilis, Am. J. Syph. 6: 481, 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXIX. Methods for Establishing a Uniform and Standard- ized Unit of Antigen, Am. J. Syph. 6: 651, 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXX. A New Complement-Fixation Test for Syphilis Based Upon the Results of Studies in the Standardization of Technic, Am. J. Syph. 6: 82, 2 pi., 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXXI. The New Complement-Fixation Test for Syphilis Conducted with Antihuman, Antichicken and Antiox Hemolytic Systems, Am. J. Syph. 6: 667, 1922. Kolmer, J. A.: Studies in the Standardization of the Wassermann Re- action. XXXII. A Comparative Studv of the New Complement-Fixa- tion Test for Syphilis with Other Methods, Am. J. Syph. 6: 680, 1922. Kolmer, J. A.: Quantitative Complement-Fixation Test in Syphilis, Am. J. Syph. 6: 496, 1922. Kolmer, J. A., and Kast, C: Experimental SjTphilis of Mice, Rats, and Guinea Pigs, Am. J. Syph. 16: 535, 1932. Kline, B. S.: Mechanism of the Microscopic Slide Precipitation Tests for Syphilis, J. Lab. & Clin. Med. 16: 1202, 1931. Landsteiner, K., Miiller, R., and Potzl, O.: Zur Fragc dcr Koniplement- binduugsreaktion bei Syphilis, Wien. klin. Wehnschr. 20: 1565, 1907. Landsteiner, K., and Stankovic, R.: Ueber die Bindung von Komplement durch suspendierte und kolloid geloste Substanzen, Centralbl. f. Bakteriol. 42: 353, 1906. Landsteiner, K., and van der Scheer, J.: Experiments on the Production of Wassermann Reagins by Means of Trypanosomes, J. Exper. Med. 45: 465, 1927. Levinson, A.: Cerebrospinal Fluid in Health and Disease, St. Louis, 1919, The C. V. Mosby Co. k 442 IMMUNOLOGY L'Esperance, E. S., and Coca, A. F.: Further Experiences with the Isolated Organ Lipoids as Antigen in the Wassermann Test, J. Immunol. 1: 129, 1916. Marie, A., and Levaditi, C: Les anticorps syphilitiques dans de liquide cephalorachidien des paralytiques gen«raux et des abetiques, Ann. de I'Inst, Pasteur 21: 138,' 1907. Cited by Citron in Handbuch der Tek. u. Meth. der Immunitats., Kraus und Levaditi, ]909. Zweiter Band, p. 1093. Also Cited by Gilbert in Newer Knowledge of Bacteriology and Immunology, 1928, p. 838. Mitchell: Third Generation Syphilis, Am. J. 8yph. 27: 492, 1933. Nicole, J. E., and Fitzgerald, E. G. : Serologic Results in IMalarially Treated General Paralysis, Am. .L S>Tph. 15: 496, 3931. Neymann, C. A., and Gagor, L. T. : A New Method for Making Wasser- mann Antigens from Normal Heart Tissue, J. Immunol. 2: .'573, 1917. Nigg, Clara, and Larsen, N. P.: Some Observations on the Wassermann and Kahn Reactions, J. Lab. & Clin. Med. 13: 843, 1928. Noguchi, H.: The Spirochetes, Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press, p. 4.52. Noguchi, H., and Bronfenbrenner, J.: Biochemical Studies on So-called Syphilis Antigen, J. Exper. Med. 13: 43, 1911. O'Leary, P. A., Bruetsch, W. L., Ebaugh, F. G., SiTupson, AV. M., Solomon, H. C, Warren, S. L., Vanderlelir, R. A., Usilton, L. J., and SoUius, I. v.: Malaria and Artificial Fever in the Treatment of Paresis, J. A. M. A. 115: 677, 1940. Olscn, R. E., and Weller, C. V.: Method for Staining Spirochetes and Molds With Aniline Dyes, Am. .1. Syph. 16: 113, 1932. Roby, .1., and Lembcke, P. A.: The Meaning and Reliability of Umbilical Cord Wassermann Tests, Am. J. Syph. 17: 473, 1933. Sams, Wilev M.: Third Generation Svphilis, Am. J. Svph. 17: 492-498, 1933. Schamberg, ,T. F., and Butterworth, T.: Diathermy in the Treatment of General Paralvsis and in Wassermann-Fast Syphilis, Am. J. Syph. 16: 519, 1932.' Schaudinn, F., and Hoffmann, E.: Arbeiten aus den Kaiserlichcn Gesund- heitsamtc. Epoch-Making Contributions to the Study of Syphilis: Preliminary Report on the Occurrence of Spirochetes in S^-philitic Disease Products and in I'apillnmata, Am. .1. Syph. 2: .'363, 1918. Sherwood, N. P., Bond, G. C, and Clark, H. F.: Results Obtained with Kolmer, Kahn, Kline, and Eagle Tests on Animal Sera, Am. J. Syph., Conor. & Ven. Dis. 25: 93, 1941. Sherwood, N. P., Bond, G. C, and Canuteson, R. I,: On the Possible Presence of a Reagin-Iike Factor iii Normal Human Serum, Am. J. Syph., Conor. & Yen. Dis. 25: 179, 1941. Supplement No. 9: The Scrodiagnosis of Svphilis, U. S. P. H. Service, 1938, p. 207. Supplement No. 11: Technics of Serodiagnostic Tests for Syphilis, U. S. P. H. Service, 1940, p. 56. Tobias, N.: Wassermann-Fast Syphilis, Am. J. Syph. 12: 396, 1928. Tschernog-ubow, N. A. : Cited by Noguchi, H. : Serum Diagnosis of Syphilis, Philadelphia, 1911, J. B. Lippincott Co., p. 226. Wassermann, Neisser, Bruck, and Schucht (1906): Cited by Citron, 1907. Wassermann and Plant (1906): Cited by Citron, 1907. Wells, H. Gideon: The Chemical Aspects of Immunity, New York, 1929, The Chemical Catalog Co. The Wassermann Reaction, p. 191. COMPLEMENT FIXATION IN SYPHILIS 443 Williams, H. U., Kice, J. P., and Lacayo, J. E.: The American Origin of Syphilis With Citations From Early Spanish Authors Collected by Dr. M. Y. Robledo, Arch. Dermat. &"Syph. 16: 683, 1927. Zinsser, Hans, Enders, J. F., and Fothergill, L. D.: Immunity, New York, 1939, The Macmillan Co. Supplementary References Lynch, F. W., Boynton, Ruth E., and Kimball, Anne C: False Positive Serological Reactions for Syphilis Due to Smallpox Vaccinations (Vaccinia), J. A. M. A. 117: 591, 1941. Moore, J. E., Eagle, H., and Mohr, C. F.: Biologic False Positive Serologic Tests for Syphilis, .T. A. M. A. 115: 1602, 1940. Moore, J. E., and Eagle, H. : The Quantitative Serologic Test for Syphilis; Its Variability, Usefulness in Routine Diagnosis, and Possible Sig- flcance; A Study of 1665 Cases, Ann. Int. Med. 14: 1802, 1941. CHAPTER XXIV PRECIPITIN TESTS IN SYPHILIS Introduction. — Many precipitation tests applicable to syphilitic seniin have been proposed since the observation by INtichaelis (1907) of a precipitate when heated syphilitic serum and lipoidal tissue extracts were mixed. Tliis method lias l)een described by various authors as a precipitation, floccvilation or a clarification reaction. In the serum of syphilitics thei-e is usually present an antibodj^-like substance, reagin, which is directly or indirectly responsible for chanoes in the dispersion of the lipoidal antigens. These aggregates may vary in size from small microscopic to large macroscopic clumps. In normal human serum reagiu is rarely present, hence the ''reagin-lipoid" complex cannot be formed. Thus there is no increase in the size of the dispersed particles of lipoidal suspension. The precipitation tests, like complement fixation methods, are a qualitative or a quantitative estimation of "reagin" resulting from the infection and not a test for the pres- ence of the causative oi-ganism. Historical Development. — The successful development of the precipitation methods began with the work of Sachs and Georgi, and Meinicke, hence their practical application has been within the last twenty years. It is of historical interest to note that the first attempt to apply precipitation methods followed the Was- sermann, Neisser and Bruck (1906) complement fixation method by one year. Michaelis (1907) using the Wassermann antigen originated this early test, believing he was using a specific extract of spirochetes and securing a specific jn-ecipitin test for the spirochete. This was followed by Klau.sner's (1908) method of precipitation with distilled water and the taurocholate suspensions. These early precipitation tests were macroscopic test tube reac- tions. Introduction of Cholesterol. — Jacobstahl made a long step forward by introducing an alcoholic extraction of syphilitic liver as an antigen in place of the lipoid-poor saline extracts. His test was also the first microscopic reaction. This test depended upon 444 PRECIPITIN TESTS IN SYPHILIS 445 the i'orniation of an ultramieroscopic precipitate. While the eoni- })lieated procedure involved in the Jacobstahl method was im- practical, it stimulated extensive research in this field. Bruck and Hidaka (1911) modified this method so that the precipitate was microscopic rather than ultramieroscopic in character. Mein- icke (1917, 1918, 1920) followed with several modifications. In the same period Sachs and Oeorgi (1920) suggested cholesterol as an added substance to increase the sensitivity of the tissue extracts. Although the Sachs-Georgi reaction has been generally discarded, their principle of adding cholesterol or a similar sensitizer to rein- force the tissue extract has made the practical development of the flocculation test possible. I\Iany tests have been and are being pro- posed with the objective of increasing specificity or sensitivity or botli, but thus far they are modifications of this original method. Simplicity of Flocculation Method. — The simplicity of the floc- culation methods contrasts sharply with many of the proposed com- plement fixation procedures. The methods combine high sensitivity and specificity, rapid results and stable standardized reagents at a low cost per test. It further permits the use of several methods as a check on each other with economy of time. Status of Flocculation Tests. — The value of the precipitation nu'thod for tlie detection of sypliilitic reagin is still a contro- versial subject. However, the widespread acceptance of such tests as the Kahn, Meinicke, Kline and others is an expression of approval by most syphilologists. From the present state of confusion generally standardized precipitation methods will de- velop which have adequate confirmation and of a specificity and sensitivity consistent with clinical practice. While many of the modifications are commendable, the process of standardizing has ]u-ogressed slowly, due to the many technical modifications in use. It is difficult to pick an ideal test witli such an assortment from which to choose when each offers one or more characteristic ad- vantages. Principle of Flocculation Tests. — The basic mechanism of all precipitation tests is essentially the same. They differ in points of technique, sensitivity, and interpretation. A positive reaction is characterized hy the formation of aggregates which result from the combination of the lipoid suspension and "reagin" found in syphilitic serum but not present in nonsyphilitic serum. The 446 IMMUNOLOGY "antigen'' used to detect syphilitic reagin consists of a disper- sion of lipoidal substances in an aqueous suspending fluid (con- tinuous phase). The reaction between the reagin in the serum and the particle of the "antigen" (the discontinuous phase) oc- curs at the surface interface of the particle. There is deposited a film of "antibody globulin" around the particle with a corre- sponding alteration in solubility of the interfacial film through an "antigen-antibody complex" formation with behavior like a denatured protein. This results in a reduction of the potential difference between the particles,, when electrolytes are present in the aqueous phase. The mixture is agitated in some mechanical manner so that tlie particles come in contact with one another. These particles arc held together, after contact is once made, by their coliesive force in the absence of a high potential difference. A high potential difference Avonld cause the charged particles to repel each other. In the negative reaction no clumping of particles occurs. There are no changes in the P.I), and the like charged particles repel one another with no resulting clianges in dispersion. The agglutinated particles may vary from a finely granular microscopic precipitation to a dense macroscopic precipitate, de- pending upon the composition of the tissue extract, the reagin titer of the syphilitic serum and the technique of the test. Thus precipitation tests may vary from a microscopic slide test to a macroscopic test tube reaction. Role of Ingredients. — The sensitivity and specificity of the pre- cipitation reaction is largely determined by the following: (1) Fraction of total lipoids extracted from heart muscle. (2) Synergistic (or sensitizing) substances added to tissue extract. (3) Electrolyte concentration. (4) Lipoidal concentration. (5) Lipoidal-cholesterol ratio. (6) Alcohol- water ratio. With the data available, it is impossible to evaluate the impor- tance of each, nor can the relative interdependence of all or several be calculated Avith success. In order to simplify an ele- PRF.CIl'ITIX TKSTS IxV SYPHILIS 447 mentary presentation of proeipilalion lesls, the various ingredi- ents and ratios of ingredients will be discussed as separate topics. Antigens. — The term "antigen" is a misnomer applied to the concentrated lipoidal tissue extracts used as a reagent in this test. However, in keeping with usage, the word antigen has been used in this chapter to designate tissue extracts. Tiie antigen used in flocculation tests is composed of a portion of tlie lipoids of normal, rapidly dried, fresh heart muscle. Beef heart, freed of connective and fatty tissue, is generally used for this purpose. Any chemical compound in which lipoids are soluble and proteins are insoluble can be used as an extracting fluid. Several extracting fluids may be used as the total lipoid content is not soluble in any one solvent. Further, solvents may be chosen to remove undesirable lipoidal fractions (as ether in preparation of Kahn antigen) and the dried residue extracted with other solvents for the '^antigenic lipoids." In general, the ether-soluble lipoids are characterized by their in- stability in water dispersions, while the ether-insoluble, alcohol- soluble lipoids give relatively stable suspensions. The extracted lipoids may be separated into acetone-soluble and acetone-insoluble fractions. The acetone-insoluble fractions of the alcohol-soluble portion have given very satisfactory results in microscopic tests. The character of the extract can again be modified by chang- ing the temperature of the solvents during extractions. Each of the fractions of lipoids appears to differ slightly in chemical com- position and activity but they grade into one another like colors of the spectrum. Hence any one fraction represents a series of lipoids. Chemical analysis has failed to reveal the structural formula or even the active compounds. The extractives appear as a light yellow to a dark brown wax.v substance composed of lipoids of the cholesterol and lecithin series along with a larger undetermined fraction known as tissue extractives. Synergistic Substances. — Substances added to the alcoholic lipoidal extract which increase the sensitivity of the finished antigen are known as sj'iiergizing (or sensitizing) agents. These substances are compounds of known chemical purity, organic in nature and soluble in fat solvents but insoluble in water. The most important of this group is cholesterol. Cholesterol in alco- holic solution is precipitated by addition of water, giving crystals 448 IMMUNOLOGY of uniform dispersion for any set of standard conditions. The size of the cholesterol particles may be controlled by the relative volume of alcohol, concentration of cholesterol in the alcohol and the amount of water used as a dispersing fluid. Solution of lipoids gives suspensions of extreme variability, even when mixed under apparently identical conditions. The addition of cholesterol ap- pears to stabilize the dispersion. Production of antigen particles of identical size under laboratory conditions is of great importance. Role of Cholesterol. — Eagle (1930) and others believe the role of cholesterol is purely physical in that it determines the size of the dispersed particle. The cholesterol forms the core and the antigenic lipoids cover it witli a thin film. As the size of the particles increases, the total surface area is decreased, requiring less reagin to film the particle. This results in less reagin per unit volume of serum necessary to produce aggregates and auto- matically increases the sensitivity of the test according to Eagle. Other substances which may be substituted for cholesterol in pre- cipitation tests are: solid sterols, lecithins, solid alcohols, balsam of Tolu, salicylic and benzoic acid. In some of the new microscopic tests, the synergistic substance is dispersed before the lipoidal extract is added (Kline, Rosen- thal, etc.). The finely precipitated lipoids are adsorbed on the surface of the cholesterol particles. Suspensions made in this manner have particles of greater consistency in size and behavior. The type of suspension differs from those made ])y precipitating the same amounts of lipoids and cholesterol from the same solution. All of the synergistic substances used are insoluble, forming suspensions in the finished alcoholic-aqueous menstruum used as a reagent. Such suspensions follow the laws of physicochemical flocculation. This nonspecific chemical flocculation imposes limita- tions on the concentration which can be used to synergize (or sensitize) the lipoids. These substances are without antigenic activity when used alone. The exact chemical and physical role of cholesterol or similar substances in the precipitation has not been determined. Water. — Water may be added separately as distilled water or as a solvent of some of the reagents, such as salt solution. The water serves as a dispersing medium for the particles of the anti- PRECIPITIN TESTS IN SYPHILIS 449 i>eii and synergistic substances. It also affects the potential dif- ferences indirectly through its effect on the dissociation of elec- trolytes. Moreover, the reagin-filmed particles change from a liophylic to a liophobic surface interface resulting in precipitation. The water apparently has a physical role. Electrolytes. — The function of electrolytes in the precipitation test is identical with their action in the agglutination reaction. The reagin-lipoid complex acts as a denatured protein. This re- sults in a lowered P.D. between the particles with drastic reduc- tion in their repulsive forces. When such particles come in contact through agitation or other physical means, sticky aggregates are held together by the cohesive force. From this it is evident that the concentration of electrolytes has an important bearing on the sensitivity of the test. Precipita- tion occurs with greater ease as the concentration of the elec- Irolytes is increased. It also becomes more sensitive as the valence of the metallic ion is increased. The various electrolyte concentrations which have been used in precipitation tests are listed in the following table : Kahn 0.90 per cent sodium chloride Kline 0.85 per cent sodium chloride Meinicke 3.00 per cent sodium chloride and 0.01 per cent sodium carbonate Hinton 5.00 per cent sodium chloride and 0.022 per cent salicylic acid Rosenthal 0.40 per cent (only serum electrolytes) Citochol 0.85 to 3.00 per cent sodium chloride Mazzini 1.00 per cent salt concentration (buffered saline) Deissler and Baker (lOSf)) and others favor the increase in the electrolyte concentration as a means of increasing sensitivity and velocity of the reaction. From two to three per cent sodium chloride has given more satisfactory citochol reactions in the laboratories of the above workers and in many other laboratories. Alcohol. — The lipoids are usually dissolved in alcohol or some similar lipoid solvent. The alcohol has no role in the precipitation of the particles. It serves as a solvent for the lipoids until disper- sion is accomplished from the solution by diluting it with water. It is an inert ingredient in the finished antigen since the changes in ionization constants are negligible. 450 IMMUNOLOGY Ratio of Components. — The ratio of concentrations between in- gredients is of great importance. To appreciate this it would seem best to discuss separately the ratio between the various in- gredients. It must be remembered, however, that they are definitely interdependent. Concentration of Lipoids. — Tlie sensitivity of the reaction i.s dependent upon the number of milligrams of lipoids per cubic centimeter of extract. An optimum range is determined by empirical titration, using strongly positive, weakly positive, and negative sera. The preceding statement must be qualified, since it holds only in general for the lipoids wdthin limited concentra- tions. The lipoidal solids obtained by the extraction methods used are crude mixtures of antigenic and nonantigenic lipoids, while the activity of the extract is due only to the antigenic lipoids. Until new methods of separation are devised, an exact quantitative ratio cannot be reached. (1) Fraction of total lipoids extracted from heart muscle Cholesterol-Lipoid Ratio. — From the viewpoint of sensitivity of a newl}^ prepared tissue extract, the cholesterol-lipoid ratio is of still greater importance. As the percentage of cholesterol in ratio to a given concentration of extractives increases, the precipitation value of this complex by human sera, especially syphilitic sera, increases to a maximum. Opinions differ con- cerning the cholesterol-lipoid ratio. Eagle (1931) believes the sensitivity of the antigen can be increased u]) to the cholesterol saturation point and recommends 0.8 per cent cholesterol and 0.6 per cent sitosterol in antigens. Levine (1932, 1933), on the other hand, believes these statements unwarranted. He contends that the cholesterol concentration required for maximum sensi- tivity depends upon the lipoid concentration of the individual antigen. According to the Levine theory the sensitizing activity increases as the cholesterol concentration increases and after reaching a maximum (optimum lipoid-cholesterol ratio), addi- tional cholesterol will reduce the sensitivity of the antigen, fol- lowing the so-called ''colloidal type curve." It has been the experience in this laboratory that excessive cholesterol concentra- tions reduce the specificity of the test. Until more data are ob- tained, an intermediate view between the two extremes appears reasonable. PRECIPITIN TKSTS IX SYPHILIS 451 Alcohol-Water Ratio — Tlie ratio of water to alcoliol is rela- tively unimportant. Sufficient alcohol must be used to carry in a stable solution the lipoidal substance. The Avater-alcohol ratio must be high enough to prevent solution of the lipoids in the finished suspension. After the suspension is prepared, the alco- hol becomes inert so far as activity of the test is concerned. The speed of "shaking" or "'rotation" of the test is important since the velocity of precipitation is chiefly determined by this factor. It is assumed that the increased speed of precipitation is due in part to a more rapid filming of the antigen particles with reagin and partly to the increased number of contacts made between the filmed particles during mixing. The filmed particles have lost their high P.D. and are sticky; hence, once in contact, they tend to precipitate in clumps. The shaking principle w-as introduced into reagin tests by Gaehtgens and Holn and studied in detail by Kahn. According to the latter author, insufficient shaking results in negative reactions from sera low in reagin titer and excessive shaking leads to weak positive reactions in sera con- la ining no reagin. Miscellaneous Factors.- — Factors that also affect the size and shape of the dispersed particles are the speed of mixing, the ratio of masses of lipoids and water, the final alcohol or ether content, order of mixing, concentration of electrolytes, temperature of mixing and the age of the emulsion. In the precipitation test, as in the complement fixation test, the state of dispersion of the antigen is an important consideration. Finished Antigen. — The finished antigen is a suspension of alcohol-soluble substance precipitated in an aqueous medium. According to Eagle the sensitivity increases, within certain limits, with the coarseness of the dispersed particles. Kline has modified this view, believing that sensitivity increases with size and shape only when all other factors of the test are held constant. Kline considers the shape equally important with surface area. However, on final analysis, as the size of the particle increases, the surface area is decreased per unit volume of suspension, which is the same as reducing the amount of antigen in the test. As the sur- face area decreases, the amount of reagin necessary for precipita- tion is correspondingly reduced. 452 IMMUNOLOGY The type of reaction depends upon the kind of antigen prepared. Macroscopic reactions require larger particles than microscopic reactions. The larger particles undergo a pseudoagglutination that is easily converted into larger aggregations in the presence of syphilitic serum. Such larger clumps give an easy end-point in reactions like the Kahn. On the other hand, microscopic reactions must have antigens with finely divided particles. Such reactions are made on a mi- croscopic slide, and the clumps are magnified from eighty to one hundred diameters. Minute clumps in the negative serum give pseudopositive reactions which interfere seriously with the test. Only slight clumping is necessary for a positive reaction with this magnification. Choice of Test. — Numerous tests have l)een ])ublis]ied, and al- though tliey differ in method, the mechanics of each is similar. The choice of a standard flocculation test awaits the accumulation of laboratory and clinical correlation over a long period of time. Until that time the choice of the flocculation test depends chiefly upon personal preference and individual success. Several of the mo.st used of the flocculation tests will be briefly described. Kahn Precipitation Reaction (1940). — The antigen consists of ether-insoluble, alcohol-soluble lipoid fractions of beef heart with the addition of 0.6 per cent cholesterol. The antigen is standard- ized by a series of titrations. Salt Titration. — This represents the minimum amount of 0.90 per cent saline necessary for dispersion of the antigen and that will also permit resolution on addition of 1 c.c. or less of isotonic saline after the emulsion has been shaken for three minutes witli 0.15 c.c. of saline and incubated for fifteen minutes in a 37° C. water bath. This is known as the titer of the antigen and repre- sents the amount of saline solution which must be added to 1 c.c. of lipoid solution. The procedure is best illustrated by the accompanying protocol of an actual titration (Tables XII, XIII). The protocol is modi- fied after Kahn. This gives a heavy white precipitate in each dilution vial. Three tubes containing respectively 0.05, 0.025, and 0.0125 c.c. of anti- gen are prepared for each dilution as in Table XIII. PRECIPITIN TESTS IN SYPHILIS 453 Table XII DISPERSION OF ANTIGEN DISPERSION OF ANTIGEN Number 1 2 3 4 5 Antigen (cholesterinized alcoholic) Salt sol. (0.90 per cent) 1.0 c.c. 0.8 c.c. 1.0 c.c. 1.0 c.c. 1.0 c.c. 0.9 c.c. 1.0 c.c. 1.1 c.c. 1.0 c.c. 1.2 c.c. Lsotonic saline, 0.15 e.c, is added to each tube. The tubes are shaken for three minutes and incubated in a 37° C. water bath for 15 minutes. Salt solution is added after incubation. In this titration, 1 c.c. of antigen plus 1 c.c. of isotonic saline is the titer. It is 1 c.c. of the antigen plus a minimum amount of salt solution giving a precipitate wliich dissolves in an excess of salt solution. St.vndardization of ANTKiEN. — After the salt titer of the antigen luis been found, comparison wilh tlie standai'd antigen is made as shown in Table XIV. Antigens numbers 1 and 2 were freshly prepared antigens. In the comparison, antigen number 1 was almost identical in sen- sitivity and specificity with the standard antigen. Antigen num- ber 2 was unfit for laboratory tests, since it was deficient in sensitivity. Except in the strongly positive serum, the reactions were consistently weaker. In the two very weakly positive sera, negative results were obtained. An antigen, standardized as described, consists of coarse particles which, under conditions of the test, are dissolved by an excess of isotonic saline in the presence of normal serum but, wlien filmed with reagin of syphilitic serum, are insoluble and produce coarser macroscopic aggregates. Inactivation. — The serum is inactivated by heating at 56° C. for thirty minutes. According to Kahn, sera low in reagin titer which give negative or weakly positive reactions before inactiva- tion, give stronger reaction after heating. No satisfactory ex- planation has been offered. Shaking. — After mixing tlie serum and antigen, a three-minute shaking period has been found to give good precipitates with 454 IMMUNOLOGY in (M iH >o q iq o d d 1 in »o q o o d lO 1-1 d lO I-H d lO d q I— 1 1 1 LO -; d q d o "—1 1 lO i^ Cl K 0-5 r-i >o t> O r-l c« lO d d s d 1 ^ lO g fO OJ lO 3 ^• q r-< a lO ^_, d d "a d 1 lO >o E-H fl o i-< 0) q d d f-i 1 lO s r-( lO O r-i iq d d d + LO CJ O LO r-l LO d d d + LO LO C' r-i q d d rH + 1 lO C-4 >o O r-l lO d d d + lO I— 1 q "O >o. d d d + lO O o r-l q d d --5 + 1— 1 ,.c. Alcoholic extract of beef heart 0.10 c.c. Isotonic saline 2.45 c.c. The distilled water is placed in a small cork-finish bottle. Choles- terol solution is slowlj^ added down the neck of the bottle and the mixture is rotated for twenty seconds. After addition of the antigen, the mixture is shaken for one minute, throwing the liquid from bottom to stopper each time. The saline solution is added and the shaking repeated, less vigorously than previously, for one minute. The antigen is "cured" for fifteen minutes in a 37° C. water bath. Technique of Testing. — With the end of the pipette touching the glass, 0.05 to 0.06 c.c. of inactivated serum is placed in the center of the paraffin ring. A drop of antigen containing between 0.0075 and 0.0085 c.c. is pla<'ed on top of the serum. The mix- ture is stirred with a toothpick. After four minutes' rotation, the test is completed. Reading Test. — A microscopic examination, using a lOx ocular and a 16 mm. objective, is made. Distinct agglutination con- sisting of coarse clumps, is a strongly positive reaction. Finer clumps are interpreted as moderately or weakly positive reac- PRECIPITIN TESTS IN SYPHILIS 459 tioiis. Ill this laboratory, very fine clumps have been considered as doubtful reactions and without diagnostic value. Typical microreactions are shown in Figs. 20-23. Fig. 20. Fig-. 21. Fig. 22. Fig. 20. — Photoinicrogiapii of a .strongly positive Kline test. (Dark-flcld illu- mination. ) Fig. 21. — Photomicrograph of a moderately positive Kline test. (Dark-fleld illumination.) Fig. 22. — Photomicrograph of a weakly po.sitivo Kline test. (Dark-field illu- mination.) Fig. 23. — Photomicrograph of a negative Kline test. (Dark-flcld illumina- tion.) The strongly positive reaction (Fig. 20) has large microscopic clumps. As the reagin titer decreases, the clumps become smaller (Figs. 21 and 22). A typical negative reaction (Fig. 23) has a number of needlelike crystals equally distril)uted over the field. 460 IMMUNOLOGY A strongly positive serum usually produces clumps of sufficient size for macroscopic diagnosis. This leads to the omission of the microscopic examination — a practice to be condemned. Weakly positive reactions are easily missed when such a practice is fol- lowed. It is also recommended that a positive and a negative serum control be included in each series of tests (on each side). Anti- gen (0.0070 c.c.) plus saline (0.05 c.c.) is also included in this laboratory as an additional control. In addition to the routine diagnostic test described above, Kline has an elimination test, comparable in purpose to the presumptive test of Kahn. This test differs in the preparation of the antigen. It is as follows : Distilled water 0.85 c.c. Cholesterol 1 per cent ab. alcohol 1.0 c.c. Antigen 0.1 c.c. yodium chloride, C.P., 0.85 per cent 2.45 c.c. Elimination Test. — The emulsion is prepared as for the diag- nostic test. After being incubated in a water bath at 56° C. for 15 minutes, it is centrifuged for fifteen minutes (eighth setting rheostat, centrifuge size 1, type S. B.). The supernatant fluid is removed and the sides of the tube are dried with blotting paper. The sediment is resuspended in 1.0 c.c. of isotonic salt solution. The technique for the test is like that of the diagnostic reaction. More details pertaining to the Kline tests are given in Supplement No. 11. Use of Elimination Test. — The elimination test is useful in cases in which it is desired to rule out syphilis as a possible com- plication. It has been successfully used in testing blood donors, in health examinations, and in routine examination of patients be- ing admitted to institutions or hospitals for other than venereal treatment. Sources of Error. — A common source of difficulty resides in the cholesterol solution. To give satisfactory results, the cholesterol must be weighed accurately. The error should not exceed 0.005 grams per gram of cholesterol. The alcohol used in preparing the solution should be free from traces of water. Such alcohol will not color anhydrous copper sulphate. This reagent rapidly absorbs PRECIPITIN TESTS IN SYPHILIS 461 moisture from the air after preparation. Thus fresh cholesterol reagents should be prepared frequently. The sensitivity of the emulsion is more dependent upon the cholesterol content than on the antigen content. The antigen extract can be varied over a range of 400 per cent while the cholesterol content has a range of 60 per cent in satisfactory emulsions. (Kline, 1931.) Dirty glassware and slides may result in spontaneous aggluti- nation. Principle of the Meinicke Reaction. — In the Meinieke reaction the reagin in the syphilitic serum disturbs the colloidal balance of beef (or horse*) heart lipoids dispersed in three and one-half per cent sodium chloride solution. The result is a microscopic to a macroscopic flocculation, depending upon which of the several modifications of Meinicke is followed. Negative .serum, lacking reagin, does not change its uniform turbidity. The precipitate is composed of a lipoidal fraction and balsam of Tolu filmed with reagin and a denatured g]o])ulin fraction. This reaction, accord- ing to Meinicke, is a ty])ical lipoid-binding reaction. It is equiva- lent to agglutination or complement fixation in specificity ; and, with an appropriate antigen, can be applied to the diagnosis of any infectious disease or differentiation of a specific protein. It is Meinicke 's conception that the antigen-antibody complex binds lipoids (1926). Specificity.— Meinicke (1917, 1918, 1926-27), Jantzen (1921) and Epstein and Paul (1921) claim equal specificity with the complement fixation methods in general use. They believe this flocculation method is slightly more sensitive in general syphilis and decidedly more sensitive for the serological diagnosis of lues congenita. Antigen. — The antigen is an ether-insoluble, alcohol-soluble extract of beef heart. To this extract is added 1.4 per cent balsam of Tolu and 0.01 per cent Victoria blue. The antigen is titrated with weakly positive and negative serum by an arbitrary method. (Meinicke, 1934.) Mixing of Antigen. — Reagents in the test are antigen extract and three and one-half per cent sodium chloride solution. A *ln the earlier modification of the Meinicke reaction tlie antigen wa.=i prepared from horse heart lipoids, but in his recent modification (1934) he uses beef heart lipoids. 462 IMMUNOLOGY dispersion is prepared by heating the ingredients to 55° C. in a water bath and pouring the saline solution into the antigen. Thorough mixing is accomplished by pouring back and forth ten times. The diluted antigen is ready for immediate use. Serum.^ — The serum is obtained in the usual manner. It mud not be inactivated. Technique of the Test. — Place 0.2 c.c. of active (raw) serum in a small test tube of the Kahn type and add 0.5 c.c. of freshly prepared suspension (M.K.R.II suspension). Mix the ingredients thoroughly and incubate at room temperature. Interpretation of Results. — According to Meinicke the test can be read by any of the four methods outlined in the following discussion. It has been our experience that the macroscopic floc- culation method is the safest procedure to follow when testing weakly positive sera. In our hands the test has no distinct ad- vantage over the Kalm and Kline tests. The four metliods of i-eading the test are : 1. Macroscopic flocculation reaction: The flocculation is I'cad like an agglutination reaction after one and one-half hours of incubation. Using a hand lens distinct clumps are present in positive serum which are absent in a negative reaction. 2. Microscopic reaction : A few drops of the liquid are removed from the serum-suspension mixture when it is set up and placed in a paraffin ring on a slide. This is incubated for one hour in a moist chamber. When magnified sixty diameters, clumps indicate a positive reaction which are absent in the negative serum. 3. Clarification reaction: Incubate tubes overnight at room temperature. Negative reaction remains uniformly turbid. The degree of clearing of the supernatant liquid indicates the strength of the positive reaction with a strong positive reaction becoming completely clear. 4. Centrifugal technique : TJie tubes are centrifuged at low speed for ten minutes immediately after setting up the test. The speed can be determined by trial for each centrifuge, which will exclude nonspecific reactions (Dombrowsky, 1933). All super- natant liquid is poured out and the tubes are inverted for half an PRECII'ITIX TKSTS IN SYPHILIS 463 lioiir ill a Wassermann rack. Unaltered sediment is positive, while tlie reaction is negative if the sediment has run down the side of tlie tube. Hinton Test.— Hinton Antigkx.— This test is known as the sec- ond modification of Hinton (1930, 1931, 1940). The reaction is a macroscopic floccuhition test for reagin. Tlie lipoid extract con- sists of the elher-insoliiblc, alcohol-soluble lipoids of beef heart with 0.4 per cent cholesterol added. Glycerated Indicator Solution. — The antigen used in making the test is called a " glycerinated indicator solution." A one hundred c.c. Erlenmeyer flask that has a ridge across the bottom dividing it into two equal compartments is used. The reagents used in preparing the antigen are : 1. Cholesterolized beef heart extract. 2. Salt-salicylic acid mixture containing 5 per cent sodium chloride and 0.00222 per cent salicylic acid. 3. Fifty per cent neutral glycerol (glycerol diluted with dis- tilled water). Technique of Test. — One cubic centimeter of cholesterolized antigen is placed on one side of the ridge and 0.8 c.c. of the salt- salicylic acid mixture on the other side. The reagents are mixed by rotating for one minute and allowed to stand at room tem- perature for five minutes. Thirteen and two tenths c.c. more of salt-salicylic acid mixture are added to the suspension in the flask and after thoroughly mixing, 15 c.c. of the 50 per cent glycerol solution are added. This makes a 1 :30 dilution of the original beef heart extract. The indicator solution keeps one week at ice box temperature. Serum is inactivated at 55° C. for thirty minutes. Five-tenths c.c. each of serum and indicator are pipetted into a test tube (10 mm. X 100 mm.). The rack is inclined at an angle of 45° and shaken in such a manner that the liquid travels halfway up the side of the tube with each forward thrust. The shaking period is three minutes. The tubes are incubated in a 37° C. water bath for sixteen hours. In the positive reaction there is a ring or band of course gran- ules at the top of the tube, accompanied by complete clearing of 464 IMMUNOLOGY the fluid. Gentle shaking causes the particles to disperse and ap- pear as a definite precipitate throughout the liquid. In the negative reaction there is no band and no precipitate. The liquid usuallj^ has a faint opalescence. Hinton claims greater speed in testing and equal specificity with the Wassermann reaction. Although salicylic acid and glycerol have been added apparently as preservatives, the occasional growth of bacteria in the mix- ture, when sterility is not accomplished, is a decided disadvantage. Such contamination in tubes may be easily mistaken for positive reactions. Citochol Reaction. — The citochol reaction of Sachs and Witeb- sky appears to be gaining popularity in America. Many labora- tories find it to possess a high degree of specificity and sensitivity checking closely Avith other precipitation and complement fixa- tion tests (Beintema, 1934, Stern, 1933, Deissler and Baker, 1935 and others). This reaction is similar in principle to the reac- tions discussed, bearing a closer resemblance to the Kahn and Meinicke. It differs from the newer precipitation tests in that the authors (Sachs and Witebsky) consider the preparation of the antigen comparatively unimportant. The antigen is a con- centration of the Sachs-Georgi antigen used in the slower cito- chol reaction. Cholesterol in concentrations of three- to six-tenths per cent is used as a synergizing agent, being dissolved in the con- centrated alcoholic lipoidal extract. Antigen.^ — The lipoidal extract is prepared by extracting one part of moist beef heart with five parts of alcohol. The extract is evaporated to dryness and dissolved in three volumes of alco- hol. To the finished extract is added 0.3 to 0.6 per cent choles- terol. A suspension is made by mixing one part of cholesterolized extract with two parts of saline solution and after standing five minutes, adding nine parts of saline solution, making the final antigen dilution 1 :12. Nine-tenths per cent sodium chloride solu- tion was originally used as a diluting fluid but the test is more sensitive if 2.5 per cent sodium chloride is sul)stituted (Deissler and Baker, 1935). Serum. — The serum is inactivated by heating at 55° C. for thirty minutes. Inactivation in quantities of less than half a PRECIPITIN TESTS IN SYPHILIS 465 cubic centimeter gives unsatisfactory results and one cubic cen- timeter quantities should be used for inactivation whenever pos- sible. Test. — A mixture of two-tenths cubic centimeter of suspension and an equal volume of inactivated serum is shaken three minutes in a Kahn shaking machine. One c.c. of saline solution is added. Particles can be oljserved in tlie strong positive tests, while the doubtful positives can be read by means of the agglutinoscope. After standing twenty-four hours, it can be read as a clarification reaction. An alternative method of reading is microscopic exam- ination (eighty diameters magnification) of the mixture for clump- ing before addition of the final saline solution. Advocates of the test claim specificity and sensitivity equal to the Kahn over which it has no apparent advantage. The dis- advantage of the test is the lack of quantitative data as to reagin titer of the positive serum. Bruck's Nitric Acid Reaction. — Bruck (1917) precipitated serum with nitric acid. The precipitate formed in syphilitic serum was less soluble in dilute nitric acid than that of nor- mal serum. Its agreement with complement fixation methods was about 70 per cent. The reaction appears to have little practical application. Formol Reaction. — Gate and Papacostas (1920) reported that two drops of commercial formalin added to syphilitic serum pro- duced a solid gel in 24 hours. Normal serum was unchanged. In their paper, they claim 85 per cent agreement with comple- ment fixation test. Ecker (1921), Pauzat (1920) and others re- port the tast as giving inconsistent results. Sachs-Georgi Reaction. — Sachs-Georgi (1920) reaction is a floc- culation test using a cliolesterolized lipoid antigen from beef heart. A precipitate forms in syphilitic serum which does not appear in normal serum under the conditions of testing. Vernes. — Vernes' reaction is based on the difference in turbidity produced by colloidal substances. Colloidal ferric hydroxide, along with other inorganic colloids, will produce greater flocculation in syphilitic than in normal serum. Eagle Flocculation Test. — Eagle (1932, 1940) has described a new flocculation test for syphilis claiming a greater sensitivity than 466 IMMUNOLOGY existing tests. Tliis increased sensitivity is secured by addition of both cholesterol and sitosterol to the antigen and a modification in manipulation. Rosenthal Test. — Rosenthal (1929) has reported a new sensi- tive flocculation test. This increased sensitivity is accomplished through an increase in the cholesterol content of the reacting mixture. The antigen is essentially a Kahn antigen without the addition of cholesterol. Equal parts of cholesterol solution (2 per cent in acetone) and antigen are mixed. Fifty milligrams of methylene blue are added to 10 c.c. of the mixed reagent. This dye stains the particles. A drop of the stained antigen is added to four drops of inactivated serum. Examination is made with a magnification of 80 to 100 diameter. Clumping occurs in syph- ilitic serum. Mazzini Microflocculation Test. — In the Mazzini (1939) test the antigen contains acetone insoluble, alcohol soluble lipoids from both dehydrated beef powder and powdered egg yolk, and also cholesterol and buffered saline. The student is referred to Mazzini 's publication for a description of the test. According to Ratcliffe (1940) the Mazzini test is superior to "certain other tests in common use." It has been impossible to discuss in the space of one chapter all of the flocculation reactions used in the serological diagnosis of syphilis. Outstanding types of reactions have been cited as examples to establish general principles. Thi.s policy has, per- haps, led to the omission of many valuable and efficient procedures. Importance of Using- Both Complement Fixation and Precipita- tion Tests.- — None of the methods described will replace the com- plement fixation methods entirely. There are sera which will give positive flocculation tests and negative complement fixation tests. The reverse is also true. It would seem better practice to use both the flocculation and complement fixation tests as routine procedures. It will greatly increase the accuracy of the results and reduce the number of false reactions to a minimum. Hypersensitive Tests of Doubtful Value. — The use of hyper- sensitive tests, either complement fixation or flocculation, is con- demned by Levine (1533). Transitions from primary to early and to later secondary stages are associated with definite changes PRECIPITIN TESTS IN SYPHILIS 467 in the intensity of the serological reactions. (Moore and Kemp, 1926.) For this reason the sensitivity of any reaction should be correlated with a great number of clinical cases before acceptance. In considering the choice of a precipitation test, the following points should be considered. The test should be : 1. Highly specific. 2. Sensitive enough to detect very small amounts of syphilitic reagin. 3. Sufficiently quantitative to show changes in reagin during antisyphilitic therapy. 4. Decisive in results giving a very small zone of doubtful reactions. 5. Of simple technique, adequately described, giving consistent results in various laboratories and not requiring special ti'aining other tlian tliat of a qualified technician. The precipitation method and complement fixation method are described as an aid to clinical diagnosis but not a substitute for it. They should be iLsed accordingly. Several tests should be made on each serum. Such a procedure is of distinct value both to the laboratory technician making the lest and the physician. In this laboratory a quantitative Kolmer complement fixation test, a routine Kahn, and a diagnostic Kline are made on each serum. Irregular reactions can be investigated carefully with such a routine. The time required for the multiple- type tests is little more than that required for one type of test. References Beintema, K. : El valor practico de un sistema de euatro reacciones modernas de floculacion para el serodiagnostico de la siflis comparado con la prueba Wassermann, Biol. Asoc. nied., Puerto Rico 26: 9, 1934. Bruck, C, and Hidaka, S. : Ueber Fallungserscheinungen beim Vermisehen von Syphilisseren mit alkoliolischen Luesleberextraken, Ztschr, f. Immunitatsforsch. u. exper. Therap. 8: 476, 1911. Bruck, C: Eiiie sero-chemische Reaktion bei Syphilis, Miinchen. raed. Wchnschr. 64: 25, 1917. Deissler, K., and Baker, A. B.: The Citochol Reaction for the Diagnosis of Syphilis, Am. J. Syph. & Neurol. 19: 48, 1935. Dombrowsky, K. H.: Beitrag zum serologischen Leusnachweis, Deutsche med. Wchnschr. 59: 1283, 1933. Eagle, H.: An Explanation of the Mechanism of the Was.sermann and Precipitation Tests for Svphilis, Johns Hopkins Hosp. Bull. 47: 292, 19;i0. 468 IMMUNOLOGY Eagle, H.: Studies in Serology of Syphilis. VIII. A New Flocculation Test for the Serum Diagnosis of S^^)hilis, J. Lab. & Clin. Med. 17: 787, 1932. Eagle, H.: The Eagle Flocculation and Wassermann Technics. Technics of Serologic Serodiagnostic Tests for Svphilis, Supplement No. 11, U. S. Public Health Service, 1940, p. 1. Eagle, H.: Studies in Serology of Sj'philis: A More Sensitive Antigen for Use in Wassermann Keaction, J. Exper. Med. 53: 605, 1931. Ecker, E. E.: Comparison of Formol and Wassermann Reactions in Diag- nosis of Syphilis, .J. Infect. Dis. 29: 359, 1921. Elias, H., Neubauer, and Solomon: Theoretisches iiber die Serum-reaktion auf S>T)hilis. Wien. klin. Wchnschr. 21: 718, 1908. Epstein, H., and Paul, F.: Zur Theorie der Serologie der Sj^hilis, Arch. Hyg. 90: 98, 1921. Gate, J., and Papacostas, G. : line nouvelle reaction des serums syphili- tiques; formol-gelification, Compt. rend. Soc. de biol. 83: 1432, 1920. Hinton, W. A.: Choosing a Serum Test for Syphilis, J. Lab. & Clin. Med. 19: 275, 1933. Hinton, W. A., and Berk, A.: The Hinton Glvcerol Cholesterol Reaction for SjT)hilis, New England J. Med. 202: 1054, 1930. Hinton, W. A., and Davies, J. A. V.: The Hinton and Davies-Hinton Tests for Svphilis. Technics of Serodiagnostic Tests for Syphilis, Supple- ment "No. 11, U. S. Public Health Service, 1940, p. 17". .Tacobstahl, E.: Versuche zu einer optischen Serodiagnose der Sj'philis, Ztschr. f. ImmunitLitsforsch. u. exper. Therap. 8: 107, 1910-11. Jantzen, W.: Theoretische und praktische Ergebnisse mit den Flockungs- reaktionen nach Meinicke, Ztschr. f. Tmnninitiltsforsch. u. exper. Therap. 33: 156, 1922. Kahn, R. L.: Serum Diagnosis of Syphilis by Precipitation, Baltimore, 1925, Williams & Wilkins Co. Kahn, R. L.: Outline of Standard Kahn Test with Appendix for Special Kahn Procedures. Technics of Serodiagnostic Tests for Syphilis. Supplement No. 11, U. S. Public Health Service, 1940, p. 29. Kahn, R. L.: A Serologic Verification Test in the Diagnosis of Latent Syphilis, Arch. Dermat. & Syph. 41: 817, 1940. Kahn, R. L., McDermott, E. B., and Marcus, S. : Effect of Temperature on Kahn Reaction. Studies I to IV inclusive. Am. J. Syph., Gonor. & Ven. Dis. 25: 151, 157, 162, and 173, 1941. Klausner, E.: Klinische Erfahrungen liber das Prazipitations-phanomen mit distilliertem Wasser im Seium S\'philitischer, Wien. klin. Wchnschr. 21: 214, 1908. Kline, B. S.: Mechanism of the jMicroscopic Slide Precipitation Tests for Syphilis, J. Lab. & Clin. Med. 16: 1202, 1931. JMicroscopic Slide Precipitation Tests for the Diagnosis and Exclusion of Syphilis, Ibid., p. 186. Microscopic Slide Precipitation Tests for the Diagnosis and Exclusion of Syphilis, Brit. J. Ven. Dis. 7: 32, 1931. Microscopic Slide Tests for the Diagnosis and Exclusion of S^-philis. Technics of Serodiagnostic Tests for Syphilis. Supplement No. 11, U. S. Pub- lic Health Service, 1940, p. 45. Kline, B. S., and L«vine, E.: One Thousand Precipitation Tests for Syphilis with Small Quantities of Defibrinated Finger Blood. (Clinical and Serological Comparison.) J. Lab. & Clin. Med. 15: 768, 1930. Kline, B. S., and Littman, S.: Clinical and Serological Comparison of the Microscopic Slide Precipitation Test for Sj'philis and the Wasser- mann Test with the Same Antigen, J. Lab. & Clin. Med. 15: 1008, 1930. PRECIPITIN TESTS IN SYPHILIS 469 Kline, B. S., and Rein, C. B.: A Microscopic Slide Precipitation Test for Syphilis with Spinal Fluid, J. Lab. & Clin. Med. 116: 398, 1930. Kline, B. S., and Young, A. M. : A Microscopic Slide Precipitation Test for Syphilis, J. A. M. A. 86: 928, 1926. A Microscopic Slide Pre- cipitation Test for Syphilis, J. Lab. & Clin. Med. 12: 477, 1927. Leathes, J. B.: The Fats. Monographs on Biochemistry, New York, Lon- don, Toronto, 1910, Longmans, Green and Co., p. 147. Levine, B. S. : The Value of Sitosterol as a Fortifying Agent for Beef Heart Antigen Used in the Precipitation Test for Syphilis, J. Lab. & Clin. Med. 18: 87, 1932. Levine, B. S.: Supersaturation of Antigenic Beef-Heart Extracts with Cholesterol and Its EfiPect on the Sensitivity and Specificity of the Complement-Fixation Eeaction, J. Lab. & Clin. Med. 18: 958, 1933. Mazzini, L. Y.r A Eeliable, Sensitive. Simple and Rapid Slide Flocculatiou Test for Syphilis. Am. J. Clin. Path. 9: 163, 1939. Meinicke, E. : Flocculatiou Tests in the Serology of Syphilis, J. Lab. & Clin. Med. 12: 891, 1927. ITeber eine neue Methode der sero- logischen Luesdiagnose, Berl. klin. Wchnschr. 54: 613, 1917. Ueber Theoi-ie und Mcthodik der serologischen Luesdiagnostik, Ibid. 55: 83, 1918. Meinick«, E.: Zur Methodik der serologischen Luesdiagnostik, Miinchen. med. Wchnschr. 65: 1379, 1918. Die Lipoidbindungsreaktion, Ztschr. f. Immunitatsforsch. u. exper. Therap. 29: 306, 1920. Meinicke, E.: A New SAphilis Reaction, the M. K. R. II in Cerebrospinal Fluids, J. Lab. & Clin. Med. 19: 518, 1934. Michaelis, L. : Pracipitinreaktion bei Syphilis, Berl. klin. Wchnschr. 44: 1477, 1907. Moore, J. E., and Kemp, J. E.: The Treatment of Early Syphilis. III. The Wassermann Reaction in Treated Early Syphilis, Johns Hopkins Hosp. Bull. 39: 36, 1926. Northrop, J. H., and DeKruif, P. H.: The Stability of Bacterial Suspen- sions. II. The Agglutination of the Bacillus of Rabbit Septicemia and of Bacillus Typhosus by Electrolytes, J, Gen. Physiol. 4: 639, 1921. Pauzat: Note sur la reaction de precipitation du benjoin colloidal dans le liquide cephalorachidi«n (Guillain, Guy Laroche et Lechelle) et sur la formol-gelification des serums svphilitiques (Gate et Papacostas), Compt. rend. Soc. de biol. 84: 503, 1921. Porges, O., and Meier, G.: Ueber die Rolle der Lipoide bei der Wassermann- schen SjT^hilis-reaktion, Berl. klin. Wchnschr. 45: 731, 1908. Porges, O., and Neubauer, E.: Physikalisch-chemische Untersuchungen iiber das Lecithin und Cholesterin, Biochem. Ztschr. 7: 152, 1908. Ratcliife, A. W.: The Mazzini Test: A Greater Aid in the Serodiagnosis of Syphilis, J. Lali. & Clin. Med. 25: 1224, 1940. Rosenthal, L. : A Rapid Precipitation Test for Syphilis, Proc. Soc. Exper. Biol. & Med. 27: 61, 1929. Sachs, H., and Georgi, W.: Zur Methodik des serologischen Luesnach- weis.ses mittals Ausfloekung durch cholesterinierte Organextrakte, ]\runchen. med. Wchnschr. 67: 66, 1920. Sherwood, N. P., Bond, Glenn C, and Canuteson, R. I. : On the Possible Presence of a Reagin-like Factor in Normal Human Sera, Am. J. Syph., Conor, and Yen. Dis. 25: 179, 1941. Stern, C: Positive Seroreaktion auf S^-philis nach Diphtherie und Plaut- Vineentscher Angina, Dermat. Wchnschr. 97: 1379, 1933. Wassermann, A., Neisser, A., and Bruck, C: Eine serodiagnostische Reak- tion bei Sj-philis, Deutsche med. Wchnschr. 32: 745, 1906. CHAPTER XXV HYPERSENSITIVENESS Anaphylaxis Discovery of Anaphylaxis. — In 1902 Richet and Portier under- took the study of the toxic substances present in the tentacles of Acthiaria with an idea of comparing this substance witli a similar one present in Pliysalia found in the South Seas. They prepared glycerin extracts and injected graded doses into dogs in order to determine the toxic dose. The animals which recovered were saved for further experiments. When they attempted to use these an- imals in later experiments, they discovered that the dogs had become intensively sensitive to the poison. Richet says that, "The most typical experiment, that in which the result was in- disputable, was carried out on a particularly licalthy dog. It was given at first 0.1 c.c. of the gljT'crin extract without becoming ill ; twenty-two days later, as it was in perfect health, I gave a sec- ond injection of the same amount. In a few seconds it was ex- tremel}^ ill, breathing became distressful and panting; it could scarcely drag itself along, lay on its side, was seized with diar- rhea, vomited blood, and died in twenty-five minutes." Early Studies of Anaphylaxis. — In reviewing the literature Richet (1913) and also Doerr (1909) state that in 1839 Magendie observed that one injection of a nontoxic dose of albumin ren- dered rabbits, after several days had elapsed, sensitive to a simi- lar dose of the same material. In 1890 Koch reported that tuber- culous animals were hypersensitive to normally nontoxic doses of tuberculin. Flexner in 1894 found that rabbits that had sur- vived a fir.st injection of dog serum succumbed when given a similar or even smaller dose several days later. Von Behring (1893) noted, in his studies of diphtheria toxin, that one injec- tion of toxin rendered guinea pigs more intensely sensitive to diphtheria toxin. Arloing and Courmont (1894) found that in- dividuals receiving several injections of donkey serum showed definite reactions to later injections of donkey serum. 470 HYPERSENSITIVENKSS 471 RiCHET AND Portier's Work. — Ricliet and Hericourt (1898) ob- served that when dogs were injec?ted with eel serum, they became sensitive to this material. Richet (1913) says that neither he and Herieonrt, nor any of the othei-s who had in the past observed sensitiveness, appreciated the sionificance of their observations. It was not until he and Portier rediscovered and investiojated the phenomenon in 1902 that its significance was appreciated. They named this particular kind of hypersensitiveness "anaphylaxis" since in their opinion it was the opposite of ''phylaxis" or pro- tection. Arthus Phenomenon. — The following year (1903) Arthus ren- dered rabbits hypersensitive to horse serum. He injected the antigen subeutaneously into the animals at definite intervals and observed that whereas the material comprising the first three in- jections was rapidly absorbed, such was not true when later in- jections were made. ?^ollowing these injections the material was not a])sorbed and there developed at the site of inoculation definite induration and not infrequently gangrene. Arthus also observed general systemic reactions in rabbits that had received several subcutaneous injections and finally an intravenous injection of horse serum. He noted also that the reaction is specific since he found that animals sensitized to horse serum are not sensitive to milk and, conversely, animals sensitive to milk are not sensitive to horse serum. The Theobald Smith Phenomenon. — In America, Theobald Smith (1903) observed anaphylaxis in guinea pigs used to test diphtheria antitoxin. He advised Ehrlieh of his observations and the latter had Otto investigate the phenomenon. They were un- aware of the earlier work of Richet and Portier. Rosenau's and Anderson's Studies. — Coincident with Otto's invastigation, Rosenau and Anderson (1906) reported extensive studies of anaphylaxis. The results of both investigations were published almost simultaneously. Onh^ a few facts of funda- mental importance relative to anaphylaxis in animals have been added since these studies appeared. They determined that one injection of a nontoxic dose of horse serum will render guinea pigs hypersensitive to a second injection of the antigen provided an interval of almost ten days is allowed 472 IMMUNOLOGY to intervene l)etween the injections. While both injections may- be given snbcntaneonsly, intraperitoneally, or intravenously, the second, i.e., the one to elicit shock, is given as a rule either intra- peritoneally or directly into the blood stream, since it is difficult to produce severe reactions and death wilh even large doses ad- ministered aubcutaneously. They found, also, that the reaction is specific, that the specific hypersensitiveness is transmissible from the mother to the off- spring, that the state of hypersusceptibility may be induced by any antigen and persists, when once acquired, for a long time. They observed that the animals which recovered from anaphy- lactic shock are immediately refractory to another injection of antigen. A sensitive animal can be made refractory by injecting, usually subcutaneously, a dose of antigen too small to cause symp- toms of shock. When the animal is thus made refractory to the antigen, it is said to be "desensitized'' and the process of render- ing it refractory is called ''desensitization. " The duration of the refractory state, when once established, varies with the species of animal used in the experiment. In guinea pigs it is said to last two or more weeks, while in rabbits it is very short. Our own experience has indicated that rabbits may become sensitive again within twelve hours. Discovery op Passive Sensitization. — In 1907 Nicolle found that when blood from a rabbit that had been sensitized with horse serum was injected into a normal rabliit, the latter became sen- sitive within the next twenty-four hours. The process of trans- ferring to a normal animal specific hypersensitiveness to an antigen by injecting blood taken from a sensitized animal is called ipatisivc sensitizatio7i. The information presented thus far enables one to offer a few definitions that may enable the student to better understand the .subject. Sensitizing Substance. — Any true antigen as defined in pre- vious chapters can be used as the sensitizing agent. This suggests what is now well established, that sensitization depends upon the development of antibodies. Landsteiner and Jacobs (1936) report success in producing anaphylaxis in guinea pigs sensitized with p-chlorobenzoyl chloride HYPERSENSITIVENESS 473 and arsphenamine respectively. Landsteinor and Chase (1937) report upon anaplniaxis in animals induced by picryl chloride and 2 A dinitrochlorobenzene. In their opinion their results offer strong evidence that antigenic conjugates are formed fol- lowing the application of substances of sim]ile chemical constitu- tion. That is to say that the simple clicmical substances used probably formed conjugates Avith \ho animals' own proteins, forming ncAV antigens. The ])resence of these new antigens resulted in sensitization. Sensitizing Dose of Antigen. — The initial or sensitizing dose or doses of antigen may be introduced by injecting the material into the tissues, blood stream or body cavity. Sensitization through the respiratory and alimentary tracts has also been dem- onstrated. This indicates that under certain conditions sufficient antigen may be al)sorbed through either the respiratory or in- testinal epithelium to bring about sensitization. Rosenau and Anderson found that the sensitization of one guinea pig was ac- complished by the injection of 0.000,001 c.c. (one millionth) of horse serum. Coca (1927) says that guinea pigs have been actively sensitized with as little as 0.000,000,5 gram of crystalline egg albumen. He further states that it requires from 1,000 to 10,000 times as much antigen to sensitize rabbits as guinea pigs. Incubation Period in Active Sensitization. — The incubation period in active sensitization is the period of time between the administration of the sensitizing dose and the development of hypersensitivene.ss. In guinea pigs this varies from five to ten days. It has been observed that the smaller the sensitizing dose employed the longer tlie incubation period. Coca (1927) says that it may l)e as long as nineteen to twenty-five daj's Avhere 0.000,1 lo 0.000,01 c.c. of ox serum is used. Rabbits and dogs arc more dit^cult to sensitize than guinea pigs. Auer (1915) recommends that rabbits be given four to eight injections of antigen sub- cutaneously four to eight days apart. He quotes Arthus as find- ing the incubation period to ])e from eight to fifteen days after the last injection. Bally (1929) working in this laboratory succeeded in sensitizing ra])bits to horse serum by administering 0.5 c.c. per kilogram of body weight subcutaneously followed in forty-eight hours by an intravenous injection of the same amount. He gave the shock- 474 IMMUNOLOGY ing dose eighteen or more days later and found all of the rabbits actively sensitized. The technique recommended by Manwaring is, perhaps, the best for sensitizing dogs. Bally employed it in his work in rabbits mentioned above. Using this method, Sherwood and Stoland (1929) found the incubation period in dogs to vary from nine to fourteen days after the second injection of horse serum. Duration of the Hypersensitive State. — The duration of hyper- sensitiveness in guinea pigs probably persists throughout the life- time of the animal. Coca (1927) states that rabbits remain sensi- tive, following a single injection of antigen, for about three weeks. Bally (1929) observed the hypersensitive state to persist in rab- bits as long as seventj'-seven days after two sensitizing doses of horse serum. Shocking' Dose of Antigen, — The shocking dose of antigen is the dose of antigen administered to produce symptoms of anaphy- laxis. When given subcutaneously, large doses must be em- ployed. The best results are obtained when it is administered intraperitoneally or intravenously, although the subdural and intracerebral routes may be employed. The shocking close should be considerably larger than the minimum sensitizing dose of the antigen. While it is necessary to employ a complete antigen to sensitize an animal, it has been shown by Tomcsik and Kurotchkin (1928), and by Avery and Tillett (1929) that anaphjdactic shock can be produced in guinea pigs passively sensitized to specific bacteria by injecting the carbohydrate specific for the bacteria in ques- tion. In this work it has been found important to use antibac- terial serum from rabbits since immune serum from horses yields negative results. Goodner and Ilorsfali (1937) inferred from their studies on anaphylactic shock in guinea pigs rendered passively sensitive to pneumococcal capsular polysaccharide that the ratio of antigen to antibody is very important. If the proportion of carbohydrate is slightly in excess of the amount necessary to satisfy the avail- able antibody, a fatal response is possible. If the antigen is present in excessively large proportion, the result will be neg- ative as is the case when the amount of antigen is too small. HYPERSENSITIVENESS 475 Passive Sensitization. — Passive sensitization is the transference of liypersensitiveness to a normal animal by injecting blood con- taining specific antibodies obtained from an actively sensitized animal. To be successful it is necessary that blood be obtained from the donor (sensitized animal) at a time when antibodies are ])resent in the general circulation; that a sufficient amount be employ ed to convey hypersensitiveness (this depends upon the concentration of antibodies in the blood) ; and that a normal animal be chosen as a recipient wliose tissues can combine with the donor's antibodies and become hypersensitive to the specific antigen. While passive sensitization is best accomplished where the donor and recipient are of the same species, it is well known that blood or serum from different species may be employed. Doerr (1909) and Sherwood and Downs (1928) observed that the sen.sitizing antibodies of one species may in some instances be incapable of sensitizing another species. Sherwood and Stoland (1930) found a variation in the recipi- ents of the same species. They transfused four small, healthy dogs of about the same age and weight with equal amounts of defibrinated blood obtained from a large sensitized dog. One of the recipients was rendered extremely sensitive, one was re- fractory (not sensitized) and two were moderately sensitive as judged by the drop in arterial blood pressure. The duration of hypersensitiveness produced by passive sen- sitization varies in different species. It persists longer when liomologous immune blood or serum is used than when the anti- bodies are obtained from a different species. Coca (1927) says that wlien a normal guinea pig is sensitized by immune serum from another guinea pig, the former may remain sensitive for sixty to seventy days. If heterologous immune serum is used, the hypersensitivity persists for about ten days only. Coca quotes Friedmann (1909) as saying that passive hypersensitiveness in the rabbit disappears within twenty-four hours, and Richet (1908) as finding that it persists in the dog for twenty days. In connection with passive hypersensitiveness, various species exhibit interesting differences in the time between the receipt of antibodies and the development of hypersensitiveness to the 476 IMMUNOLOGY specific antigen. Manwaring (1910) and Scott (1910-11) report that dogs and rabbits respectively become sensitive immediately. In the case of guinea pigs, it is well established that at least four to six hours must intervene before hypersensitiveness can be demonstrated.* It is customary to test animals twenty-four to forty-eight hours after they receive immune serum. Desensitization. — An animal can be desensitized usually by one injection of an amount of antigen that either produces mild shock or produces no symptoms. Large amounts can be employed where the antigen is administered subcutaneously and in frac- tional doses. The phenomenon of desensitization is important in the study of anaphylaxis in the experimental animal or when the ''Dale" reaction is being employed. It should be remembered that practically all native proteins such as egg white, blood serum, etc., are more or less toxic for laboratory animals. This is true also for tlie iiterine liorns of virgin guinea pigs used in the Dale technique. An animal or the excised uterine hoi'us ])ecome de- sensitized following an anaphylactic response. While the systemic symptoms of toxic reactions and anaphylactic shock can be dif- ferentiated as a rule, the uterine horn response to toxic doses of protein is identical in appearance with the anaphylactic response. To prove that the response of a uterine horn is anaphylactic rather than toxic, it is customary to repeat the shock dose. This should produce no reaction if desensitization has occurred. If a reaction occurs, it suggests that the original response might have been toxic rather than anaphylactic. The failure to observe such a criterion, described by Dale, has led to much confusion in the literature. Silva (1941) has suggested that trypsin may play an indirect role in anaphylaxis. The Refractory State. — Animals supposedly rendered sensitive may not develop symptoms following the injection of a shock dose of antigen for several reasons which may be enumerated as follows : 1. They may not have been made sensitive either by the injec- tion of antigen or by passive transfer. Antigens vary in their sensitizing capacity, and animals vary in their capacity to re- spond and become sensitive. Crystalline egg albumen is superior to native egg white as an antigen. ♦Bronfenbrenner maintains that immediate sensitization occurs if liomologous rather than heterologous immune serum is used. HYPERSENSITIVENESS 477 2. Animals are refractory following shock, or the injection of desensitizing doses of antigen. This is due probably to the fact that the antibodies attached to the tissue cells are exhausted or saturated with antigen. This refractory state is called "imti- atiaphylaxis" by Besredka and Steinhardt. 3. AVlien the antibody content of the blood stream is high, the animal may not develop symptoms following the injection of antigen. It is thought that this is due to the union of antigen and antibody in the blood stream and that, for this reason, the former is not available to unite with antibody bound by the tis- sues. This is called ''masked anaphylaxis." 4. Animals may be protected by drugs. The literature on this subject is reviewed by Aucr (1915). According to him, Fried- l)crger' and Hartocli protected guinea pigs by injecting about 1 c.c. of saturated sodium chloride intravenously before the anti- gen was administered. Biedl and Kraus prevented anaphylaxis in dogs by injecting barium chloride. They also state that ani- mals recovering from peptone shock are refractory to anaphy- lactic shock. Likewise, atropine, adrenalin, chloral hydrate, and even ether when administered to guinea pigs tend to reduce the severity of the symptoms and prevent death in a fairly high per- centage of cases. They have no effect upon the antigen-antibody reaction, but affect tissue mechanisms only. 5. Bronf enbrenner 's (1914, 1915) experimental results suggest that a refractory state may occur when the antitryptic index is high. While the symptoms of acute or protracted anaphylactic shock exhibited b}^ all members of any one species are the same, it is interesting to note that because of anatomical and physiological differences in species each exhibits its own characteristic symp- toms. These, along with a number of additional facts concern- ing anaphylaxis, will be presented in the following brief dis- cussion. Anaphylaxis in the Guinea Pig. — Acute Shock. — Symptoms of acute shock in the guinea pig develop within one or two minutes folloAving the intravenous or intracardial or intraperitoneal in- jection of an adequate amount of antigen. The animal is quiet for a few moments, then is restless and becomes excited, there is a roughening of the hair, the pig sneezes, rubs its nose or ears 478 IMMUNOLOGY and may discharge urine and feces, the nioveiuents of the ala nasi and of the respiratory muscles indicate difficulty in breath- ing. The animal usually coughs, jumps, staggers, falls over, and makes rhythmical movements of the extremities and violent move- ments of the muscles of respiration. It dies of asphyxia. If the thorax is opened immediately, it will be observed that the lungs are distended and cannot be made to collapse. The heart Avill continue to beat for some time although Auer and Lewis showed that heart block is present. They observed two or three auricu- lar to each ventricular beat. The principal pathological finding is the distention of the lungs with air. This was first observed by Gay and Southard. Auer and Lewis conclude that this is the immediate cause of death. In their opinion it results from a tetanic contraction of the smooth musculature of the broncliioles, producing stenosis. According to Schultz and Jordan, tliis is localized in the secondary and tertiary bronchi. Auer says that they claim: "the tetanic contraction of the nuiscle coat.s folds the mucous membrane of this area into a plug which occludes the lumen and thus brings about asphyxia." Protracted or Subacute Shock in the Guinea Pig. — When the pig dies following prolonged or protracted shock, the lungs are onlj^ partly distended. While this condition may be a contribut- ing factor to death, it is possible that a drop in blood pressure is also important. Another fairly constant finding in protracted shock is a marked fall in body temperature. This is regarded as a characteristic symptom by Braun, Friedberger, and others. In addition to these findings, a prolongation of the clotting time of the blood, a definite leucopenia, and a diminution in comple- ment titer have been reported. The Dale Phenomenon. — In 1910 Schultz reported that if one removes short segments of the small intestine from a sensitized guinea pig and suspends them in a bath of Locke's or Ringer's solution kept at body temperature, they will undergo specific tetanic contraction when the homologous antigen is added to the bath. Dale (1913) studied this phenomenon and found that al- though Schultz used toxic doses of antigen, nevertheless, a specific contraction can be demonstrated when nontoxic doses are em- ployed. He suggests that excised uterine horns from young virgin sensitized guinea pigs be employed instead of the loops HYPERSENSITIVENESS 479 or segments of small intestine. The contractions of the uterine horn are recorded hy means of a light heart lever attached at one end by means of a thread to the upper end of the uterine horn, while the other end of the heart lever produces a kymo- graphic tracing on smoked paper. As previously mentioned, it is necessary to demonstrate desensitization lo differentiate between toxic and anaphylactic reactions. The latter phenomenon is called quite frequently "the smooth muscle reaction of Dale." Stoland and Sherwood (1923) showed that when adequate doses of atropine are added to the bath, specific reactions are prevented. In their opinion Tyrode's solution containing dextrose and half the usual amount of calcium is superior to either Locke's or Ringer's solution. Anaphylactic Shock in the Rabbit. — The first extensive studies of anaphylaxis in rabbits were carried out by Arthus (1903). When a sensitized rabbit is given an adequate intravenous injec- tion of antigen, it may develop symptoms before the injection is completed or it may remain quiet for one or two minutes and then become excited, run aimlessly about, and die suddenly with its head retracted and eyes in exophthalmus. This is acute fatal anaphylactic shock in the rabbit. The cause of death has been studied by Auer, Coca, Airila and others. They have apparently shown that it is due to right heart failure caused by an increase in resistance to blood flow through the pulmonary circuit. The lungs are observed to be completely collapsed when the chest is opened. Arthus observed a prolongation of the clotting time of the blood. Bally (1929), working in our laboratory, carried out extensive physiological studies of anaphylaxis in rabbits. PTe summarizes some of his results as follows: ''1. The characteristic blood pressure response is an increased pressure followed by a decreased pressure which slowly returns to normal in the case of recovery. ''2. There is a peripheral circulatory response shown by the 'blanching reaction' of the ear to be much more pronounced than in either peptone or histamine shock. "3. There is a tachycardia during the blood pressure increase which gives way to a bradycardia during the blood pressure fall and persists after the ])lood ]>ressure has reestablished itself. 480 IMMUNOLOGY These phenomena resemble tlie various ones produced by hista- mine more closely than peptone at similar points on the blood pressure curve. "4. A definite increase in coagulation time develops soon after the shocking dose of antigen is administered. "5. A marked drop in the precipitin content of the blood oc- curs after intoxication, but there is not a complete loss of pre- cipitins. ".6. A markedly engorged right heart with the lungs well col- lapsed in rabbits dying of shock is observed. "7. The results of intestinal smootli muscle, kidney volume, in- tracystie and intracranial pressures are variable and so frequently negative as to be of no importance as an index of sensitization. "8. A definite fall occurs in body temperature during the ana- phylactic shock. ' ' Some of his other observations have been mentioned earlier in the chapter. Protracted Anaphylactic Shock in the Rabbit. — Coca (1927) gives an excellent description of this phenomenon. The animal may survive for varying lengths of time. Those that survive for days or weeks show marked loss of weight (cachexia). Local Anaphylaxis or Arthus Phenomenon. — This has been extensively studied by Opie (1924). He concludes that it is an inflammatory reaction initiated by the meeting of antigen and antibody in the tissues. His studies include observations on reversed anaphylaxis. His published papers on anaphylaxis should be read by the student. Opie's work on the Arthus Phe- nomenon has been confirmed by Cannon and Marshall (1941). Anaphylaxis in the Dog. — Acute anaphylactic shock was first observed in the dog by Richet and Portier (1902). Their descrip- tion is given at the beginning of this chapter. The subject was investigated extensively by Biedl and Kraus (1909) and by Pearce and Eisenbrey (1910). They found that the characteristic physi- ological picture in dogs under ether anesthesia is a profound drop in blood pressure accompanied by extreme engorgement of the liver and splanchnics. They also reported a prolongation of the clotting time of the blood. A few other characteristic findings were observed. In 1913 Auer and Robinson observed heart block as occurring during acute shock. Manwaring et al. (1923, 1924) HYPERSENSITIVENESS 481 and Siinoiids (1923, 1925) noted definite changes in ])ern)ea- ])ility which Manwaring regards as characteristic of anaphylaxis in the dog. In Manwaring's opinion, the explosive liberation of a toxin by the liver is responsible for canine anaphylactic shock. Simonds (1923) and Simonds and Brandes (1924) have offered a different explanation of the hepatic phenomena. They find that the walls of the hepatic veins contain a large amount of nonstriated mus- cle. They conclude from their experimental work that the drop in blood pressure and the engorgement of the liver observed in canine anai^hylaxis result from increased pressure in the hepatic veins caused by tlie tetanic contraction of the smooth muscle present in the vessel walls. Sherwood and Stoland reported that while animals injected with horse serum showed increased permeability changes, the phenomenon is observed in animals tliat have not become sen- sitized to horse serum; it can be demonstrated by perfusion with Locke's solution without antigen and is present in desensitized animals. The chronaxie studies of Stoland, Sherwood, and Wood- bury (1931) indicate that following the injection of horse serum there develops an increase in irritability of the vagus which cor- relates with increased permeability of the tissues but does not always indicate that the animal is sensitive. They note also, that about 10 per cent of the sensitized dogs show no prolongation in the clotting time of the blood during shock. When the clotting time is prolonged, it is due, according to Stoland and Haughey (1932), to the liberation of a heparin-like substance by the liver. The role of heparin as a factor in blood coagulation has been studied extensively by Howell and Holt (1918) and more re- cently by Howell (1924). Anaphylaxis in the Cat. — In 1900 Brodie called attention to the extreme sensitivity of normal cats to natural foreign proteins administered intravenously. Doses of foreign blood serum, egg white, etc., that are entirely nontoxic for guinea pigs, rabbits, dogs and other animals, caused a marked fall in arterial blood pressure in the normal anesthetized cat. For this reason, Man- waring (1910), Schultz (1911), Edmunds (1914), and later Drinker and Bronfenbrenner (1924) have encountered difficulty 482 IMMUNOLOGY in studying anaphylaxis in this anhnal. Kabler and Sherwood (1933) reinvestigated the subject after discovering that the Brodie reaction is not elicited by the intravenous injection of relatively large doses of crystalline egg albumen. They attempted to sensitize actively twenty adult eats and obtained entirely nega- tive results. They were, however, able to sensitize passively 40 per cent of a series of eats to the same antigen by means of high titered immune rabbit serum. As a result of further work, they conclude that they were unable to sensitize cats actively because the latter did not produce sufficient antibodies. The results of their physiological studies of anaphylaxis in the passively sen- sitized cat under ether anesthesia are partly summarized as follows : 1. The characteristic blood pressure response is a profound drop followed by a slow return to normal. There may be a temporary- return toward or to normal followed by a second drop and final return. This is spoken of as the "three-phase" curve. 2. During the period of low blood pressure there is a marked slowing of the heart rate. 3. There is a progressive decrease in the body temperature throughout the experiment. 4. There is an increase of intestinal pressure in the cannulated loop of small intestine. This suggests an active participation of the intestinal smooth muscle in the anaphylactic phenomenon. 5. The intracystic (bladder) responses are variable. 6. The kidney volume is consistently decreased during shock. This suggests an active constriction of the renal blood vessels. 7. There is no characteristic prolongation of the clotting time of the blood such as is observed in the dog. 8. The smooth muscle reaction can be demonstrated in vitro when excised strips are removed from animals that can be shown to be sensitive by the intravenous injection of antigen. Anaphylaxis in Rats. — Arthus (1903) was the first to study the effects of repeated injections of a foreign antigen into white rats. He claimed that he was able to produce specific sensitiza- tion. Novy and De Kruif (1917) were unable to confirm his re- sults. In 1924 Parker and Parker, working in Zinsser's laboratory, succeeded in producing definite anaphylactic shock in white rats. IIYPERSENSITIVENESS 483 and demonstrated typical Dale reactions with the excised uterine horns. They state that anaphj-lactic phenomena observed in the white rat are similar to those described for the dog. Spain and Grove (1925) report that they were unable to sensitize guinea pigs passively with precipitating rat serum. Zinsser, Enders, and l^'othergill (1939) suggest that this is due not only to the low antibody titer of the serum used, but, perhaps, also to tlie heterogeneity of the rat serum. Anaphylaxis in Frog's. — In 1911 Friedberger and Mita inocu- lated a few frogs with 0.1 c.c. of sheep serum intravenously and after a period of seven to ten days gave a second injection of 0.25 to 0.5 c.c. of the same antigen. They report a definite de- crease in pulse rate and note that the pulse is also weaker. Within two hours the animals were too weak to respond when stimu- lated mechanically. Kritchevsky and Birger (1924) were unable to confirm these results. Goodncr (1926), working in our labora- tory, was likewise unable to confirm the clinical findings but he Avas able to confirm, by means of in vitro studies, the findings of Friedberger and Mita as to the sensitization of the frog heart. AVhen specific antigen is added to a bath of Ringer's solution in Avliich the excised heart from a sensitized frog is suspended, there "will occur an abrupt fall in amplitude and some decrease in rate" (Goodner, 1926, p. 338). Anaphylaxis in Turtles. — ^The phenomenon of anaphylaxis in turtles was investigated by both Sherwood and Downs (1928) and Downs (1928). The results of the former deal wdth passive sensitization and may be summarized as follows : 1. Passive sensitization of turtles (ten out of a series of thirty- six) was accomplished by injecting them with high titered im- mune rooster serum. 2. When a shock dose of antigen was injected into the ventricles of the heart, a specific reaction occurred. It consisted of a marked slowing of the heart, increase in diastole, a decrease in amplitude, cardiac engorgement, and a sinking of the heart to a lower level. There was an apparent rise in tone probably due to the latter phenomenon. 3. In two of the turtles that showed positive results for pas- sive sensitization, the hearts returned to normal and survived 484 IMMUNOLOGY eighteen hours with regular rhythm. They remained desensitized throughout this period. 4. Reversed anaphylaxis was not demonstrated in seven turtles, but a doubtful positive was obtained in one. The incubation period for the latter was two hours. 5. The shortest incubation period for passive sensitization was four hours, while the average was twenty-four to forty-eight hours. 6. High titered immune serum from rabbits failed to sensitize passively in a series of sixteen turtles. The subject of active sensitization in turtles was investigated by Downs (1928). Her results may be summarized as follows: 1. Turtles may be actively sensitized to mammalian serum. 2. The heart in situ responds in a specific characteristic way to the injection of antigen used for sensitization. 3. Desensitization can be demonstrated. 4. Precipitins (titer 1:10 to 1:100) are present in the serum of a certain number of the actively sensitized animals, and this serum seems to confer passive anaphylaxis to normal turtles. 5. The specific response resembles vagus stimulation of the turtle heart. Anaphylaxis in Chickens. — In 1926 Gahringer demonstrated clinical anaphylaxis in chickens. It is characterized by profound weakness, and lacrimation. The following year Hanzlik et al. (1927) described the smooth muscle reaction of the crop in pigeons. Sherwood (1928) reported upon his attempt to sen- sitize the embryonic chick passively. He found that high titered immune rooster serum will passively sensitize a very small per- centage of 48- to 72-hour chick embryos. The characteristic response is a marked slowing of the heart with a final cessation in diastole. When the bath is changed, the heart will resume the normal rhythm. The latter is not affected by another dose of antigen. In other words, desensitization can be demonstrated. He produced also reverse anaphylaxis in a few embryos. Anaphylaxis in Monkeys.— Monkeys are apparently very re- fractory to anaphylaxis. Coca (1927) and Zinsser, Enders, and Fothergill (1939) cite the work of Auer and others who were un- able to demonstrate anaphylaxis in monkeys. Zinsser states that HYPERSENSITIVENESS 485 in liis studies one monkey developed, after prolonged sensitization, symptoms similar to those of serum sickness in man. Kopeloff and Kopeloff (1936, 1939) seem to have produced both acute anaphylactic shock and the Arthus phenomenon in the Rhesus monkey. The functional and pathological changes ob- served in acute shock were prolonged clotting time of the blood, reduction in blood platelets and presence of skin hemorrhages. There were certain variable changes observed such as "emphysema, edema and hemorrhage of the lungs, edema and hemorrhages of the intestinal tract." Hemorrhages were also observed occa- sionally in other organs. The Schultz-Dale reaction was negative with both intestinal loops and uterine strips. They say there was no correlation found between the precipitin titer of the serum and the degree of sensitivity as determined by shock. , Anaphylaxis in Man. — Coca (1927) states that in his opinion "the existence of the condition of anaphylaxis in human indi- viduals has not been demonstrated ; in other words, no human pathological change has yet been shown to be the result of an anaphylactic antibody-antigen reaction." In his opinion ana- phylaxis comparable to that observed in the lower animals has not been observed in man. He calls attention to extensive clini- cal observations that immediate symptoms of anaphylactic shock have not occurred as the result of a second injection of horse serum. He chooses to disregard the few cases that have shown symptoms of acute shock and does not classify serum disease, which occurs several days or weeks after a first injection of horse serum, as anaphylaxis. Zinsser, Enders, and Fothergill (1939) is of the opinion that the latter will ultimately be shown to be an ex- pression of anaphylaxis in man. Criteria of Anaphylaxis. — Wells (1929) calls attention to cer- tain criteria which, if ol)served, will enable one to differentiate between true anaphylaxis and the phenomena resembling it which do not depend upon an antibody-antigen mechanism such as is described in the preceding pages. These criteria he lists as fol- lows: "1. The observed toxicity of the injected material must depend upon the sensitization of the animal ; i.e., the substance must not produce similar symptoms in non-sensitized animals. 486 IMMUNOLOGY "2, The symptoms produced must be those characteristic of anaphylactic intoxication as observed in the usual reactions with typical soluble proteins, being therefore the same for all antigens with the same test animal, but differing characteristically with each species of animal. ''3. It should be possible to demonstrate typical reactions in the nonstriated muscle tissue of the sensitized animal. "4. The possibility that the observed symptoms are caused by capillary thrombosis or embolism must be excluded. "5. After recovery from anaphylactic shock there should be ex- hibited a condition of specific desensitization to the same anti- gen under proper conditions. "6. In addition to the above, it is usually, but not always, pos- sible (a) to demonstrate passive sensitization with the serum of sensitized animals; and (b) to demonstrate amelioration or pre- vention of the bronchial spasm in guinea pigs by proper use of atropine and epinephrine." Zinsser (1931) is of the opinion that criteria numbers 4 and 6b may be omitted, since he regards the others as adequate. He sug- gests that the criterion relative to passive sensitization should be modified since it has been shown that the species origin of pas- sive sensitizing serum is important. He refers to observations previously cited that guinea pigs may be passively sensitized with antibacterial serum from rabbits, but not with antibacterial serum containing antibodies from a horse. It might also be added that Sherwood and Downs (1928) succeeded in passively sen- sitizing turtles to sheep and human blood with immune sera obtained from chickens, but were unsuccessful when they employed similar immune sera obtained from rabbits. Zinsser also calls attention to the newer work which has shown that clinical bac- terial anaphylactic shock or the specific response of sensitized uterine horns can be demonstrated when the specific bacterial hapten (carbohydrate) is used. The whole antigen is, however, necessary for sensitization. The Sensitizing' Antibody. — It has been shown by Doerr and Russ (1909) that the sensitizing property of immune rabbit serum for guinea pigs parallels its precipitin content. Weil (1916) determined that the precipitate formed in the precipitin reaction could be washed free of serum and used to sensitize guinea pigs 11 YPEK.SENSITIVENESS 487 passively. Those who hold to the plurality of antibodies for any one antigen have offered certain evidence against tlie assumption that precipitin and sensitizing antibody are the same. They point out that Longcope (1913), Spain and Grove (1925) and others were unable to sensitize guinea pigs with precipitating serum ob- tained from rats. This may be due to species differences as pointed out elsewhere in this chapter. Zinsser remarks that perhaps the low titer of precipitins in immune rat serum may be a factor. Furthermore, he does not regard certain quantitative discrepancies observed between the precipitating and sensitizing power of a serum, as significant since the formation of a visible precipitate depends not only upon the union of antigen and antibody but also upon many additional factors which have been discussed in preceding chapters. In fact one makes use of the failure of antigen-antibody mixtures to develop precipitates when he em- ploys the "suppression phenomenon" of Landsteiner in the study of specificity. Opie (1924) in his study of the Arthus phenomenon noted definite correlation between the sensitizing and precipitat- ing property of immune serum. According to Zinsser (1931), Ward and Enders carried out studies in his laboratory which indicate that the complement fixing titer of a serum is a more accurate measure of its sensitizing property than the precipitin titer. Since we accept as a working hypothesis for the present the unitarian concept of antibodies, it is obvious that we regard precipitin and anaphylactic antibody as the same. Specificity of the Reaction. — The specificity of the reaction has been investigated extensively by Wells and also by Dale and others. They liave found the reaction to be not only very specific Init far more delicate than any biochemical tests known. The Changes in Metabolism During- Shock. — Wells (1929) in an excellent condensed discussion of anaphylaxis reviews the literature on the pathological, physiological and metabolic changes in anaphylactic shock. Major (1914) reports an increase in non- coagulable nitrogen, creatinine and urea in the blood; Abder- halden and Wertheimer note a decrease in the gas metabolism as a whole; Eggstein and others describe a marked acidosis asso- ciated with the asphyxia in guinea pigs, while McCullough and O'Neill report a marked increase in lactic acid. Among other 488 IMMUNOLOGY important papers cited by Wells are those of Ilaiizlik and De Eds (1926), who observed a marked change in endothelial permea- bility in anaphylactic shock, and Paterson and Levinson, who noted not only an augmented flow but an increased protein con- tent of thoracic lymph during severe shock. More recently Kuslmarjew (1930) calls attention to changes in the blood cal- cium and potassium during shock. We have suggested that since electrolj'tes enter into antigen-antibody reactions in general, the disturbance of electrolyte balance in or on the tissue cells may be an important factor. The Nature of Anaphylaxis. — The evidence presented in the preceding pages, which seems to prove conclusively that the type of hypersensitiveness, discovered independently by Richet and Portier and by Theobald Smith, respectively, and named by the former anaphylaxis, is mediated by an antigen-antibody mecha- nism, may be summarized as follows : 1. Active sensitization can be produced by introducing into the tissues a true antigen only, 2. The incubation period which must elapse before specific hypersensitiveness develops corresponds to the required incuba- tion period for the development by the body of specific antibodies and their fixation by the tissues. 3. Anaphylactic shock, as previously defined, is elicited by nor- mally nontoxic doses of the specific antigen only, or, in the case of bacterial anaphylaxis, it may also be elicited by the specific bacterial polysaccharide used likewise in amounts nontoxic for the normal animal. 4. Blood from a sensitive animal is capable of passively sensi- tizing a normal animal only when it is obtained from the former at a time at which experience has indicated antibodies should be present. Where blood from sensitized rabbits is employed, it has been shown that its sensitizing power parallels its precipitin (antibody) content. 5. Weil showed that the washed precipitate obtained in the precipitin reaction can passively sensitize as well as actively sen- sitize a normal guinea pig. This suggests the identity of sensitizer and precipitin, since the precipitate is known to contain the latter bound to antigen. HYPERSENSITIVENESS 489 Site of Reaction. — Tlie socoiid important point concerning the nature of anaphylaxis is that the site of the antigen-antibody reaction which leads to shock is in or on the tissue cells and not in the blood stream. This is supported by the following ob- servations : 1. The introduction of sensitizing antil)odies into the blood stream of a normal guinea pig docs not render it immediately sensitive to antigen such as would be the case if the site of the reaction were in the blood.* Instead there must elapse four or more hours after the antibodies are introduced before the animal becomes sensitive to antigen. This is best explained by assuming that this time is required for the tissues to take up antibody. 2. Manwaring has shown that the blood of a sensitized dog may be replaced by the blood of a normal dog without impairing the sensitiveness of the former. 3. Doerr and others have shown that during passive sensitiza- tion, the animal becomes progressively more sensitive as the anti- bodies disappear from the blood. 4. Weil has shown that the injection of large amounts of anti- body into a sensitive guinea pig interferes with the reaction. The reason is presumably that the circulating antibodies meet and react with the antigen before the latter can react with antibodies in the tissues. 5. Finally both Schultz and Dale have shown that segments of small intestine and also the uterine horns of sensitized guinea pigs perfused free of l)lood, removed from the body and properly suspended in Locke's or Ringer's solution, will give a specific anaphylactic response when a nontoxic dose of antigen is added to the bath. Likewise Friedberger and Mita and also Goodner have siiown that the excised sensitized frog heart gives a specific reaction when brought in contact with antigen. It should be stated, however-, that there are a number of in- vestigators who have observed symptoms of anaphylaxis immedi- ately after antigen and sensitizing serum were injected separately, but simultaneously, or mixed immediately before injection. Zins- ser and also Wells do not regard these observations as invalidat- ing the conclusion that in anaphylaxis the reaction occurs in or on the tissue cells. The}^ suggest that the symptoms might be either anaphylactoid or toxic phenomena of undetermined cause. •According to Bronfennbrenner iniinetUate .sensitization occurs if homologous instead of heterologous antiserum is used for passive sensitization. 490 IMMUNOLOGY While it is quite generally agreed that anaphylaxis is an anti- gen-antibody reaction occurring in or on the tissue cells, there is disagreement among immunologists as to what tissues are in- volved and how antigen and antibody cause the reaction. Pearce and Eisenl)rey were unable to rule out the participation of intrinsic nervous mechanisms in canine anaphylaxis. Tissues Involved in Anaphylaxis. — At the present time little Is known as to how extensively the various tissues other than smooth muscle and, perhaps, the reticulo-endothelial system and the hepatic parenchyma are primarily involved in anaphylaxis. It is easy to recognize smooth muscle contraction but difficult to detect reactions or changes in other tissues and exceedingly diffi- cult to be certain that any changes observed are primary and not secondary. Manwaring has long held that the hepatic paren- chyma is primarily involved in anaphylaxis. P'reund and others have shown that in immune animals antibodies are widely al- though unevenly distributed throughout the body, but, as to what cells other than those previously mentioned are involved and the kind as well as degree of relationship that exists between the various tissue cells and antibodies, nothing is known. Perhaps because there is considerable evidence that the reticulo-endothelial system is an important source of antibodies, there have been a number of attempts made to show that it is also an important primary site of anaphylaxis. Standenath (1923) and others re- port that blockade of the reticulo-endothelial system does not prevent shock. It is doubtful whether the observation of Klenge that he was able to prevent the Arthus phenomenon in sensitized rabbits by preliminary local injections of trypan blue into the area, can be regarded as proof of the primary participation of the reticulo-endothelial system in the Arthus phenomenon. It might be that the reaction between antigen and antibody in or on the clasmatocytes, which are part of the reticulo-endothelial system present in the skin, leads to the liberation of toxic sub- stances, which cause changes in the endothelium of the skin capillaries (which do not belong to the reticulo-endothelial sys- tem), but proof is apparently lacking. Since the endothelium of the ordinary lymph vassels, blood capillaries, arteries, veins and heart is not regarded as a part of the reticulo-endothelial system, the observation of changes in capillary permeability does not HYPERSENSITIVENESS 491 prove that the rcticulo-endothelial system is the primary site of anaphylactic shock. All that can be definitely stated is that Opie in his studies of the Arthus phenomenon showed that when anti- gen is injected into the skin of a sensitized rabbit there develops an inflammatory process. This, like all inflammation, is accom- panied by local changes in vascular permeability along with other phenomena described in Chapter III. Manwaring, Chilcote and Hosepian (1923) reported that when they perfused heart-lung preparations from sensitized dogs with Locke's solution containing antigen (horse serum), there was a 50 to 75 per cent reduction in the rate of perfusion flow, the lungs, which had been half inflated, failed to collapse when the tracheal clamp was removed, and a tracheal exudate appeared during the fourth minute which frequently amounted to 1,000 c.c. in seven minutes. These together with studies of the hepatic phenomena in dogs have led Manwaring to conclude that in- creased capillary permeability is tlic principal physiological change in anaphylaxis to which all other phenomena are secondary. It is interesting to note, however, that neither Manwaring nor Bally was able to demonstrate a similar pulmonary permeability change in the sensitized rabbit. Furthermore, Sherwood and Stoland (1930, 1931) found that the permeability change, reported by Manwaring, Chilcote and Hosepian, develops as a result of the first injection of horse serum, can be demonstrated by perfusing with Locke's solution without as well as with antigen, is present before sensitization as judged by fall in blood pressure can be demonstrated, and is not affected by desensitization. Whether antigens other than horse serum will produce a similar change has not been determined. It would appear that the pulmonary permeability change described by ]\Ian- waring is at least not due entirely to an antigen-antibody-cellular reaction occurring at the time of the experiment, but results from the first injection of horse serum. Whether or not a similar funda- mental change in permeability occurs in the canine liver as a re- sult of sensitizing doses of horse serum is not kno-\m nor has it been investigated. It is conceival)le, however, since horse serum has been used almost exclusively in the study of canine anaphylaxis that such a phenomenon may occur and be partly responsible for 492 IMMUNOLOGY the exi)losive edema of the liver that is observed in canine anaphylaxis. Since cells lining the liver sinusoids belong to the reticnlo-endothelial system, it has been suggested that the explosive, hepatic edema points to these cells and the hepatic parenchyma as the primary site of anaphylaxis. On the other hand, Wells (1929) calls attention to evidence which indicates that the primary site of the antigen-antibody reaction is upon or within the cells of nonstriated muscle tissue. The evidence may be summarized as follows : 1. Smooth muscle removed from the body of sensitized guinea pigs will give specific anaphylactic contractions when nontoxic doses of the antigen used in sensitization arc added to the bath. Desensitization can be demonstrated. 2. The iuflalion of the lungs of the guinea pig, which is the cliaract eristic i)lienomenon of ana])]iylaxis in that animal, is due to the tetanic contraction of the smooth muscle of the secondary and tertiary 1)ronchioles. 3. Simonds has shown that the walls of the hepatic veins of the dog are endowed witli a large amount of smooth muscle. He offers experimental evidence which indicates that the characteristic drop in arterial blood pressure results from the increased pressure in the hepatic veins due to the tetanic contraction of the smooth muscle in their walls. 4. It luis been estaljlished by Coca and others that the most important physiological anaphylactic phenomenon in the sensi- tized rabbit is a tetanic contraction of nonstriated muscle present in the walls of the pulmonary artery. Mechanisms of the Reaction. — While it is definitely estab- lished that anapliylactic shock is due to an antigen-antibody re- action occurring upon or within ti.ssue cells of tlie body, the exact mechanism which elicits the physiological responses is unknown. At the present time there are two theories either of which ap- pears to offer an adequate explanation of most of the observed phenomena. One theory assumes that the toxic stimulus results from physical (colloidal) changes, either at the surface or within the tissue cells, which result from antigen-antibody union. This explanation is either a part of or implied by the physical theory of Weil, the membrane hypothesis of Doerr, and the inflammation theory of Opie. A second theory formulated by Lewis and sup- HYPERSENSITIVENESS 493 ported by certain experimental studies of Dale postulates the existence of an histamine-like substance (H-substance) in loose combination with the tissue cells. According to this theory the H-substance is released as a result of the antigen-antibody reac- tion. The symptoms of anai^liylaxis are due to the H-substance thus liberated. There are a number of observations whicli support tlie physical theoiy just mentioned. These may be summarized as follows: 1. It is definitely established by experiment that changes in the colloidal dispersion of both antigenic substance and antibody globulin accompany antigen-antibody reactions in vitro. 2. Specific agglutination and precipitation phenomena have been observed within animal tissues. 3. The results of Bronfenbrenner's (1914, 1915) investigations of the Abderhalden reaction suggest that the union of antigen and antibody is accom])anied l)y physicochemical change which lowers tlie antitryptic index. Tliis leads to the liberation of toxic l)i'oducts by the action of proteolytic enzymes. 4. Wells has called attention to experiments wliich show that slight changes in colloidal disjiei-sion of phisma proteins may render them exceedingly toxic. It would appear that such a theory might explain the smooth muscle reaction of Schultz and Dale as well as the clinical symp- toms of shock. One can also offer strong evidence in support of the histamine theory of Lewis or some modification of his theory which does not restrict tlie toxic sul)stance to histamine. The evidence which has been offered in support of Lewis' theory may be summarized as follows : 1. The symptoms of histamine shock in the guinea pig, rabbit, dog, and cat are very similar to those of an;T])hylactic shock in tlie respective animals. 2. Histamine will produce a contraction of the intestinal loop or uterine horn which gives a kymograph tracing identical in ap- pearance with the specific anaphylactic response. 3. It is generally agreed that histamine and histamine-like sub- stances are widel}" distributed throughout the tissues of the body. Dale has shown that histamine is present in apparently loose combination with various fresh normal tissues (1939) and Drag- 494 IMMUNOLOGY stedt and Meade (1936) offer confirmatory evidence that the anaphylatoxin previously described by Dragstedt et al. is histamine. Farmer (1939) reports that the injection of histamine into serum-sensitized guinea pigs renders the uterine horns less sensitive to antigen. Sherwood, Stoland and Nelson (1941) have shown that the liistamine reaction in turtles resembles the anaphylactic response and that turtles, unlike dogs, cats and rabbits, become refractory to histamine following recovery from the intracardiac injection of liistamine. It is yet to be determined whether the refractory state is due to the liberation of histaminase. Another report which points to the similarity beween histamine and anaphylactic reactions is given by Andrus and Wilcox (1939). They report that the response of the coronary artery of hearts per- fused witli histamine was the same as tlie anaphylactic response. In 1930 Best and McHenry demonstrated a histamine inactivat- ing substance in horse lung and later in beef lung, liver and kidney. They noted that the inactivating substance was specific for his- tamine and have designated it histaminase. The early work on histaminase appeared to show that the inactivation of histamine oc- curred only in vitro but subsequent work by Karady and Browne (1939) indicates that inactivation occurs in vivo. They report that histaminase injected into guinea pigs fifteen minutes before the injection of a lethal dose of histamine prevented histamine shock in a majority of the animals. They also report that anaphy- lactic shock in sensitized guinea pigs is almost entirely prevented by an injection of histamine prior to the administration of the shock dose of antigen. Since it would appear from the evidence submitted that either theory may be adopted to explain the anaphylactic response, it might be profitable to extend somewhat the discussion of each. Further Discussion of the Physical Theory. — 1. The physical theory assumes that physical changes occur either at the cell surface or within the cell and that such physical change supplies the toxic stimulus. It would seem that neither of these assumptions can be proved directly by experimental ob- servation although they are in harmony with our present chemi- cal and physiological concepts. HYPER6ENSITIVENESS 495 2. Desensitization is satisfactorily explained by assuming that the antibodies are either exhausted or neutralized during the re- action, hence, after recover}^ when a second dose of antigen is administered there are no antibodies available to react with antigen, 3. The specific tetanic contraction of smooth muscle does not explain the prolongation of clotting time frequently, although not invariably, observed in anaphylaxis. If one pastulates physical changes occurring at the surface of or within the hepatic paren- chyma cells or perhaps in the endothelium of the liver sinusoids, one might explain the appearance of a heparin-like substance which would cause the prolongation of the clotting time of the blood. 4. The inflammatory response observed in the Arthus phe- nomenon might be explained as due to similar changes in the skin. Not infrequently it is explained by assuming that the spe- cific precipitate formed acts as a foreign body in causing inflam- mation. The exact mechanism involved is not understood. Further Discussion of the Histamine Theory. — Certain ob- jections to Lewis' theory that the reaction between antigen and antibody causes an explosive liberation of histamine which acts locally and perhaps always systemically have been summarized and discussed by Wells and others. Among the objections are listed the failure of histamine to desensitize animals, its inability to produce coagulation changes of the blood similar to those observed in anaphylactic shock, and finally its ability to produce strong contractions in desensitized uterine horns. Wells calls attention to the fact that the first and third objections arc not necessarily valid. Since the histamine is theoretically liberated as a result of an antigen-antibody reaction, the phenomenon of desensitization Avould mean an exhaustion of antibody and not an acquired tolerance for histamine. If antibody is exhausted during shock, then the second injection of antigen will find no antibody to unite with it and therefore no histamine will be liberated. A similar explanation is applicable to the third objection that histamine will produce a reaction in a desensitized uterine horn. If the antibodies present in the uterine horn are ex- hausted, then an antigen-antibody reaction cannot occur when antigen is added. Therefore no histamine will be liberated. The 496 IMMUNOLOGY capacity of the .smooth muscle to respond to histamine need not be altered. In fact, one would expect the desensitized horn to react to histamine if it is added to the bath. In regard to the second objection tluit liistaniine will not pro- duce prolongation of the clotting time of the blood as is com- monly ob.served in anaphylactic shock, this may be a real objec- tion and again it may be found either that a toxic substance possessing properties of both histamine and peptone is liljerated or that both chemical and physical mechanisms are involved. Rous and Gelding (1930) have offered experimental evidence which casts doubt upon the contention of Lewis that local vaso- dilatation after different tissue injuries is due to a single factor such as histamine. From their work they draw the following conclusions : ' ' Experiment shows that the vascular contraction responsible for Bier's spotting ]>revaiLs over the local vasodilata- tion caused by liistamine pricked into the skin. The results raise doubts concerning tlie validity of the hypothesis referring all local vasodilatations to the action of a single chemical substance or set of substances (H-snbstance) lil)erated within the tissues." The only serious physiological objections to regarding his- tamine as the toxic factor in anaphylactic shock have been raised as the result of studies carried out in our laboratory. These may be summarized as follows : 1. In histamine shock in the dog and rab])it each i)hase of the blood pressure response is decidedly sliorter than in anaphylaxis. In the rabbit histamine frequently does not produce a drop in blood pressure below normal such as occui's almost invariably in anaphylactic shock. When histamine ]n'oduces a lowering of the l)lood pressure in the rabbit, both the drop in pressure and the return to normal occur in much shorter time than in anaphylaxis. In histamine shock in the cat the blood pressure tracing re- sembles that observed in anaphylaxis except that the phase of low pressure is longer (20 min.) in histamine shock than in anaphy- lactic shock (12.5 min.). It will l)e observed that this is the opposite of the findings in the dog and rabjjit. 2. There is in histamine and anaphylactic shock a noticeable difference in the heart rate following the drop in blood pressure. In histamine shock in the rabbit 62.5 per cent showed an increase HYPERSEXSITIVENESS 497 in heart rate, whereas in aiia|)hylaetie slioek 100 i)er eent of the rabbits sliowed a decrease in heart rate (Bally, 1929). There is also a differenee in Ihe dej^-ree to which tlie heart is slowed. Quile similar results were obtained by Kabler and Sherwood in their study of anaphylaxis in the cat. In the latter, when histamine slows the heart, the rate is reduced only modei-ately, while in anaphylactic shock the slowing is usually profound. 3. When one com])ares tlie vascular clianges in the ear ol' the ralj])it in histamine and anaphylactic shock, he ol)serves definite diffei'ences in the intensity of the blanching reaction described by Bally. The injection of histamine, even though a fatal dose is given, never results in a complete obliteration of both capil- laries and larger vessels since histamine is a capillary dilator. On the other hand, one notes complete constriction of both capil- laries and larger vessels ol" the ear in ral)bits during mild anaphy- lactic shock. 4. A comparison of kidney volume changes in the rabbit is also of interest. In the series of rabbits which Bally injected with histamine, none showed an increase, while 60 per cent of the in- jections resulted in a decrease of kidney volume. In a compa- I'able series in Avhich anaphylactic shock was studied, he reports that 39 per cent showed an increase and only 13 per cent a de- crease in kidney volume. Similar differences in intracystic pres- sure in histamine and anaphylactic shock in the cat arc reported by Kabler and Sherwood. 5. If human idiosyncrasies are mediated l)y the same mechanism as exi)erimental anaphylaxis, then another reason presents itself for doubting the identity of histamine and the exciting agent causing shock. It is quite well established that when one injects a snuill amount of histamine into the human body an increase in gastric acidity results. Tliis phenomenon is used in the diagnosis of pernicious anemia where an aehylia is a characteristic finding. Since in asthma and hay fever the gastric acidity varies from zero to normal (Criep and Wechsler, 1931), it would appear that his- tamine is not liberated in tlie body in these allergic conditions. 6. Fein])erg and Bernstein (1940) in a review^ of the literature on asthma and hay fever for 1939 conclude that more research is needed before the histamine mechanism of allergy can be accepted. 498 IMMUNOLOGY References Anderson, J, F., and Rosenau, M. J.: Further Studies Upon Anaphylaxis, J. Med. Ees. 19: 37, 1908. Further Studies Upon Immunity and Hyper-Susceptibility, J. Med. Res. 16: 381, 1907. Andrus, E. C, and Wilcox, H. B.: The Effects of Anaphylaxis, and of Histamine Upon the Coronary Arteries in the Isolated Heart, J, Exper. Med. 69: 545, 1939. Arthus, M.: 1903. Cited by Doerr (1909). Auer, J.: Forchheimer's Therapeusis of Internal Diseases, New York, 1915, D. Appleton-Century Company 5: 39-120. Auer, J., and Lewis, P. A.: The Physiology of the Immediate Reaction of Anaphylaxis in the Guinea Pig, J. Exper. Med. 12; 15, 1910. Auer, J., and Robinson, G. C: An Electrocardiographic Study of the An- aphylactic Rabbit, J. Exper. Med. 18: 450, 1913. Avery, O. T., and Tillett, W. S.: Anaphylaxis with the Type-Specific Carbohydrate of Pneumococeus, J. Exper. Med. 49: 251, 1929. Bally, L. H.: Anaphylaxis. XI. Physiological Studies of the Hyper- sensitive Rabbit, J. Immunol. 17: 223, 1929. Anaphylaxis. X. Physiological Studies of Peptone Reactions in the Rabbit, Ibid. 17: 207, 1929. Anaphylaxis. IX. Studies on Histamine Reactions in Rabbits, Ibid. 17: 191, 1929. Best, C. H., and McHenry, E. A.: Inactivation of Histamine, J. Physiol. 70: 349, 1930. Biedl, A., and Kraus, R.: Experimentelle Studien iiber Anaphylaxis, Wien. klin. Wchnschr. 22: 363, 1909. Brodie, T. G. : The Immediate Action of an Intravenous Injection of Blood Serum, J. Physiol. 26: 48, 1900. Bronfenbrenner, J.: Studies on So-called Protective Ferments. III. The Abderhalden Reaction Is Not an Adsorption Phenomenon, Proc. Soc. Exper. Biol. & Med. 12: 4, 1914. Bronfenbrenner, J.: The Mechanism of the Abderhalden Reaction, Studies in Immunity. I, J. Exper. Med. 21: 221, 1915. Bronfenbrenner, J.: On the Present Status of the Abderhalden Reaction and of the Theory of the So-called Abwehrfermente, J. Lab. & Clin. Med. 1: 79, 1915. Cannon, P. R., and Marshall, C. E.: Studies on th« Mechanism of the Arthus Phenomenon, J. Immunol. 40: 127, 1941. Coca, A. F. : Hypersensitiveness. Practice of Medicine, Hagerstown, Mary- land, 1927, W. F. Prior Co., pp. 107-131. Dale, H. H.: The Anaphvlaetic Reaction of Plain Muscle in the Guinea Pig, J. Pharmacol. & Exper. Therap. 4: 167, 1913. Doerr, R.r Die Anaphylaxie (Ch. 34) Hand, der Tek. u. Meth. dor Im- munitat., Kraus u. Levaditi (1909), Zweiter Band, p. 856. Doerr, R., and Russ, V. K. : Studien iiber Anaphylaxie: 3. Der anaphylak- tische Immunekorper und seine Beziehunger zum Eiweissantigen, Ztschr. f. Immunitatsforsch. u. exper. Therap. 3: 181, 1909. 2. Die Identitat der anaphylaktisierenden und der toxischen Substanz artfremder Sera, Ibid. 2: 109, 1909. Downs, C. M. : Anaphylaxis. VII. Active Anaphylaxis in Turtles, J. Im- munol. 15: 77, 1928. Dragstedt, C. A., and Gebauer-Fulnegg, E.: Studies on Anaphylaxis. I. The Appearance of a Physiologicallv Active Substance During Anaphylactic Shock, Am. J. Physiol. 102^: 512, 1932. Dragstedt, C. A., Meade, F. B., and Eyer, S. W.: Further Studies in the Mechanism of Peptone Shock, J. Pharmacol. & Exper. Therap. 63: 400, 1938. ttYl>ERSENSlTlVENESS 499 Dragstedt, C. A., and Meade, F. B.: Further Observations on the Nature of the Active Substance (Anaphylatoxin) in Canine Anaphylactic Shock, J. Immunol. 30: 319, 1936. Dragstedt, C. A.: Anaphylaxis and Allergy, Ann. Int. Med. 13: 248, 1939. Edmunds, C. W.: Anaphylaxis in the Cat and Opossum, J. Pharmacol. & Exper. Therap. 5: 518, 1914. Physiological Studies in Anaphylaxis, Ztschr. f. Immunitatsforsch. u. exper. Therap. 22: 18, 1914. Farmer, L.: Experiments on Histamine-Refractoriness: II. Non-specific ' ' Desensitization ' ' Through Oral Application of Histamine, J. Im- munol. 37: 321, 1939. Farmer, L. : Non-specific "Desensitization" Through Histamine, J. Im- munol. 36: 37, 1939. Feinberg, S. M., and Bernstein, T. B.: Asthma and Hav Fever, J. Allergy 11: 281, 1940. Freund, J.: Eole of Reticulo-Endothelial System in Tuberculin Hyper- sensitiveness, J. Immunol. 11: 383, 1926. Friedberger, E., and Hartoch, O.r Ueber das Verhalten des Komplements bei der aktiven und passiven Anaphylaxie, Ztschr. f. Immunitats- forsch. u. exper. Therap. 3: 581, 1909. Friedberger, E., and Mita, S. : Ueber Anaphylaxie. XIX. Mitteilung. Die Anaphylaxie des Frosches und die Einwirkung des Anaphylatoxins auf das isolierte Froschherz, Ztschr. f. Immunitatsforsch. u. exper. Therap. 10: 362, 1911. Oahringer, J. E.r Sensitization of Pigeons to Foreign Proteins, J. Im- munol. 12: 477, 1926. Gay, F. P., and Southard, E. E.: Further Studies in Anaphylaxis. I. On the Mechanism of Serum Anaphylaxis and Intoxication in the Guinea Pig, J. Med. Res. 18: 407, 1908. Gay, F. P., and Southard, E. E.: On the Chemical Separation of the Sensitizing Fraction (Anaphylactin) From Horse Serum, J. Med. Res. 18: 433, 1908. Gay, F. P., and Southard, E. E.: Further Studies in Anaphylaxis. II. On Recurrent Anaphylaxis and Repeated Intoxication in Guinea Pigs by Means of Horse Serum, J. Med. Res. 19: 1, 1908; III. The Relative Specificity of Anaphylaxis, J. Med. Res. 19: 5, 1908. Goodner, K.: Studies in Anaphylaxis. IV. Allergic Manifestations in Frogs, J. Immunol. 11: 335, 1926. Goodner, K., and Horsfall, F. L., Jr.: Passive Anaphylactic Sensitivity to Pneumococcal Capsular Polysaccharides, J. Immunol. 33: 259, 1937. Hanzlik, P, J.: Basis of Allergic Phenomena, J. A. M. A. 82: 2001, 1924. Hanzlik, P. J., Butt, E. M., and Stockton, A. B.: Reciprocal Action of Crop Muscles in Anaphylactic Shock, With Notes on Effect of Heparin, J. Immunol. 13: 409, 1927. Howell, W. H., and Holt, E.: Two New Factors in Blood Coagulation, Heparin and Pro-Antithrombin, J. Physiol. 47: 328, 1918. Howell, W. H. : The Purification of Heparin and Its Presence in Blood, J. Physiol. 71: 553, 1924. Kabler, P.: Anaphylaxis XVI. Studies in Histamine and Peptone Shock in the Cat, Univ. Kansas Sc. Bui. 25: 149, 1938. Kabler, P., and Sherwood, N. P.: Anaphylaxis. XVII. Studies on Anaphy- laxis in Cats, Univ. Kansas Sc. Bui. 25: 159, 1938. Karady, S., and Browne, J. S. L.: Effect of Histaminase Treatment on Histamine and Anaphvlactic Shock in Guinea Pigs, J. Immunol. 37: 463, 1939. Karsner, H. T., and Ecker, E. E.: Colloidal Inhibition of Anaphvlactic Shock, J. Infect. Dis. 34: 636, 1924. 500 IMMUNOLOGY Kopeloflf, L. M., and Kopeloff, N.: Anaphylaxis in the Ehesus Monkey. I. Horse Serum as Antigen, J. Immunol. 36: 83, 1939. II. Egg-white as Antigen, Ibid. 36: 101, 1939. Kopeloff, N., Davidson, L. M., and Kopeloff, L. M.: General and Cerebral Anaphylaxis in the Monkey (Macaeus rhesus), J. Immunol. 30: 477, 1936. Kritchevsky, I. L., and Birger, 0. G.: A Contribution to the Cellular and Humoral Theories of Anaphylaxis and Similar Processes, J. Immunol. 9: 339, 1924. Kuschnarjew, M. A.: Kalium und Kalzium bei Anaphylaxie, Ztschr. f. Im- munitatsforsch. u. exper. Therap. 67: 9, 1930. Kalium und Kalzium bei Anaphylaxie unter Bloekadebedingungen, Ztschr. f. Immunitats- forsch. u. exper. Therap. 68: 299, 1930. Landsteiner, K., and Jacobs, J.: Studies on the Sensitization of Animals With Simple Chemical Compounds, J. Exper. Med. 64: 625, 1936. Landsteiner, K., and Jacobs, J.: Studies on the Sensitization of Animals With Simple Chemical Compounds. III. Anaphylaxis Induced by Arsphenamine, J. Exper. Med. 64: 717, 1936. Landsteiner, K., and Chase, M. W. : Studies on the Sensitization of An- imals With Simple Chemical Compounds. IV. Anaphylaxis Induced by Picryl Chloride and 2:4 Dinitrochlorobenzene, J. Exper, Med. 66: 337, 1937. Lougcope, W. T.: The Production of Experimental Nephritis by Kepeated Proteid Intoxication, J. Exper. Med. 18: 678, 1913. Major, E. H.r Ueber den Einfluss der Anaphylaxie auf den Stickstoffwech- sel bei Kanenchen, Arch. f. klin. Med. 116: 248, 1914. Manwaring, W. H.r Der physiologische Mechanismus des anaphylaktischen Shocks, Ztschr. f. Immunitjitsforsch. u. exper. Therap. 8: 1, 1910. Manwaring, W. H.r Technique of Experimentation in Anaphylaxis, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, Uni- versity of Chicago Press, pp. 989-1003. Manwaring, W. H., Chilocote, E. C, and Hosepian, V. M.: Capillary Permeability in Anaphylaxis, .1. A. M. A. 80: 303, 1923. Hepatic Eeaction in Anaphylaxis; Anaphylactic Eeactions in Isolated Canine Organs, J. Immunol. 8: 233, 1923. Types of Canine Anaphylaxis, Proc. Soc. Exper. Biol. & Med. 20: 274, 1920. Manwaring, W. H., Brill, S., and Body, W. H.: Hepatic Eeactions in Anaphylaxis; Hepatic Mechanical Factor in Peptone Shock, J. Im- munol." 8: 121, 1923. Manwaring, W. H., Hosepian, V. M., Enright, J. E., and Porter, D. F.: Hepatic Eelations in Anaphylaxis; Effects of Dehepatization on Eeactions of Certain Smooth Muscle Structures in Canine Anaphy- laxis, J. Immunol. 10: 567, 1925. Manwaring, W. H., and Marino, H. D.: Eeactions of Urinary Bladder in Eabbit Anaphvlaxis, J. Immunol. 13: 69, 1927. Nicolle, M.: 1906. Cited by Eiehet, 1913, p. 11. Novy, F. G., and De Kruif, P. H. : Trypanosome Anaphylatoxin. Anaphyla- toxin and Anaphylaxis, J. A. M. A. 68: 1524, 1917. Specific Anaphy- lactic Shock, J. infect. Dis. 20: 776, 1917. Opip, E. L. : Inflammatory Eeaction of the Immune Animal to Antigen (Artlius Phenomenon) and Its Eelation to Antibodies, J. Immunol. 9: 231, 1924. Desensitization to Local Action of Antigen (Arthus Phenomenon), Ibid. 9: 247, 1924. Acute Inflammation Caused by Antibody in an Animal Previously Treated With Antigen, Ibid. 9: 255, 1924. Pathogenesis of the Specific Inflammatory Eeaction of Immunized Animals (Arthus Phenomenon), Ibid. 9: 259, 1924. Anaphylactic Shock Caused by Antibody in Animals Sensitized by Antigen, Eeversed Passive Anaphylaxis, J. Exper, Med. 43: 469, 1926. HYPERSENSITIVENESS 501 Parker, J. I., and Parker, F., Jr.: Anaphylaxis in White Kat, J. Afed. Res. 44: 263, 1924. Pearce, R. M., and Eisenbrey, A. B.: The Physiology of Anaphylactic Shock in the Dog, J. Infect. Dis. 7: 565, 1910. Robinson, G. C, and Auer, .J.: Disturbances of the Heart Beat in the Dog Caused by Serum Anaphylaxis, J. Exper. Med. 18: 556, 1913. Rous, P., and Gelding, H. P.: Is Local Vasodilatation After Different Tissue Injuries Referable to a Single Cause.' J. Exper. IMcd. 51: 27, 1930. Scott, W. M.: Anaphylaxis in the Rabbit; the Mechanism of the Symptoms, J. Path. & Bact. 15: 31, 1910-11. Schultz, W. H.: Physiological Studies in Anaphylaxis. I. The Reaction of the Smooth Muscle of the Guinea Pig Sensitized "With Horse Serum, J. Pharmacol. & Exper. Therap. 1: 549, 1909-10. II. Reaction of Smooth Muscle From Guinea Pigs Rendered Tolerant to Large Doses of Serum, Ibid. 2: 221, 1910-11. IV. Reaction of the Cat Toward Horse Serum, Ibid. 3: 299, 1911-12. Schultz, W. H., and Jordan, H. E.: Physiological Studies in Anaphylaxis. III. A Microscopical Study of the Anaphylactic Lung of the Guinea Pig and Mouse, .1. Pharmacol. & Exper. Therap. 3: 375, 1911. Sherwood, N. P., and Stoland, O. O.: Anaphylaxis. XII. The Pulmonary Permeability in Normal and Sensitized Dogs and Its Relation to Anaphylactic Shock, J. Immunol. 20: 101, 1930. Sherwood, N. P., and Stoland, O. O. : Anaphylaxis. XV. A Theory as to Its Mechanism, University of Kansas Science Bull. 20: 103, 1931. Sherwood, N. P., and Stoland, O. O. : Bacterial Anaphjdaxis, .T. Immunol. 8: 141, 1923. The Prophylactic Action of Atropin Sulphate Upon the AnaphA'lactic and Allergic Reactions of the Excised Uterus of Mrgin Guinea Pigs, Ibid. 8: 91, 1923. Sherwood, N. P., and Downs, C. M.: Anaphylaxis. VI. Passive Sensitiza- tion of Turtles and Guinea Pigs Using Immune Serum From Chickens and Rabbits, J. Immunol. 15: 73, 1928. Sherwood, N. P.: Anaphvlaxis. V. Allergic Responses of the Embrvonic Chick Heart, .1. Immunol. 15: 65, 1928. Sherwood, N. P., Stoland, O. 0., and Nelson, H. G. : A Comparison of Histamine and Anaphylactic Reactions in Turtles. (Proceedings of the Missouri Valley Branch of the Society of American Bac- teriologists, Columbia, Mo., May 2, 1941, J. Bact. 42: 287, 1941.) Silva, M. Rocha E. : Concerning the Mechanism of Anaphylactic and Tryptic Shock, J. Immunol. 40: 399, 1941. Simonds, .1. P.: Anaphylactic Shock in Dogs, J. Infect. Dis. 19: 746, 1916. A Study of the Low Blood Pressures Associated With Anajihylactic and Peptone Shock and Experimental Fat Embolism With Special Reference to Surgical Shock, .1. Exper. Med. 27: 539, 1918. The Fundamental Physiologic Reaction in Anaphylactic and Peptone Shock, J, A. M. A. 73: 1437, 1919. Simultaneous Changes in Blood Pressure in Carotid Artery and .Tugular and Portal A'eins in Anaf)hy- lactic and Peptone Shock in Dog, Am. J. Physiol. 65: 512, 1923. Simond.s, J. P., and Ranson, W. S.: The Effect of Pe[);one on the Peripheral Circulation, J. Exper. Med. 38: 275, 1923. Simonds, J. P., and Brandes, W. C: Effect of Obstruction of Hepatic Veins on Systemic Circulation, Am. J. Physiol. 72: 320, 1925. An- aphylactic Shock and Mechanical Obstruction of Hepatic Veins in Dog, J. Immunol. 13: 1, 1927. Effect of Mechanical Obstruction of Hepatic Veins Upon Outflow of Lvmph From Thoracic Duct, J. Im- munol. 13: 11, 1927. 502 IMMUNOLOGY Smith, T.: 1903. Cited by Auer, 1915, p. 42. Spain, W. C, and Grove, E. F. : Studies in Specific Hypersensitiveness, Study of Eat Precipitin, J. Immunol. 10: 433, 1925. Standenath, F.: Untersuchungen iiber die Bildungsstate der Prazipitine, Ztschr. f. Immunitatsforsch. u. exper. Therap. 38: 19, 1923. Stoland, 0. O., Sherwood, N. P., and Woodbury, R. A.: Anaphylaxis. XIV. Chronaxie of the Cardiac Vagus Nerve in Dogs, Sensitized to Horse Serum and in Anaphylactic Shock, J. Immunol. 21: 393, 1931. Stoland, O. O., and Haughey, C. F.: 1932. Unpublished data. Tillett, W. S., Avery, O. T., and Goebel, W. F.: Chemo-Immunological Studies on Conjugated Carbohydrate-Proteins. III. Active and Passive Anaphvlaxis With Synthetic Sugar-Proteins, J. Exper. Med. 50: 551, 1929. Tomcsik, J., and Kurotchkin, T. J.: Eole of Carbohydrate Haptenes in Bacterial Anaphylaxis, J. Exper. Med. 47: 379, 1928. Weil, E.: Studies in Anaphylaxis. XIV. On the Eelation Between Pre- cipitin and Sensitizin, J. Immunol. 1: 1, 1916. XV. Equilibrium in Precipitin Eeaction. Equilibrium in Combination, Ibid. 1: 19, 1916. XVI. Equilibrium in Precipitation Eeactions. Dissociation, Ibid. 1: 35, 1916. XVII. On the Coexistence of Antigen and Anti- body in the Body, Ibid. 1: 47, 1916. Wells, H. Gideon: Chemical Aspects of Immunity, New York, 1929. The Chemical Catalog Co. Zinsser, H.: Resistance to Infectious Diseases, New York, 1931, The Macmillan Co. Zinsser, H., Endcrs, J, H., and Fothcrgill, L. D.: Imnuinitv, New York, 1939, The Macmillan Co. Supplcmcntanj References Arloing, S., and Courmont, J.: 1894. Cited by Eichct, Anaphylaxis. The Univ. Press, Liverpool, 1913, p. 6. Arthus, M. : La Sero-anaphylaxie der lapin. Arch. Internat. de Physiol. 7: 471, 1909. Ibid. 9: 156, 1910. von Behring, E.: (1893.) Cited by Eichet. Anaphylaxis, Liverpool, 1913, The University Press, p. 5. Doerr, A., and Euss, V. K.: Studien iiber Anaphylaxie. II. Die Identitat der anaphylaktisierenden und der toxischen Substanz artfremder Sera, Ztschr. f. Immunitatsforsch. u. exper. Therap., 1909, 2 Orig. p. 109. III. Der anaphylaktische Immunkorper und seine Beziehungen zum Eiweissantigen, Ztschr. f. Immunitatsforsch. u. exper. Therap., 1909, 3 Orig. p. 181. Flexner, S. : (1894.) Cited by Zinsser. Eesistance to Infectious Diseases, New York, 1931, The Macmillan Co., p. 355. Gay, F. P., and Southard, E. E.: Further Studies in Anaphylaxis. IV. The Localization of Cell and Tissue Anaphylaxis in the Guinea Pig, With Observations on the Cause of Death in Serum Intoxication, J. Med, Ees. 19: 17, 1908. Karady, S., Selye, H., and Browne, J. S. L. : The Influence of the Alarm Eeaction on the Development of Anaphylactic Shock, J. Immunol. 35: 335, 1938. Karsner, H. T.: Anaphylaxis and Anaphylactoid Eeactions. Newer Knowl- edge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, pp. 960-988. Koch, E.: (1890.) Cited by Eichet. Anaphylaxis, 1913, The University Press, Liverpool, p. 4. HYPERSENSITIVENESS 503 Kopeloflf, N., and Kopeloff, L. M. : Blood Platelets in Anaphylaxis, J. Immunol. 40: 471, 1941. Landsteiner, K., and van der Scheer, J.: Anaphylactic Shock by Azodyes, J. Exper. Med. 57: 633, 1933. Landsteiner, K., and Jacobs, J.: Studies on the Sensitization of Animals With Simple Chemical Compounds, J. Exper. Med. 61: 643, 1935. Landsteiner, K., and Di Somnia, A. A.: Studies on the Sensitization of Animals With Simple Chemical Compounds. V. Sensitization to Diazomethane and Mustard Oil, J. Exper. Med. 68: 505, 1938. Landsteiner, K., Rostenberg, A., and Sulsberger, M. B.: Individual Differences in Susceptibility to Eczematous Sensitization With Simple Chemical Substances, J. Invest. Dermat. 2: 25, 1939. Landsteiner, K., and Chase, M. W.: Studies on the Sensitization of Animals With Simple Chemical Compounds. VI. Experiments on the Sensitization of Guinea Pigs to Poison Ivy, J. Exper. Med. 69: 767, 1939. Landsteiner, K., and Chase, M. W.: Studies on the Sensitization of Animals With Simple Chemical Compounds. VII. Skin Sensitization by Intraperitoneal Injections, J. Exper. Med. 71: 237, 1940. Magendie: (1839.) Cited by Richet. Anaphylaxis, 1913, The University Press, Liverpool, p. 4. Ohlmacker, A. P.: The Reaction of Hypersusceptibility as Produced by Bacterial Inoculation, J. Med. Res. 19: 113, 1908. Otto, R.: Das Theobald Smithsche Phenomenon der Serum-iiberempflnd- lichkeit, Bedenksch. f. Rudolph V. Leuthold. 1: 155, 1906. Portier, P., and Richet, C: The Anaphylactic Action of Certain Poisons, Bull. French Biol. Soc, 1902, pp. 170-172. Cited by Richet, 1913, p. 1. Pottenger, F. M.: Symptoms of Visceral Disease, ed. 2, St. Louis, 1923, The C. V. Mosby Co. Richet, C: Anaphylaxis, 1913, The University Press, Liverpool, p. 4. Richet, C, and Hericourt, J.: (1898.) Cited by Richet. Anaphylaxis, 1913, The University Press, Liverpool, p. 6. Rosenau, M. J., and Anderson, J. P.: A Study of the Cause of Sudden Death Following the Injection of Horse Serum, Hyg. Lab. Bull., 1906 No. 29 (U. S. Gov't Print. Off.). Smith, Theobald: (1906). Cited by Zinsser, Resistance to Infectious Disease, New York, 1931, The Macmillan Co. Vaughan, V. C, Vaughan, V. C, Jr., and Vaughan, J. W.: Protein Split Products in Relation to Immunity and Disease, Philadelphia, 1913, Lea and Febiger. CHAPTER XXVI HYPERSENSTTIVENESS DUE TO INFECTION Allerg-y in Tuberculosis Tuberculosis and Tuberculin Hypersensitiveness. — In 1891, Koch made the remarkable discovery that active tuberculosis in guinea pigs renders them extremely sensitive to extracts of old autolyzed glycerin broth cultures of the tubercle bacillus. He concluded that the active substance to which they become sen- sitive is a protein fraction of the bacterial cell and accordingly named it "tuberculin." Tuberculin OT, BE, and TR. — In his original work, Koch em- ])loyed a filtrate of a glycerin broth cultui-e of the tubercle bacil- lus that had been incubated at 37° C. for six or eight weeks, sterilized and concentrated to one-tenth its volume. In an attempt to obtain a better yield of the specific active substance, Koch pre- pared a bacillary emulsion of the tubercle l)acillus in glycerol and water and also a combined extract of finely pulverized bacteria. The former tuberculin he designated as BE and the latter as TR. It is interesting to note that of all Kocli 's preparations his original or old tuberculin, OT, is the one that lias been most generally ac- cepted. It is used quite extensively at the present time in the diag- nosis and to a limited extent in the treatment of tuberculosis. Objections to OT. — Certain objections to Koch's old tuberculin have been made by numerous investigators. It has been pointed out (1) that the meat broth employed as a culture medium con- tains protein derivatives and perhaps protein from the peptone and meat used in its preparation; (2) the old glycerin broth culture contains products of bacterial metabolism and bacterial autolysis other than the active tuberculin fraction. Long's Synthetic INIedium. — The first objection has been met by growing the tu])ercle bacillus in a synthetic medium. While a great many methods and formulae for preparing a medium of known chemical constitution have been used, the synthetic me- dium devised by Long is regarded in America as the first satis- factory one. It contains glycerol, asparagine, acid potassium 504 HYPERSENSITIVENESS 505 phosi)liat(\ aininonium citrate, sodium carbonate, sodium chloride, maiinesium sulphate, and ferric ammonium citrate. The propor- tion and concentration of the constituents are such that the mix- ture is neutral and at the same time well buffered. Long states that growth in this medium yields 100 to 150 grams of moist tubercle bacilli per liter. Synthetic iMediuji of the Bi-reau oe Animal Industry. — An- other synthetic medium which Seibert (1934) says yields a heavier groAvth of tubercle 1)acilli than Long's medium has been developed by Dorset in the Bureau of Animal Industry. According to Seibert it differs from Long's medium in that it contains ap- proximately two and one-half times more asparagine and twice as much glycerol as the latter. It contains also 10 per cent dex- trose but no carbonate or ammonium ion except that which might 1)C derived from tlie aspai-agine. This is the medium which Seibert (1934) and Long (1934) recommend for the production of a standard tuberculin which is now called PPD (Purified Protein Derivative of tuberculin). The second objection lias been overcome by Long and Seibert (1926) and Seibert (1932, 1934), although Long states that the greater part of the chemical work was carried out by Seibert. They grew the tubercle bacillus in Long's protein-free synthetic medium and studied the chemical changes that occurred. From these and other studies the following important results were ob- tained : The Active Substance, Its Recovery and Properties. — 1. That the tuberculin activity of filtrates develops simultaneously with the appearance of protein in the medium. The protein is derived apparently by autolysis or extraction of the tubercle bacillus. 2. If the tuberculin is subjected to dialysis, there remains in the dialyzing sac only the active substance together with protein and the polysaccharide peculiar to the tubercle bacillus. 3. The protein and active substance are completely precipi- tated out of the filtrate by full saturation with ammonium sul- phate or by tricliloracetic acid. 4. The ammonium .sulphate precipitate contains both eoagulable and noncoagulable protein and in addition proteose. The activity is destroyed by pepsin which splits the protein, but the activity 506 IMMUNOLOGY is not affected when the proteose is destroyed by trypsin or erepsin. This indicates that the active substance is a protein. 5. The active substance, in the presence of N/10 hydrochloric acid, is not destroyed when heated under fourteen pounds pres- sure at a temperature of 120° C. for four hours. It is hydro- lyzed by acids having a strength of N/6 or above. 6. Seibert attempted crystallization of the protein and suc- ceeded in inducing a portion of the active substance to crystallize in needle form. This active crystalline substance possesses the property of an albumin. 7. They observed that a greater yield of active substance is ob- tained in filtrates of the old synthetic broth culture than from extraction of tubercle bacilli. This probably explains why tuber- culin OT is generally preferred to BE or TR. 8. More recently Seibert and Munday have combined ultra- filtration with trichloracetic acid precipitation in the preparation of a purified active protein fraction. 9. Masucci and McAlpine (1930) made use of the suggestions of Seibert in preparing a tuberculin which Funk and Huntoon (1930) have designated as MA-100 in accordance with the John- son chart for the chemical analysis of the tubercle bacillus. TPT. OF Seibert and Munday. — The new tuberculin protein pre- pared by Seibert and Munday (1932) is designated by them as TPT. These letters indicate that the product is tuberculin pro- tein precipitated by trichloracetic acid. In their opinion it is superior in some respects to TPA which they prepared in 1931 by precipitating old tuberculin with ammonium sulphate. Seibert and Munday describe their method of preparing TPT as follows: ''Virulent tubercle bacilli (Saranac Lake Strain H37 has been used in our work) are grown upon Long's synthetic medium for eight weeks at 37.5° C. (more protein is obtained by allowing the culture to grow for eight to twelve weeks instead of the usual four to six weeks). The bacilli are removed by filtration through China silk or a Buchner funnel and then through a Mandler filter. The clear yellow tuberculin, preserved with 0.5 per cent phenol, is then concentrated by ultrafiltration on a 12.5 per cent gun- cotton-glacial-acetic-acid membrane, and washed with 0.5 per cent phenol solution by continued ultrafiltration until the filtrate is chloride- and iron-free, and then filtered. (It has not yet been HYPERSENSITIVENESS 507 determined whether for routine preparations this additional washing process is absolutely essential before precipitation. In order to obtain the quantitative results given in this paper, it was, of course, necessary.) The protein is then precipitated from the ])ure colloidal solution in a final concentration of 10 per cent trichloracetic acid until the wash is clear and colorless. The drained precipitate is partly dried in vacuo and then ground to a fine powder under larger volumes of ether, which simultaneously dehydrates the product and removes the trichloracetic acid. A fine cream-colored powder, TPT, is thus quickly obtained." The product is rendered completely soluble in water by cau- tiously adding a few drops of N/10 alkali and immediately neu- tralizing it with HCl. They prepare stock solutions in 0.9 per cent saline containing 0.5 per cent phenol. The underlying principles of the above technique may be sum- marized as follows : 1. Using a standard virulent culture of the tubercle bacillus and a synthetic medium, a tuberculin is produced. 2. The bacteria are removed by filtration. 3. By means of ultrafiltration the resulting tuberculin is con- centrated and at the same time rendered free of all substances except the active colloidal tuberculin protein and the ever present polysaccharide found in old tuberculin. 4. The active tuberculin protein is separated from the poly- saccharide by precipitation with trichloracetic acid. 5. The precipitate is washed, dried, and dissolved in saline to which phenol is added as a preservative. The above treatment does not denature or in any way injure the tuberculin protein. Seibert's sot and SOTT or PPD.— Seibert prepared also an old tuberculin (SOT) from cultures grown in a synthetic medium. She next concentrated and purified SOT by ultrafiltration and precipitated the active principle with trichloracetic acid. The pre- cipitate was treated with ether to remove the acid and to dehydrate the protein. This purified protein derivative of the tubercle bacillus she calls SOTT (synthetic medium old tuberculin pre- cipitated with trichloracetic acid). In 1932 she prepared SOTT from cultures grown on Long's medium, but later she has adopted the .synthetic medium used by the Bureau of Animal Industrv. 508 IMMUNOLOGY This product is now called Purified Protein Derivative or PFD. The method of its manufacture is described in an excellent paper by Reichell and Clark (1934). MA-100 OF Funk and Huntoon. — To prepare MA-100, a culture is grown in Long's medium, rendered bacteria-free by filtration through a Berkefeld filter, and the active tuberculin protein pre- cipitated from the medium at a given isoelectric point by am- monium sulphate. The precipitate is purified by eight successive precipitations. The precipitate obtained in the operation is dis- solved in distilled water and the protein precipitated by ethyl alcohol. Subsequent purification of the protein is accomplished by a series of isoelectric precipitations. Mariette and Fenger working in collaboration with Funk, Huntoon and White (1932) have carried out extensive studies of MA-100 proteins olitained from the human, bovine and avian tubercle liacillus and from a culture of the timothy bacillus. In preparing the purified tubercle l)acillus protein they apparently employed a modification of IVLasucci's and McAlpine's method, since they say tluit precipitation at a given isoelectric point was ac- complished by means of glacial acetic acid. They recommend the use of MA-100 tul)ercle bacillus (human) protein in diagnostic work. The results of their studies and also those of Funk and Huntoon (1930) are discussed later in this chapter. Relationship of Molecular Weight to Sensitizing Property, — In 1933 Seibert reported upon the relationship between the sen- sitizing properties of tuberculin and its molecular weight. Numer- ous .studies had indicated that while OT, TPA, TPT, and SOTT or PPD are all capable of eliciting specific allergic reactions in tuber- culous animals, they differ markedly in the degree of sensitization to tuberculin produced by repeated injections of the respective products into the animal body. Since Svedberg had emphasized the fact that in dealing with purified proteins the final molecular size of the latter will depend upon the method of isolation, it occurred to Seibert that differences in the molecular weight of OT, TPA and TPT might result from the methods used in their preparation and that some correlation between the molecular weight and sensitizing property might exist. Her experimental investi- gation led her to believe until recently (1941) that such is the ease. HYPERSENSITIVENESS 509 These earlier results of Seibert have been reinvestigated by Seibert, Pedersen and Tiselius (1938) by means of the ultra- centrifuge and cleetrophoresis. They have obtained from TPA an antigenic protein with a molecular weight of 32,000 and from PPD a protein with a molecular Aveight of 17,000 to 18,000. These two proteins would elicit both local and systemic reactions in tuberculous guinea pigs. Positive skin reactions were obtained with substances of smaller molecular weight obtained from old tuberculin. Seibert (1941) has since modified the method of PPD production by carrying out the procedure at 4° to 5° C, using less heat and weaker acid solution and drying the final product by the lyophile process. The i)roduct obtained by this pi'ocedure is twice as potent biologically and much purer than pi-evious preparations. Th(! surprising thing is that while the molecular weight is only al)ou1 10,500 this more i)otent tuberculin is more antigenic than the previous PPD protein preparations, having a molecular weight of 17,000 to 18,000. This has led Seil)ert to think that the size of the molecule is not so important in antigenicity as she previously tliought. She is now of the opinion that the potency is inherent in one part of the protein molecule while the antigenicity is de- pendent upon another part of the same molecule. The advantage of the purified protein derivative of tuberculin (PPD) lies in the fact that it can be injected repeatedly into the same person without causing skin allergy to develop. The method of preparation of the new PPD is given by Seibert and Glenn (1941). Just why some of these highly purified fractions give a higher percentage of reactors in the second injection than previous preparations Ls unknown. Seibert (1941) says that this point is being investigated. Chemicai> Nature of PPD. — Seibert (1941) says that the new, (.Iried PPD is almost colorless, quite soluble in water, and contains approximately 1.2 per cent nucleic acid and 5.9 per cent polysac- charide. Its potency is twice that of the previous PPD. This is indicated by the fact that 0.00001 mg. produces as strong reactions in sensitive patients as 0.00002 mg. of the previous product. According to Chase and Landsteiner (1939) the separation of tuberculin into two fractions, one responsible for skin reactions and the other for systemic reactions, is reported by ]\Iaschmann 510 IMMUNOLOGY (1937). The skin reacting fraction is broken down by trypsin or papain while the other fraction is not acted upon by proteolytic enzymes. Classification of Standardization Methods. — An excellent dis- cussion of present methods used in the standardization of tuber- culin is included by Long* (1925) in his report of a new method of assaying the strength of tuberculin by means of the "spermatocyte reaction" in tuberculous guinea pigs. Long classifies the methods in use at the present time as follows : "I. Hypersensitiveness of the Tuberculous Guinea Pig. 1. The lethal dose. Method of : Koch The Institute for Experimental Therapy, Frankfurt a. M. The United States Bureau of Animal Industry. 2. The skin test. Method of Lewis and Aronson. "II. Antigenic Capacity of Tuberculin in Serum Reactions. 1. The precipitin test. Method of Dreyer and Vollum. 2. The complement-fixation test. Method of Watson and Heath. ' ' The following description of these methods is based upon Long 's report. Koch's Method. — The discoverer of tuberculin according to Long, "considered a preparation of tuberculin satisfactory if 0.5 c.c. or less would kill in six to thirty hours, with characteristic pathologic change, a guinea pig infected one month previously with tuberculosis. ' ' Method Used ix the Institute of Experimental Therapy, Frankfurt. — This is a modification of Koch's method sug- gested by Doenitz (1921). A large number of guinea pigs, fifty or more, are infected with tubercle bacilli. After defi- nite symptoms of the disease develop two series of six guinea pigs each are selected. One series is injected subcutaneously with varying amounts of Koch's OT while the second series is injected with corresponding amounts of the tuberculin to be standardized. ♦Long: J. Infect. Dis. 37: 368, 192.5. HYPERSENSITIVENESS 511 The smallest dose oi" each that kills within twenty-four hours is determined. The minimum lethal dose of Koch's OT is the stand- ard with which the M.L.D. of the new product is compared. If the latter is larger than the former, then the new product is below standard, while if it is smaller it must be dilnlod 1o correspond to the strengtli of Die old tiil)crculin used as a standard. Method of U. S. Bureau ob^ Animal Industry. — Apparently this is also a modification of Koch's method suggested by Schroeder and Brett (1919). Tuberculous tissue from guinea pigs is inocu- lated into a series of normal guinea pigs. As soon as these latter become sensitive to the extent that 0.25 gram of OT per 500 grams body weight will kill 4 out of 6 pigs within twenty-four hours, the remaining pigs are ready for use in standardizing a new product. They are divided into groups of six. Each animal of group I is inoculated intraperitoneally with 0.25 gram of the standard OT per 500 grams l)ody weight and each of the other groups is inoculated with a corresponding amount of the tuberculin to be tested. Long says that ' ' a preparation of tuberculin is considered as passing the test if under the conditions of the experiment it kills at least one- half as many guinea pigs as the standard tuberculin, with the characteristic lesions of tuberculin death. ' ' Skin Test Method of Lewis and Aronson. — In this method tu- berculin hypersensitiveness is produced in guinea pigs by inoculat- ing intraperitoneally a series of animals, ranging in weight from 200 to 300 grams each, with 0.1 mg. of virulent tubercle bacilli. After three weeks they are ready for use in the standardization of tuberculin. They are divided into groups of six and decreasing amounts of a standard tuberculin are injected intracutaneously after the method of Roemer. Each of the other series is injected in a like manner with corresponding amounts of tuberculin of un- known potency. In the series cited by Long the amounts used for intracutaneous injections were 0.02, 0.01, 0.005, 0.002 and 0.001 mg., brought up to a total volume of 0.1 c.c. by the addition of a di- luting fluid. The results were read after forty-eight hours and re- corded as 1, 2, 3, or 4, the last named figure (4) indicating the strongest reaction. Standardization of Tuberculin by the Precipitation Method OF Dreyer and Vollum. — Immune serum for use in the test is pre- pared by injecting defatted tubercle bacilli in amounts varying 512 IMMUNOLOGY from 0.1 iiig. to 225 mg. into a horse. A period of three montlis is employed for immunization. When the precipitin titer is suffi- ciently high, the horse is bled and the serum obtained for use in the test. The authors find it necessary to employ the optimum proportion technique of Dean which is discussed in the chapter on Precipitins. They choose the Frankfurt standard tuberculin as the standard for comparison, and arbitrarily assign to it a value of 100 units per cubic centimeter. "The value of any other tu])erculin is inversely proportional to the smallest amount re- quired to produce the same degree of precipitation as the stand- ard with the same quantity of serum under the same conditions. ' ' In the opinion of Dreyer and VoUum the strength of a tuberculin determined by the precipitin method compares favorably with tlie strength determined by skin tests on human subjects. Complement Fixation Method of Watson and Heath. — Stand- ardization of Tuberculin by Complement Fixation : This method is credited to Watson and Heath. They prepare an antiserum by injecting intravenously into a horse 1 mg. of virulent, moist tubercle bacilli followed in fourteen days and twenty-one days by 2 mg. and 3 mg., respectively, of the same kind of material. The concentration of complement fixing antibody reaches a peak during the seventh week. At this time the titer is said to be about 0.0066 or 150 units per c.c. when titrated against a fixed dose of standard tuberculin. They state that a fixed dose of 20 units of antibody is the most suitable one for titration of tuber- culin. In standardizing commercial tuberculins they determine the minimum amount of the tuberculin which, when mixed with 20 units of antiserum, will completely fix the unit of complement used. This amount of tulierculin is said to represent one active unit. Objections to Ijethal Tests. — Criticism of tests based upon lethal doses of tuhereulin. As regards the three methods, which it will be recalled involve the death of the guinea pig, there are too many unknown factors that affect the outcome of the test. Among these is the extent of the tuberculosis within the guinea pig. This cannot be known or controlled. In Long's opinion "individual variation (in animals) is so great as to rob the test of much of its value. ' ' HYPERSENSITIVENESS 513 Tlie result of Koch's test does not establish a unit: it is quanti- tative only in a ''pass or fail" manner. Long states that the test will not differentiate between two preparations of tuberculin, one of which might be twice as potent as the other. While the PYankfurt method attempts to avoid this criticism, it does so only by introducing an extremely "laborious process of comparison with a standard tuberculin on a large series of animals." Al- though the test employed by the United States Bureau of Animal Industry is superior in certain respects to other ' ' lethal dose tests, ' ' it too requires a large number of animals and is also quite laborious. Long calls attention to the fact that while a test of this type estab- lishes a "standard which must be met before a tuberculin is ac- ceptable, it does not establish a unit which can be used in measuring doses." Advantages and Disadvantages of the Various Tests. — Criticism of the intracutaneous test. 1. The technique is simple and economical of both time and animals. 2. It would seem more logical to standardize in respect to the skin allergy of a test animal since the product is to be used to determine the skin allergy of patients. 3. Long and others feel that one serious objection to the test is that in a series of tuberculous guinea pigs the reactive capacity of the skin varies tremendously. On the other hand, Seibert and Munday (1932) find that when one pound guinea pigs are inocu- lated subcutaneously with 0.1 mg. of strain H37, they develop quite uniform skin sensitivity. In regard to the standardization of tuberculin they say "since it is possible to obtain consistent results in sensitive tuberculous guinea pigs by means of the in- tracutaneous test, and since the chief use of tuberculin at present consists in diagnostic skin reactions, it is advisable to base the standardization, so far as possible, upon the intracutaneous test as has been maintained by Aronson (1926), Okell and Parish (1927) and Funk and Huntoon (1930)." Criticisms of the complemetit fixation and precipitin methods. The chief objection to both of these methods of standardizing tuberculin is that neither complement fixing nor precipitating antibodies are known to be involved in the tuberculin reaction. 514 IMMUNOLOGY In fact, Parker and others workino- in Zinsser's laboratory have shown that so far as the precipitin reaction is concerned, it is dependent upon an entirely different substance in the tuberculin from that which elicits a positive skin reaction in tuberculous animals. Furthermore, both complement fixing and precipitating antibodies can be produced by injecting tuberculin into noi'nuil animals, but such treatment does not produce tuberculin liypor- sensitiveness such as is observed associated with active tuberculosis. Standardization by Long's Spermatocyte Reaction. — In this test 10 animals are required. Eight are infected by injecting one-half milligram of the Saranac laboratory strain R^ into the groin. This leads to the development of a caseous nodule in the regional lymph nodes within one month, but the process does not become generalized. The animals are injected in duplicate with 0.01, 0.001, 0.0001 and 0.00001 mg., respectively, with the tuber- culin to be tested. The two normal controls are inoculated with the strongest dose. The volume of each dose is made up to 0.1 c.c. Injections are made into the middle of one testicle. The reaction may be read after thirty-six hours or in from tw^o to four weeks, the animals being killed and paraffin or celloidin sec- tions prepared. Spermatocyte Tuberculin Unit. — The spermatocyte tuberculin unit according to Long ''is that quantity of tuberculin just suf- ficient to aboli.sh spermatogenesis in the majority of tubules on injections of a volume of 0.1 c.c. into the testicle of a 400 gram guinea pig with a mild localized tuberculosis of one month's dura- tion. Microscopic se-ction of the testicle (middle cross-section) one month after the injection of one unit or more shows the majority of the tubules atrophic and lined only by spermatogonia. No normal spermatocytes, spermatids, or spermatozoa are present. The test is controlled by examination of the noninjected opposite testicle and of the injected testicle of a noninfected animal, both of which should show normal spermatogenesis. ' ' In Long's opinion this unit is useful in standardizing tuber- culins for use in either therapeutic or diagnostic procedures. He states that tuberculins such as OT and BE contain 10,000 and 1,000 spermatocyte units, respectively, per cubic centimeter. hypersensitiveness 515 Dependable Methods of Tuberculin Production and Stand- ardization.— It would appear from the research so far reported in this chapter that there are at present four methods available for the production of fairly pure tuberculin. The products of these methods have been designated as MA-100, TPA, TFT, and SOTT or PPD, respectively. A new method of producing PPU has been suggested by Seibert and Glenn (1941). It is discussed briefly later in this chapter. Because all of these tuberculins except the purified protein derivative are more or less antigenic and lead to the development of skin hypersensitiveness in some individuals wlien injected repeatedly, Long (1934) recommends that the latter (PPD) be used in determining the presence of tuberculin allergy in man. There have been developed also two methods of standardiza- tion tliat give wluit appear to be consistent and dependable re- sults. These may be designated as the standardized intracutane- ous method of Aroiison (recommended by Seibert) and the spermatocyte reaction of Long. Diagnostic Tuberculin Tests. — In diagnostic Avoi-k both local and systemic reactions have been used to determine tuberculin hypersensitiveness. Among the tests based upon local allergic phenomena may be mentioned the cutaneous tuberculin test of von Pirquet, the percutaneous tuberculin reaction of Moro, the intra- cutaneous test of Mantoux and the ophtlialmo-tuberculin reaction of Calmette or a similar one called the conjunctival-tuberculin reaction of Wolff-Eisner. Koch first employed a subcutaneous tuberculin test in which a positive reaction is indicated by both a local and a systemic reaction. Tlie technique and allergic phe- nomena involved in each of the various tests may be summarized as follows : The von Pirqukt Test. — Tlie technique of the von Pirquet test consists in first washing the inner surface of the forearm with ether and applying two drops of Koch's OT to the surface of the skin about 10 em. apart. Then with a sharp instrument one scarifies by pressure and rotation a small area about midway between the two drops of tuberculin. This serves as a control. Similar scarifications are then made through each drop of tuber- culin after which the latter is allowed to dry. While dressing is 516 IMMUNOLOGY not necessary, tlie areas are frequently covered with small pieces of sterile gauze held in place by adhasive tape. The result of the test according to von Pirquet may be a trau- matic, a negative or a positive reaction. The traumatic reaction may appear as an area of hyperemia or the latter may surround a small wheal. These disappear usually within twenty-four hours. There may remain a slight redness and a small crust for a few days. The negative reaction may also resemble the traumatic reaction. According to von Pirquet the redness and swelling should not exceed 5 mm. after twenty-four hours and this should not persist. Positive reaction. This begins as a rule after a latent period of several hours. It is characterized by an area of edema (swelling) one or more centimeters in diameter accompanied as a rule by hyperemia (redness). The swelling reaches its height in about forty-eight hours and then begins to subside. The color may be- come darker and even develop a yellowish tinge. The swelling usually disappears within a few days to one week and there re- mains not infrequently an area of pigmentation. MoRo's Percutaneous Test. — To use the percutaneous test of Moro one must prepare a salve by triturating equal parts of old tuberculin and lanolin. A piece of this about tlie size of a pea is rubbed for one minute into an area of skin about 5 cm. in diameter over either the pectoral region or the epigastrium. The salve is then allowed to dry for about ten minutes. A positive re- action is indicated by the development of nodules, a rash or of small vesicles in the area tested. The test is not in use at the l)re.sent time. Wolff-Eisner Test. — The conjunctival-tuberculin test of Wolff- Eisner is made by instilling one drop of a 1 per cent solution of OT into the conjunctival sac. When positive, the reaction ap- pears as either a simple hyperemia, an enlargement of the follicles or a severe conjunctivitis coming on within sixteen to twenty-four hours. It is no longer regarded as safe for use in testing children because of severe reactions and permanent injury. Patch Test. — Pearse, Fried, and Glover (1940) state that the Volmer patch test is as reliable as the Mantoux test. Mantoux Ticst. — The intracutaneous test of Mantoux is now regarded by many as the most satisfactory method of determining tuberculin hypersensitiveness. The dosage employed by different HYPERSENSITIVENESS 517 individuals and the results which they report may be summarized as follows : Funk and Huntoon's Results With OT and MA-100.— In 1930 Funk and Huntoon* performed intracutaneous tests in 441 indi- viduals using; both Koch's old tuberculin and the new product j\[A-100. In regard to the appearance of the reaction they say, '"The reaction is usually an erythema surrounding an area of edema, although occasionally the edema is larger in area than tlie erythema, or rarely, edema without erythema. The erythema may vary from a very faint blush to a dark red, may be diffuse in character, or very sharply limited. The edema varies from a barely perceptible (to the touch) thickening to a marked indura- tion several millimeters in height. ' ' The reactions come on within twenty- four hours; about half of them begin to fade before, and all after, forty-eight hours. They state that pigmentation occurs but is less likely to follow with the protein than when OT is used. Dosage Employed. — They adopted a dosage of 0.00001 c.c. of OT and 0.0005 mg. for the MA-100. These respective tuberculins were diluted so that 0.05 c.c. contained the test dose for the Man- toux test. They used tuberculin syringes (1 c.c.) of the Luer type with a number 24-gauge % inch needle. The level of the needle was turned up, i.e., in line with the graduations. After washing the anterior surface of the forearm with alcohol and drying it, they inserted the needle into the skin until the bevel was completely covered although dimly visible. The test dose contained in 0.05 c.c. of solution was then injected, producing a small wheal about 5 to 7 mm. in diameter. The arm was then wiped with alcohol. Reading the Reactions. — They made the first reading between twenty-three and twenty-five hours after the injection and a second reading after forty-eight hours. If the first reading was negative they made a second test injection using 0.1 c.c. of the same dilution. In other words, they doubled the dose for the second test. In read- ing the reactions they noted the intensity of the color of the hyperemia and measured the longest and shortest diameters of liotli the erythema and edema. The latter information was recorded in millimeters. The measurements of the reactions o])served in 218 individuals positive to both OT and MA-100 showed an average diameter of ♦Funk and Huntoon: J. Iniinunol. 19: 237. 1930. 518 IMMUNOLOGY 17 mm. for the erythema and 13.4 mm. for the edema produced by OT as compared with 20.1 mm. for the erythema and 11.2 mm. for the edema caused by the MA-100. They observed no necrosis in any of the reactions with MA-100 although the induration per- sisted for several weeks in a few individuals. Occasionally super- ficial necrosis was observed in the reactions produced by OT. No general symptoms were noted in any of the individuals tested. Incidence of Reactions in Known and Suspected Cases of Tuberculosis. — In a series of 204 cases of pulmonary tuberculosis 94.1 per cent were positive to MA-100 while 90.6 per cent were posi- tive to tuberculin OT. In 57 cases of suspected tuberculosis the percentage of positive reactions was 92.9 with MA-100 and 91.2 with tuberculin OT. Incidence of Reactions in Clinically Nontuberculous Cases. — It is interesting to note that in a scries of 137 adult patients, all clinically ?ir>ntube7'culous, Funk and Iluntoon found 81 per cent positive reactions to MA-100 and 71.5 per cent to tuberculin OT. In another series of 25 cases also clinically nontuberculous the percentages of positives were 80 and 68, respectively. They also tested 43 children whose ages varied from one to twelve years (average 5.3 years) and found 25.5 per cent positive to J\IA-100 and 16.2 per cent to tuberculin OT. Noncorrelative Reactions. — In the series of clinically tuber- culous cases cited above there were 12 individuals who reacted to tuberculin MA-100 and not to OT and 5 who reacted to the latter but not the former. Cases of Tuberculosis That Did Not React. — There were also 13 clinically tuberculous individuals who did not react to either MA-100 or OT. Eight of these patients were quite ill, three of them were described as cachectic. The condition of the five re- maining individuals was regarded as either fair or good. In this connection Rich and McCordock (1929) as well as others call at- tention to the fact that tuberculin tests may be negative in 3 to 5 per cent of the cases of miliary tuberculosis and may be sup- pressed in certain of the exanthematous diseases as well as during labor and the puerperium. Mariette and Fenger's Results With MA-100 and OT.— More recently Funk, Huntoon and White have collaborated with Mari- ette and Fenger (1932) in an extensive study of the skin reactions HYPERSENSITH^ENESS 519 to MA-100 and OT in tuberculous and nontuberculous subjects. In this work the skin reactions with OT and the MA-100 pi'otcins of the timothy bacillus and tlie human, bovine and avian tubercle bacillus were studied in 225 individuals by Funk, Huntoon and White and in 3,454 by Mariette and Fenger. They employed the following dosage in these investigations: Tuberculin OT "Dorset": Dose 0.05 c.c. = 0.00001 c.c. commonly designated at 0.01 mg. MA-100: Human tubercle bacillus protein: 0.05 c.c. = 0.0005 mg, MA-100: Bovine tubercle bacillus protein: 0.05 c.c. = 0.0001 mg, MA-100: Avian tubercle bacillus protein: 0.05 c.c, = 0,001 mg. MA-100: Timothy bacillus protein: 0,05 c,c, := 0,01 mg. If the reaction is negative at the end of forty-eight hours, repeat with double the dose. The statistics of Funk, Huntoon and White are fairly repre- sentative of the general results and may be tabulated as follows: TEST MATERIAL Tuberculin OT "Dorset" MA-100 Human tubercle protein MA-100 Bovine tubercle protein MA-100 Avian MA-100 Timothy protein In a larger series studied by Mariette and Fenger, the ]\IA-100 human tubercle protein gave about 10 per cent more positives among adults and 2 per cent among children classified as sana- torium patients than tuberculin OT. In tlie general population i\[ariette and Fenger report that tuberculin OT gave 27.2 per cent positives among adults and 6.9 per cent positives among chil- dren, whereas ]\IA-100 human tubercle protein yielded 39.9 and 8.5 per cent positives, respectively, in the same individuals. From these studies they draw the following conclusions : 1, That MA-100 human tubercle protein is probably more sensi- tive than OT. 2, The initial and repeat doses may be administered safely to tuberculous patients. 3, Larger doses are not necessary to detect the majority of tuber- culous individuals, 4, Large doses of MA-100 proteins give nonspecific results. 5, There is a protein common to all members of the acid-fast group which when administered in large doses gives a re- action similar to a positive tuberculin (OT) reaction. G, They regard MA-100 tubercle protein as purer and better than OT for determining tuberculin hypersensitiveness. CLINICALLY CLINICALLY TUBERCULOUS NONTUBERCULOUS PER CENT PER CENT POSITIVE POSITIVE 87.4 08.5 84,6 73.1 82.0 69.5 94.0 87,0 95.7 87.9 520 IMMUNOLOGY Slater and Jordan's Studies of the von Pirquet and Mantoux Tests. — Slater and Jordan (1932) made a comparative study of the von Pirquet and Mantoux tuberculin tests in school children. For the former they employed Koch's old tuberculin and the usual von Pirquet technique. In conducting the Mantoux test they injected intracutaneously 0.1 c.c. of a 1 :1,000 dilution or approxi- mately 0.1 mg. of Koch's old tuberculin and read the results forty- eight hours later. Both tests were given at the same time, one on each forearm. In all they tested 1,006 white children of whom 134 reacted to one or both tests. It is of interest to note that a physical examination and x-ray plates of the chest of each of the 134 positive reactors revealed lesions of tuberculosis in only fifteen individuals. In regard to the relative delicacy of the two tests they report that of the 134 giving positive tests 42 reacted to the Mantoux but not to the von Pirquet, 63 reacted to both, while 29 were positive to the von Pirquet but not to the Mantoux. These children were all from schools in a district where during the last five years the death rate from tuberculosis has been less than 25 per 100,000 population. In contrast to this they tested 62 children from 19 families in which known cases of open tuberculosis were present. Fifty-one of the children reacted to one or both tests. Of these positive reactions six were to the Mantoux but not to the von Pirquet and two to the von Pirquet, but not to the Mantoux. In the author's opinion both tests are good, but not infallible, the Mantoux being more sensitive and perhaps better adapted for older children, while the von Pirquet gave good results with younger children. Survey to Determine Primary Tuberculosis Infection Attack Rate. — Stewart, Harrington, Myers, Boynton, and Streukens (1939) employed 0.1 mg. of old tuberculin intradermally to test 3,868 persons, representing five different groups. Thej^ estimate that the children tested became infected with tubercle bacilli at the rate of 0.8 per cent of the group annually. The parents had an attack rate of about 1.6 per cent annually. Among college students they report that 5.8 per cent became sensitive to tubercu- lin during their four-year course at the university. The most dis- turbing figures they give pertain to medical students. They state that of 265 who gave negative reactions to 0.1 mg. of old tuberculin at the beginning of their senior year, 118 Avere positive at the end HYPERSENSITIVENESS 521 of Ihiit scliool year. This constituted an attack rate of 44.5 per cent which was sixteen times the annual rate noted for the first three years of the course. They say that this rate of 44.5 per cent is fifty-six times the infection attack rate they found for children in the city schools who resided in private homes. Stewart and his associates also report high attack rates in private hospitals, es- l)ecially those that maintain wards for tuberculous patients. Significance of Skin Reaction in Children. — Stewart (1934) has published an interesting paper on the skin sensitiveness to tuber- culin in primary tuberculosis. He reports the results of extensive roentgenologic and clinical investigations of 188 children ranging in age from 8 to 171/) years who gave positive skin reactions to the intracutaneous administration of 0.1 mg. of old tuberculin. In 132 children of this group he found either characteristic cal- cified hilus glands or Ghon tubercles or both, twenty-one showed slight or questionable calcification of hilus glands, eight showed only pleural thickenings and twenty-one showed negative chest plates. He states that clinically the entire group showed no symp- toms of tuberculosis and could be distinguished from normal unin- fected children only by means of the tuberculin test and roentgen examinations. Tuberculin Surveys With PPD. — Long, Aronson and Seibert (1934) report on results obtained in tuberculin surveys with the purified protein derivative and a highly potent OT, i-espectively. This woi-k was done, apparentlj^ with an idea of determining the potency, uniformity, and general reliability of the new tuberculin, Tlie results of their work may be partly summarized as follows : 1. They found that an initial dose of 0.00002 mg. of the purified protein derivative detects a high percentage of total positive re- actors to OT with very few severe reactions. The remaining posi- tive reactors to 0.01 mg. and 1.0 mg. of highly potent OT are detected by a dose of 0.005 mg. of PPU. 2. They recommend that PPD be incorporated into sterile lac- tose tablets in quantities of 0.0002 mg. and 0.05 mg. per tablet for convenience in the preparation of ten first or second doses to be used in tuberculin tests. Park, Kereszturi, Mishulow (1933) also point out the superiority of the tuberculin test over the x-ray as an aid in the diagnosis of 522 IMMUNOLOGY primary tuberculosis. The roentgenologic findings are of great value, however, in the diagnosis of the reinfection or adult type of tuberculosis. Effect of Age Upon Diluted Tuberculin. — In regard to the effect of age upon dihited tuberculin Aronson says that Mantoux as early as 1909 noted that its potency decreased on standing and that Okell found that a 1 :1,000 dilution of OT shows a diminution of potency, if kept at room temperature, of 40 per cent within ten weeks, and 60 per cent within one year. Aronson has observed that a 1 :10 dilution of OT, incubated at 37° C, shows a marked loss of potency within seven to ten days and is practically inactive after several months. Undiluted tuberculin or the purified protein derivative incorporated with beta lactose in tablet form is quite stable. System of Grading Mantoux Tests. — Aronson (1934) em- ploys an initial skin test dose of 0.00002 mg. of PPD, reads the results forty-eiglit liours later and reinjects all who are negative with 0.005 mg. of PPD. These doses of the purified protein de- rivative correspond to 0.01 mg. and 1.0 rag. of OT, respectively. The system of grading which he has finally adopted is a slight modification of the one previously recommended by the National Tuberculosis Association. He says that the new system of grading enlarges the two-plus range and thus takes in some that were formerly graded three-plus. The new classification of tuberculin reactions is as follows: + Some redness and an area of edema more than 5 and less than 10 mm. in diameter. + + Area of redness and edema 10 to 20 mm. in dia- meter. + -I- 4- Area of redness and edema exceeding 20 mm. in diameter. + ^^ ^ Marked redness, edema and an area of necrosis. Doubtful Tliese show redness and traces of edema measur- reactions ing 5 mm- or less in diameter. Redness is always regarded as of less significance than edema. Long (1934) states that during the past twenty-five years there has occurred a reduction in the incidence of tuberculosis in the United States. He suggests that the antituberculosis campaign which has been carried on for many years is largely responsible for this reduction in the incidence of the disease. H YPER.SKXSITIVENESS 523 Incidence of Tuberculosis Determined at Autopsy.— In 1917 Opie reported evidence of tuberculosis in 8.3 per cent of infants dying within the first two years of life, 44 per cent in children dying between the ages of two and ten years, 66.7 per cent for ages ten to eighteen years, and 100 per cent for those dying at later ages. These percentages include many cases of healed tuberculosis as well as cases that showed activity. In 1927 Opie and Aronson studied material from 169 bodies that showed what appeared to be healed lesions. By means of guinea pig inoculation they found that 20 out of 29 apparently healed lesions contained living tubercle bacilli. In a later discussion of the epidemiology of tuberculosis, Opie (1932) calls attention to the continued high incidence of tuber- culosis in Philadelphia and to the appalling number of serious tuberculosis infections revealed by anatomical studies. He states that such studies made in St. Louis and Philadelphia indicate that one out of every six white adults who die from diseases other than tuberculosis have partially caseous lesions at the apices of one or both lungs. He seems to interpret the diminution of the death rate from tuberculosis as indicating that the infection is now following a more benign course than formerl3^ Opie points out that the skin test is the most delicate method of detecting infection, although it does not differentiate between latent and manifest tuberculosis. Robertson's Anatomic Studies. — On the other hand, the studies of Robertson (1933) and also of Terplan (1934) apparently sup- port the statement of Long that the incidence of tuberculosis is less than it was twenty-five years ago. Robertson reviews the series of 3,306 autopsies done at the Mayo Clinic over a period of six years (1926-1931) to determine the incidence of tuberculosis. He found tuberculous lesions present in 2,064, or 62.43 per cent, of the bodies examined. As a result of histological studies he con- cludes that the lesions in 1,725 of these cases weie healed and that there is evidence of active tuberculosis in 134, or 4.05 per cent. The incidence of active tuberculosis for the various age groups is interesting. For the first three decades of life it was 2.94, 6.71 and 6.84 per cent, respectively. For the decades between 30 and 70 years it was 3.78, 3.17, 4.47, and 3.99 per cent, respectively. 524 IMMUNOLOGY Terplan reports the anatomical incidence of tuberculosis among 312 white children whose ages are between one month and six years as 4 per cent; in 52 individuals ranging in age from seven to eighteen years, it is 20 per cent; while for the age groups twenty to forty and forty-one to eighty years, it is 60 and 95.7 per cent, respectively. Terplan calls attention to the close agreement be- tween the incidence of 20 per cent having tuberculous lesions de- termined at autopsy for the age group seven to eighteen years and the 20 to 25 per cent positive reactors to tuberculin (Mantoux test) among the school children of Buffalo. It should be remem- bered that the majority of the anatomical tuberculous lesions were healed. In this study Terplan found some primary lesions in all age groups. In discussing this report Long remarks that Terplan has offered definite evidence that adults who escape primary tuber- culosis infection in childhood are not likely to break down with massive primary tuberculosis as was formerly thought to occur. Apparently primary tuberculosis in adults tends to pursue a course similar to that observed in children. Tissue Response to Various Fractions of the Tubercle Bacillus. — The effect on the allergic animal of various fractions of the tubercle baciUus injected into the tissues has been studied exten- sively by Sabin and her associates. They report that the phosphatid fraction stimulates the formation of epithelioid and giant cells while the acetone-soluble fat causes the proliferation of connective 1 issue and blood vessels and also causes hemorrhages. The waxlike constituents stimulate fibroblast proliferation, and the carbohydrate fraction is both chemotactic and toxic for leucocytes. When the tubercular protein fraction is injected, both fever and the prolifera- tion of plasma cells result. The Necessary Factors for the Development of Tuberculin Allergy. — Wliile one can produce antilwdies for and render a guinea pig anai^hylactic to tuberculin protein, it is interesting to note that tuberculin hypersensitiveness or allergy as previously described in this chapter does not result from the injection of tuberculin OT or any of the others described by Koch (Baldwin, 1910, Fleischner, Meyer and Shaw, 1919, Zinsser, 1931). The necessary factor for its production is the formation of a tubercle containing living or dead tubercle bacilli or a tuberculin that is antigenic; e.g., MA-100 within the tissues of a susceptible HYPERSENSITIVENESS 52§ animal or experimental conditions described by McJunkin and confirmed by Zinsser. McJunkin injected tubercle bacilli in- to the peritoneal cavity of infected guinea pigs and 24 hours later removed the peritoneal exudate, filtered and injected it into normal guinea pigs. A number of these normal recipients showed tuberculin hypersensitiveness when tested six or eight days later. Baldwin (1910) and Zinsser (1931) suggest that per- haps the train of events leading to the allergic state in tu])erculosis may be described somewhat as follows : First tlie tubercle bacillus lodges in the tissues and the ])ody reacts to its presence by the formation of a tubercle. The action of the inflammatory tissue ui)on the bacteria, probably by means of enzymes, causes the liberation of an antigenic substance which is responsible for tuberculin allergy. It is only recently that one has been able to extract from the tubercle bacillus such an antigenic substance and no one has been able to prove that anti- bodies are involved. It is interesting to note that while tuber- culin OT is devoid of skin sensitizing power, such tuberculins as MA-100 and TPT possess it. At first Seibert believed that OT and likewise PPD were not antigenic because of their small molecular weights and conversely MA-100 and TPT were antigenic be<^ause of their large molecular weight. Subsequent work has led Seibert (1941) to alter her views. She is now inclined to think that potency is inherent in one part of the tuberculin pro- tein molecule while antigenicity is dependent upon another part of the same molecule. Apparently the work of Landsteiner (1935) (1938-1940), as well as the work of StuU and Hampton (1941), indicates that less complex substances than complete proteins can function as antigens. StuU and Hampton report that certain proteoses obtained from Witte's peptone are antigenic. It is pos- sible, as suggested by the work of Landsteiner, that these sub- stances function as haptens and unite with body proteins to form complete antigens. Seibert 's work suggests that tuberculin may function as a hapten when it is antigenic. A somewhat similar state of affairs exists in undulant fever. Fleischner, Meyer and Shaw (1919) as well as others have shown lluit while guinea pigs can be rendered anaphylactic by injecting suspensions of Brucella abortus and melitensis, skin sensitiveness does not develop unless active infection is present. 526 IMMUNOLOGY As a result of numerous investigations of the tuberculin, ty- phoidin, abortin and mallein reactions, which are of the tuber- culin t.ype, it appears that certain essential points of dissimilarity exist between protein anaphylaxis and bacterial allergy of the tuberculin type. An interesting review of the early literature along with important experimental data bearing upon this prob- lem is included in an excellent paper by Fleischner, Meyer and Shaw (1919). For a more extensive discussion of Brucellosis the student is referred to a very interesting paper by Giltner (1934). In 1921 Zinsser made an extensive study of bacterial allergy and anaphylaxis and summarized the points of difference between the two phenomena. Aronson (1933) investigated by means of tissue cultures the relation of the tul)erculin reaction to anaphy- laxis and the Artlius phenomenon. He confirmed the results of Rich and Tjcwis who found that tissue explants of l)one marrow and spleen from tul)erculous guinea pigs are killed by dilute solutions of tuberculin tliat are nontoxic for similar explants from normal animals. When lie adds dilutions of horse serum to similar ex- plants from guinea pigs sensitized to horse serum the tissue cells are not killed but grow as well as explants from normal guinea pigs used in control experiments. These findings are in harmony with the tissue culture studies of Meyer and Loewenthal (1927), cited by Aronson, although contradictory results are reported by others. The differences suggested by Zinsser have been tabulated by Dienes and Mallory (1932). Their table, with the addition of the difference suggested by Aronson (1933) and others, is shown in Table XYI. Possible Explanation of Apparent Dissimilarities Between Anaphylaxis and Tuberculin Allergy. — It is generally agreed that the union of antigen and antibody is in some manner respon- sible for anaphylactic reactions The first two points of dissimilarity between the latter and tuberculin allergy tabulated above would seem to indicate that the tuberculin reaction is not mediated by a similar mechanism. That such a conclusion is not warranted at the present time is indicated by the experimental results of Dienes and Schoenheit (1929, 1930) and of Dienes and Mallory (1932). The former report that when tuberculous guinea pigs are sensitized to crystalline egg albumen by injecting it directly in- to a tuberculous lesion and subsequently testing by injecting 1 1 YPKKSEN.SITIVEX KSS 527 Table XVI ANAPHYLACTIC TYPE TUBERCULIN TYPE Skin test Immediate Transitory Delayed Prolonged (Serum Antibodies demonstra- ble Passive transfer pos- sible Antibodies not demonstra- ble. Passive transfer not demonstrable Kesulta of intravc- Acute shock Dclajed shock nous injection Sensitizing antigen Testing antigen Tissue culture re- sponse to antigen Ordinary proteins Bacteria and some of their protein-contain- ing products Proteins Carbohydrate fraction of bacteria (appar- ently the most effec- tive) Transplants of bone marrow and spleen from sensitized ani- mals not killed by antigen Bacteria, best living but also killed if in condi- tion to produce granu- lomatous tissue responses in tuberculosis. Tuber- culins MA-100 and TPT Bacterial proteins and pro- tein fractions only Transplants of bone mar- row and spleen from tu- berculous animals killed bv tuberculin crystalline egg albumen intraeutaneonsl.y, the resulting reaction is specific and of the tuberculin tj'pc, i.e., it is delayed and prolonged. When the sensitizing dose of egg albiunen is administered elsewhere tiian directly into a tuberculous lesion, a skin test performed eight to ten days later with the homologous antigen results in an im- mediate and transitory or anaphylactic type of reaction. Certain reports in the literature of exceptions to these results suggested to Dienes and Mallory that a time factor might be im- portant. They, therefore, sensitized normal uninfected guinea pigs to egg albumen and egg globulin and noted the type of re- action when skin tests were made with the homologous antigen at four-, six-, eight-, and ten-day intervals following the adminis- tration of the sensitizing dose of antigen. They discovered that re- actions obtained on the sixth day and earlier were of the tuberculin type, while the reactions obtained later were of the anaphylactic type. When skin tests were performed upon guinea pigs pas- 528 IMMUNOLOGY sively sensitized to egg albumen, they invariably observed the anaphylactic type of response. Histological studies of the various skin reactions confirmed the gross findings. The anaphylactic reaction in passively sensitized guinea pigs is characterized by an immediate serous and poly- morphonuclear exudate. In guinea pigs actively sensitized to egg albumen and tested on the sixth day the reaction is delayed. It appears in about six hours and persists forty-eight hours or longer. Microscopically the cellular reaction is predominantly mononuclear. When the actively sensitized pigs are tested on the eighth day or later the reaction is immediate and the cellular exudate contains a predominance of polymorphonuclear cells. Similarity Between Immune Response and Tuberculin Allergy. — The authors believe that the ratio of mononuclear to polymorphonuclear cells is a valuable criterion of differentiation between the two types of hypersensitiveness. They conclude that the tuberculin type of hypersensitiveness represents the first stage of the immune response to the introduction of any antigen within the tissues of a suitable animal. In tuberculous animals there is a fixation of this early stage for tuberculin protein so that skin reactions to tuberculin are always of the tuberculin type. In protein anaphylaxis, on the other hand, there is no fixation of the early stage, but, instead, the anaphylactic type supervenes after the sixth day. The observation of Dienes that the injection of egg albumen and other protein antigens directly into a lesion causes in some manner subsequent skin reactions to the homologous antigen to be of the tuberculin type, whereas antigen injected elsewhere leads to the anaph.ylactic type of reaction, may be explained in part at least on the basis of the time factor. If the egg albumen is injected directly into a lesion, its aljsorp- tion may be delayed and the primary stage of tlie immune re- sponse therefore prolonged. Dienes' work suggests that dosage and other factors as yet unknown are involved. In his experi- ments where sensitizing injections of egg albumen were made away from the lesion it is interesting to note that skin tests were never done as early as the sixth day, hence the early stage typical of the tuberculin type of allergy was not detected. A careful reading of Dienes' papers reveals many exceptions and also nu- HYPERSENSITIVENESS 529 merous mixed reactions: hence it is probable that host variation and other factors affect the results obtained. Hanks (1935) confirmed the Dienes phenomena but he con- cluded that it is not necessary to inject the antigen into a tubercu- lous focus. Zinsser, Enders, and Fothergill (1939, p. 429) ques- tion Hanks' conclusion as to the latter point. In addition to these results which suggest that tuberculin al- lergy in tuberculosis is mediated by an antigen-antibody mecha- nism, there are the observations of Rich and his colleagues as well as others before them that the tuberculous animals can be desensitized to tuberculin. Summary of Evidence Indicating an Antigen-Antibody Mechanism. — The evidence which seems to indicate that bacterial hypersensitiveness due to infection is mediated by an antigen- antibody mechanism may be summarized as follows : 1. It results only from the presence of an antigenic complex, the whole bacterial cell, or from the introduction of a tuberculin such as MA- 100 within the tissues of the body. 2. An incubation period comparable to that in anaphylactic sen- sitization is present. 3. The reaction is specific. 4. The early skin reaction in animals sensitized to egg white and other soluble proteins is similar to the tuberculin reaction in tuberculous guinea pigs. 5. In animals sensitized to the pneumococcus a typical tuber- culin type of skin reaction can be demonstrated. 6. Desensitization to tuberculin can be accomplished. One must admit that although the evidence just cited is strongly suggestive it cannot be regarded as proof that antibodies partici- ])atc in tlie 1ul)orcu]in skin reaction o])served in tuberculous ani- mals. Aronson (1933) calls attention to a number of other explana- tions that have been offered by various investigators. He says that Bordet believes that the reaction may be due to an increased affinit}^ of the tissues of a tuberculous animal for tuberculin ; Koch assumes that tuberculin contains a necrotizing substance that is especially toxic for the white cells of tuberculous animals; Babes and Proca assume that it is a summation effect due to tuberculin 530 IMMUNOLOGY and a similar substance present in the tuberculous lesion; Selter regards tuberculin as a specific irritant for tuberculin-sensitive cells and assumes that its action is catalytic in that it promotes but does not participate in the reaction. The antigen-antibody theory was suggested by von Pirquet and Schick in 1903 and is tentatively accepted by Zinsser, Enders, and Fothergill (1939). If antibodies do not participate in the reaction, then to us the most plausible alternative would be to accept the hypothesis tliat the tissues acquire the property of reacting directly with tuberculhi without the participation of antibod.y. The differences in the severity of tissue reactions (pointed out by Aronson and others) in tuberculin allergy and protein ana- phylaxis, respectively, do not, in our opinion, invalidate the concept that the tuberculin reaction is mediated by an antigen-antibody mechanism. These results might be interpreted as indicating one or more of the following : a. A qualitative or quantitative difference in antibodies which leads to a greater degree of sensitization in tuberculin allergy than in protein anaphylaxis. Under such conditions the meeting of the antigen (tuberculin) and its antibody might produce either a more profound physical change than in protein anaphylaxis or liberate more histamine or perhaps some substance with greater toxicity than is liberated in protein anaphylaxis. b. The site of the reaction in tuberculin allergy may be intra- cellular, while in protein anaphylaxis it may occur at the cell surface. Such a difference in the field of operation might ex- plain the difference in toxicity. 7. According to Seibert a tuberculin preparation (SOTT) was able to inhibit the precipitin reaction when tubercle protein and its homologous antiserum Avere brought together. The tuberculin (SOTT) had a molecular weight of 3,800. It apparently acted as a hapten. Questions Raised by Antigen-Antibody Concepts. — For the present we are accepting the theory that the reaction of antigen and antibody is an important factor in tuberculin hypersensitive- ness. When one takes this stand he is confronted immediately with questions relating to the nature of the sensitizing and reacting substance, the site of antibody formation, the principal tissues in- HYPERSENSITIVENESS 531 volved in the reaction, how antigen and antibody elicit the delayed response and the relationship of tuberculin allergy to immunity. While it is true that most of these questions cannot be answered, yet it is possible to state a few experimentally derived facts that seem to fit into the complex picture and which may be regarded as partial answers to some of these questions. In regard to the nature of the sensitizing substance responsible for tuberculin allergy, it seems that either the entire tubercle bacillus or an antigenic tuberculin must be present in the tis- sues of the body for sensitization to occur. Zinsser believes that the sensitizing antigen is a nucleo-protein whose antigenic prop- erty is destroyed during attempted extraction. This is apparently confirmed by Seibert's work (194-1). Antigenic Factors Present in Acid-Fast Bacteria. — Typical skin reactions are elicited by either large or exceedingly small doses of tubei'culin or by large doses only of bacterial protein from other members of the acid-fast group. This suggests that the tubercle ))aeillus contains an antigenic fraction common to the timothy bacillus and other members of the acid-fast group. On the other hand, Menzel and Heidelberger (1938) have isolated several fractions of proteins that differed chemically and sero- logically in each of the following acid-fast bacteria: human, bovine and avian tubercle bacilli and the timothy-grass bacillus. In regard to the site of antibody formation one has what ap- pears to be only a few alternatives, the tuberculous lesion itself, the reticulo-endothelial and other tissues of mesenclwmal origin, and the epithelial tissues. It has been generally assumed that antibodies are produced by certain tissues of mesodermal origin. Epithelium as the Site of Antibody Production and Antigen- Antibody Reaction. — ^Since Dienes (1933) has presented evidence that in tuberculin skin allergy the cells of the epithelium rather than the subepithelial tissues are the primary sites of the reaction, and since all attempts at passive transfer of antibodies have failed, one is faced with the possibilitj^ that if antibodies are involved in the skin reaction, they may be produced and retained by the cells of the epithelium and that the systemic reaction is due to antibodies produced by other tissues of the body. This is suggested also by the tissue culture work of Rich and Lewis (1927). They report 532 IMMUNOLOGY that transplants of bone marrow and spleen from tuberculous animals are killed by dilutions of tuberculin that have no effect up- on similar culturas from normal animals. Furthermore Dorset, Henley and Moskey (1927) obtained a fraction of tuberculin which produces severe and fatal systemic reactions in tuberculous guinea pigs but is inert when administered intracutaneously. In the systemic reaction the tissues immediately adjacent to the lesions give the greatest allergic response. Studies of Corper and Cohn. — These authors compare tuberculo- anaphylaxis and tul)erculo-allergy. The former was produced by injection of tubercle protein wliile the latter was produced by infection. The differences they found between the two are: 1. Tuberculo-anaphylaxis symptoms could be elicited only when the shocking dose was given intravenously, whereas tuberculo- allergic intoxication could be induced by intravenous, subcuta- neous, or intraperitoneal injections. 2. The symptoms of tuberculo-anaphylaxis were the typical ones of all protein anaphylaxis, whereas in tuberculo-allergic in- toxication the symptoms were slow to develop, coming on in from a few hours to seventy-two hours, and did not resemble those of anaphylaxis. Corper and Cohn also report that histaminase had no retarding or neutralizing effect on either tuberculo-anaphylaxis or tuberculo- allergy. From the preceding discussion of tuberculin and other bacterial allergies, the following general conclusions can be drawn : 1. That tuberculin allergy develops from infection or vaccina- tion with the tubercle bacillus or with an antigenic tuberculin. 2. The allergic state apparently results from the effect upon the tissues of some diffusible substance formed within a tubercle, presumably by the action of certain cellular enzymes within the lesion, upon the tubercle bacilli present. 3. The safest method of detecting tuberculin allergy is by the intracutaneous administration of small doses of tuberculin. It has been demonstrated that relatively large doses may give nonspecific reactions. 4. Allergy does not persist long after recovery. hypersensitivp:ness 533 5. It may be temporarily removed by desensitizing the patient or the same may occur as a result of massive infection. 6. The reaction may be suppressed during an attack of an acute exanthematous disease or during labor and the puerperium. 7. In some 3 to 5 per cent of the cases of miliary tuberculosis the reaction to tuberculin OT is absent. 8. The typical tuberculin reaction is of the delayed type and persists for several days. It is characterized by edema, more or less hyperemia and infiltration. Microscopically there is an initial neutrophile response followed by a preponderance of mononuclear over polynuclear cells. The cells are for the most part of vascular origin. 9. A similar reaction is demonstrable in protein anaphylaxis when skin tests are made on the fourth to the sixth day after the sensitizing dose is administered. When skin tests are made after the sixth day the reaction is immediate and of the anaphylactic type. The latter reaction is characterized by hyperemia, wheal formation, and an exudate containing a preponderance of neutro- philes. 10. While antibodies have not been detected in tuberculin allergy, there is some evidence which suggests that the tuberculin reaction is mediated by an antigen-antibody mechanism. 11. It is suggested by Dienes that in tuberculosis there is a fixation of the early anaphylactic type of response. 12. In public health work the tuberculin test is of value in screening out of a large group of individuals for further study, those who have healed, potentially active, or active lesions of tuberculosis. 13. One cannot differentiate between primary and reinfection types of tuberculosis by means of the tuberculin test. 14. Likewise the severity of the reaction cannot be correlated with the type of lesion present. 15. Corper and Cohn report that the symi)toms of tuberculo- allergic intoxication are different from those of tuberculo-anaphy- laxis. They also state that histaminase does not prevent or modify the sjonptoms of either. 16. The purified protein derivative of tuberculin prepared in accordance with tlu; recommended procedure of Seibert is gener- ally regarded as superior to OT. 534 IMMUNOLOGY 17. While anaphylactic sensitization can be produced with killed suspensions of the typhoid and abortus organisms, skin allergy is produced in experimental animals only when living organisms are injected and infection established. References Aronson, Joseph D.: Tissue Culture Studies on the Relation of the Tu- berculin Reaction to Anaphylaxis and the Arthus Phenomenon, J. Immunol. 25: 1, 1933. See Twenty-Fourth Report of Henry Phipps Inst., 1932-33. Aronson, Joseph D., Zacks, David, and Poutas, J. J.: The Comparative Sensitiveness of Pirquet and Intracutaneous Tuberculin Test, Am. Rev. Tuberc. 27: 465, 1933. Baldwin, E. R.r Studies in Immunity to Tuberculosis. I. Hypersuscepti- bility or Anaphylaxis, J. Med. Res. 22: 189, 1910. Chase, M. W., and Landsteiner, K. : Immunochemistry, Ann. Rev. Biochem. 8: 579, 1939. Corper, H. J., and Cohn, M. L.: Effect of Histaminase on Histamine Intoxication, Tuberculo-anaphylaxis, and Tuberculo-allcrgy, J. A. M. A. 115: 30, 1940. Dicnes, L.: Local Hj-persensitiveness. General Considerations, J. Immunol. 14: 61, 1927. Dienes, L.: Ueber die Wirkung des tuberkolosen Krankheitsherdes auf die Immunitatsreaktionen des Organismus, Ztschr. f. Immunitats- forsch. u. exper. Therap. 68: 13, 1930. Dienes, L.: Further Observations Concerning the Sensitization of Tubercu- lous Guinea Pigs, .1. Immunol. 15: 153, 1928. Dienes, L.: Factors Conditioning the Development of the Tuberculin Type of Hypersensitivity, J. Immunol. 23: 11, 1932. Dienes, L.r The Participation of Cutaneous Epithelium in Immunity Re- sponses, J. Immunol. 24: 253, 1933. Dienes, L,: The Specificity of the Tuberculin Type of Sensitiveness Pro- duced with the Different Protein Substances of the Egg White, J. Immunol. 18: 279, 1930. Dienes, L., and Schoenheit, E. W.: Local Hypersensitiveness, J. Immunol. 14: 9, 1927. Dienes, L., and Schoenheit, E. W. : The Reproduction of Tuberculin HyipeT- sensitiveness in Guinea Pigs with Various Protein Substances, Am. Rev. Tuberc. 29: 92, 1929. Dienes, L., and Schoenheit, E. W.r Certain Characteristics of the Infec- tious Processes in Connection With the Influence Exerted on the Immunity Response, .J. Immunol. 19: 41, 1930. Dienes, L., and Schoenheit, E. W. : The Antigenic Substances of the Tubercle Bacillus. V. The Antigenic Substances of the Synthetic Culture Medium, J. Immunol. 18: 285, 1930. Dienes, L., and Mallory, T. E.: Histological Studies of Hypersensitive Reactions, Am. J. Path. 8: 689, 1932. Dorset, M., Henlej^, R. R., and Moskey, H. E.: The Relationship of the Lethal Power to the Skin Reacting Power of Tuberculin, J, Am. Vet. M. A. 71: 487, 1927. Dorset, M., Henley, R, R., and Moskey, H. E.r Precipitation of Lethal Principle of Tuberculin bv Ammonium Sulphate, J. Am. Vet. M. A. 72: 363, 1927. Dorset, M., Henley, R. R., and Moskey, H. E.: (1931) Cited by Seibert and Munday (1932). HYPERSENSITIVENESS 535 Fleischiier, E. C, Meyer, K. F., and Shaw, E. B.: A Resume of Some Ex- perimental Studies on Cutaneous Hypersensitiveness, Am. J. Dis. Child. 18: 577, 1919. Funk, E. H., Huntoon, F. M., and White, H. J.: Collaborations with Mariette and Fenger (1932). Funk, E. H., and Huntoon, F. M.: Biochemical Studies of Bacterial Deriva- tives. XI. Skin Reactions in Man with Human Tubercle Bacillus Protein MA-100. Preliminary Report, J. Immunol. 19: 237, 1930. (Jiltner, W.r (1934) lirucellosis — Memoir 1, March, li>34, Michigan Ag- ricultural Experimental Station. Karan, A. A.: Adult Type Tuberculosis in ("hildren. Am. Rev. Tubere. 26: 571, 1032. Koch, R.: (1891.) Cited by Lowenstein in Hand, der Tek. u. Meth. der Immunitat., Kraus u. Levaditi (1908), Erster Band, p. 821. Krause, A. K.: Studies in Passive or Transferred Anaphylaxis, J. Med. Res. 22: 257, 1910. Leggett, E. A., and Myers, J. A.: The Incidence of Tuberculous Infection Among the High-School Students of Morrison County, Minnesota, Am. Rev. Tubere. 26: 559, 1932. Lewis, P. A., and Aronson, J. D.: Cited by Aronson, J. D., 23rd Report Henry Phipps Inst., 1931, Studies No. 2, Am. Rev. Tubere. 7: 404, 1923.' Long, E. R.: Tuberculin and the Tuberculin Reaction. Newer Knowledge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press, p. 1016. Long, E. R.: Discussion of Article by Terplan, Am. J. Path. 10: 681, 1934. Long, E. R., and HoUey, S. W.: The Origin of the Epithelioid Cell in Ex- perimental Tuberculosis of the Cornea, Am. J. Path. 9: 337, 1933. Long, E. R. : The Inflammatorv Reaction in Tuberculosis, Am. J. Med. Sc. 185: 749, 1933. Long, E. R.r Tuberculosis in University Students, J. Outdoor Life, Nov., 1933. Long, E. R.: Standardization of Tuberculin, J. Infect. Dis. 37: 368, 1925. Long, E. R., and Seibert, F. B.r (1926) Cited by Long in Newer Knowl- edge of Bacteriology and Immunology, Jordan and Falk, Chicago, 1928, University of Chicago Press, p. 1020. Long, E. R., Aronson, J. D., and Seibert, F. B.: Tuberculin Surveys with Purified Protein Derivative, Am. Rev. Tubere. 30: 733, 1934. Mariette, E. W., and Fenger, E. P.: The Present Status of the Skin Re- action in Tuberculous and Nontuberculous Subject.s, Am. Rev. Tubere. 25: 357, 1932. Maschmann, E.r Deutsche med. Wchnschr. 63: 770, 1937. Cited by Chase and Landsteiner, p. 580. Masucci and McAlpine: (1030) Cited by Funk and Huntoon (1930). McPhedran, F. M.: Incidence of Tuberculosis in Medical Students, Twent}'- Fourth Report of Henry Phipps Inst., 1932-33. McPhedran, F. M.: The Occurrence and Progress of Tuberculous Lesions in School Children and Adults, Transactions of the College of Physicians of Philadelphia 54: 98, 19.''.2. Myers, J. A.: Progress in the Diagnosis of Tuberculosis, J. A. M. A. 100: 2004, 1933. Myers, J. A., and Wulff, M.: Eleven Years' Observations in Tuberculosis Among LTniversity Students, Am. Rev. Tubere. 26: 530, 1932. Menzel, A. E. O., and Heidelberger, M.: Cell Protein Fractions of Bovine and Avian Tubercle Bacillus Strains and of the Timothy-Grass Bacillus, J. Biol. Chem. 124: 301, 1938. 536 IMMUNOLOGY Afeiizel, A. E. O., and Heidelberger, M.: Protein Fractions of the Human Strain H-37 of Tubercle Bacillus II, J. Biol. Chem. 124: 89, 1938. Okell, C. C, Parish, H. J., et al.: (1927) Cited by Seibert, F. B., and Munday, B.: Am. Eev. Tuberc. 26: 724, 1932. Opie, E. L.: Eecognition and Control of Tuberculosis in Childhood, Am. J. Pub. Health 23: 305, 1933. Park, W. H., Kereszturi, C, and Mishulow, L.: (1933) Effect of Vaccina- tion with B. C. G., on Children from Tuberculous Families, J. A. M. A. 101: 1619, 1933. Pearse, A. J., Fried, K. I., and Glover, V. A.: The Tuberculin Patch Test and the Mantoux Intradermal Test, J. A. M. A. 114: 227, 1940. Petroff, S. A.: Immunological Studies in Tuberculosis. II. Further Ob- servations on Skin Hypersensitiveness in Experimental Tuberculosis, J. Immunol. 4: 309, 1924. Keichell, J., and Clark, L. T.: The Manufacture of Purified Protein Deriva- tive of Tuberculin, Am. Eev. Tuberc. 30: 721, 1934. Eieh, A. E., and Lewis, M. E.: Mechanism of Allergy in Tuberculosis, Proc. See. Exper. Biol. & Med. 25: 596, 1927. Eich, A, E., and McCordock, H. A.: Enquiry Concerning Eole of Allergy, Immunity, and Other Factors of Importance in Pathogenesis of Tuberculosis, Bull. Johns Hopkins Hosp. 44: 273, 1929. Eobertson, H. E.: The Persistence of Tuberculosis Infection, Am. J. Path. 9: 711, 1933. Eubin, E. H.: Pulmonary Tuberculosis in the Aged, Am, Eev. Tuberc. 26: 516, 1932. Sabin, F. E., Miller, F. E., Doan, C. A., and Wiseman, B. K.: A Study of the Toxic Properties of Tuberculo-proteins and Polysaccharides, J. Exper. Med. 53: 51, 1931. Schroeder, E. C, and Brett, C. W.: The Method of the Bureau of Animal Industry for Testing the Potency of Tuberculin, J. Am. Vet. M. A. 54: 357', 1918-19. Seibert, F. B., Pedersen, K. O., and Tiselius, A.: Molecular Weight, Electrochemical and Biological Properties of Tuberculin Protein and Polysaccharide Molecules, J. Exper. Med. 68: 413, 1938. Seibert, F. B.: The Purification and Properties of the Purified Protein Derivative of Tuberculin, Am. Eev. Tuberc. 30: 713, 1934. Seibert, F. B.: The Eelationship Between the Sensitizing Properties of Tuberculo-protein and Its Molecular Weight, Trans. Twenty-Ninth Ann. Meeting Nat. Tuberc. Ass., 1933. Also see the twenty-fourth report of the Henry Phipps Inst., 1932-33. Seibert, F. B., and Munday, B.: The Chemical Composition of the Active Principle of Tuberculin, Am. Eev. Tuberc. 26: 724, 1932. Seibert, F. B.: The Chemical Composition of the Active Principle of Tuberculin, J. Immunol. 28: 425, 1935. Seibert, F. B.: History of the Development of Purified Protein Derivative Tuberculin, Am. Eev. Tuberc. 44: 1, 1941. Seibert, F. B., and Glenn, J. T.: Tuberculin Purified Protein Derivative. Preparation and Analyst's of a Large Quantity for Standard, Am. Eev. Tuberc. 44: 9, 1941. Seibert, W. W.: Die praktische wichtigen Formen der Lungentuber- kulose, Deutsche med. Wchnschr. 59: 643, 1933. Slater, S. A., and Jordan, K.: A Comparative Study of the Pirquet and Mantoux Tuberculin Tests in School Children, Am. Eev. Tuberc. 25: 218-223, 1925. Soper, W. B., and Wilson, J. L.: The Detection of Pulmonary Tuberculosis in 3,000 Students Entering Yale University, Am. Eev. Tuberc. 26: 548, 1932. HYPERSENSITIVENESS 537 Stewart, (". A.: Skin Sensitiveness to Tuberculin in Primary Tuberculosis, J. A. M. A. 103: 176, 1934. Stewart, C. A., Harrington, F. E., Myers, J. A., Boynton, R. E., Chiu, P. T. Y., and Streukens, T. L.: Primary Tuberculosis Infection Attack Eates, J. A. M. A. 113: 2204, 1939. Svedberg, T.: Colloid Chem. 151: 67, 1928. Terplan, K. L.r (1934) Anatomical Studies on Primary and Postprimary Tuberculosis in White Children and Adults, Am. Rev. Tuberc. 10: 680, 1934. Tuft, L.: The Skin as an Immunological Organ. With Results of Experi- mental Investigations and Review of the Literature, J. Immunol. 21: 85, 1931. Webb, C. H.: Skin Reaction and Tuberculous Disease in Children, Am. Rev. Tuberc. 26: .583, 1932. Zinsser, H.: Resistance to Infectious Diseases, New York, 1931, The Mac- millan Co. Zinsser, H.r Studies on the Tuberculin Reaction and on Specific Hyper- sensitiveness in Bacterial Infections, J. Exper. Med. 34: 495, 1921. Zinsser, H., and Petroff, S. A.: Tuberculin Hypersensitiveness Without Infection in Guinea Pigs, J. Immunol. 2: 85, 1924. Zinsser, H., Enders, J. F., and Fothergill, L. D.r Immunity, New York, 1939, The Macmillan Co. Supplementary References Campbell, D. H., and Nicoll, P. A.: Studies on In Vitro Anaphylaxis and Release of an Active Non-histamine Material From Sensitized Guinea Pig Lung, J. Immunol. 39: 89, 1940. Casals, .L, and Freund, J.: Sensitization and Antibody Formation in Monkeys Injected With Tubercle Bacilli in Paraffin Oil, .J. Im- munol. 36: 399, 1939. Gottschall, R. Y.. and Bunney, W. E.: The Effect of Age on the Spread of Dye in the Skin of Normal, Antigenically Stimulated, and Tuberculous Guinea Pigs, J. Immunol. 38: 345, 1940. Myers, J. A., Harrington, F. E., Sprague, E., and Perez, J. A.: Epidemi- ology of Tuberculosis, J. A. M. A. 115: 3 609, 1940. Plunkett, R. E.r Tuberculosis Control, J. A. M. A. 113: 2288, 1939. Stadnichenko, A., Cohen, S. J., and Sweany, H. C: Stomach Lavage in the Diagnosis and Control of Treatment of Tuberculosis, J. A. M. A. 114: 634, 1940. Wong, S. C, and Ouyang, G.: Estimation of Biological Activities of Purified Tul>erculin bv the Hopkins Tube, Proc. Soc. Exper. Biol. & Med. 46: 615, 1941. CHAPTER XXVII THE SIGNIFICANCE OF ALLERGY IN TUBERCULOSIS AND A FEW OTHER DISEASES Immunity in Tuberculosis. — As a result of extensive investiga- tion it has been definitely established that the resistance of sus- ceptible animals such as man, rabbits, guinea pigs, etc., to infec- tion with the tubercle bacillus can be increased by infection with either virulent or avirulent human or bovine strains and also to a moderate extent by the injection of a suspension of killed tubercle bacilli. Numerous anatomical studies of primary and reinfection types of tuberculosis offer abundant evidence that partial immunity results from infection with the tubercle bacillus. The primary or childhood tj^pe of tuberculosis occurs when the organisms lodge in a body that is neither allergic nor immune. In primary pulmonary tuberculosis the original lesion may be anywhere in the lung, no allergic symptoms are detectable and the regional lymph glands soon become involved. In the reinfection or so- called adult type of pulmonary tuberculosis the lesion is as a rule in the apex of one or both lungs, allergic symptoms may develop and the regional lymph glands are not involved because the tissue immunity, due to the first infection, localizes the organisms and limits the pathway of spread largely to the bronchi. For a more comprehensive discussion of the occurrence and significance of the various lype,s of tuberculosis the student is referred to papers by McPhedran and Opie (1933), McPhedran (1933), Hetherington (1933) and Opie (1933). At the present time there is some controversy over the mechanisms involved and the role which allergy plays in acquired immunity to the tubercle bacillus. Krause (1910, 1916) made an extensive study of tuberculin allergy and immunity and suggested that tissue hypersensitiveness may play an important role in immunity. It will be recalled that in Chapter VII the mechanism of ac- quired resistance to infection is discussed. According to Cannon, 538 SIGNIFICANCE OF ALLERGY 539 Rich, and their respeK'tive colleagues, as well as a number of other investigators, certain factors of primary importance can be deter- mined. Cannon et al., working with staphylococci, and Rich et al., stiidying dermal pneumonia in rabbits, report that when virulent organisms are introduced into the tissues of normal animals they not only undergo multiplication but are disseminated rapidly to various places remote from the point of inoculation. In immune animals the organisms remain localized althougli a certain amount of multiplication occurs. Apparently the presence of antibodies in the tissues causes the organisms to grow in chains or in clus- ters in the manner analogous to that observed in culturas grown in immune serum. The organisms not only tend to adhere to each other but they also stick to the tissue cells. Rich says that such phenomena may be observed before evidence of inflamma- tion appears. He challenges the concept of Zinsser, Krause and many others that acquired resistance is due largely lo allergy and the allergic inflammatory response. Rich grants that the pouring out of an inflammatory exudate after the bacteria have been localized is of value in diluting toxins or toxic substances, and that neutrophiles and macrophages aid in destroying and removing bacteria, but he regards acquired resistance as consist- ing mainly of two factors : ( 1 ) the mechanism by which the or- ganisms are retained at the point of entry and (2) the creation of an environment in which they are less able to survive than in the normal animal. He and his colleagues have shown that rabbits passively immunized to type I pneumococcus are highly resistant but not allergic to the organisms. As a result of this and other experimental studies he concludes that bacterial al- lergy and immunity can be definitely separated from each other. He and McCordock feel that this is especially true in human tuberculosis largely for the following reasons : 1. Krause and Willis planted tubercle bacilli into areas of tuberculin allergic inflammation and observed that the organ- isms did not remain localized. The results led them to conclude that immunity is reduced at the site of an inflammatoi'y tuber- culin reaction for at least four days. 2. Immunization with dead tubercle bacilli produced an in- tensive allergic state but only moderate immunity. Petroff and 540 IMMUNOLOGY Stewart (1925) state that "the local cutaneous allergic reaction in animals sensitized with dead bacilli varies in no way from that in infected animals." 3. Rich and McCordock cite the statement of Calmette (1927) to the effect that in immunizing with B.C.G. allergy may disap- pear for a while although acquired immunity persists. 4. They cite the experiments of Willis who found that animals immunized with a tubercle bacillus of low virulence developed tuberculin allergy but retained it for less than two and one-half years. After allergy had disappeared the animals were found to ]iossess a pronounced degree of immunity when reinfected with virulent organisms. 5. In certain types of clinical cases of tuberculosis the admin- istration of repeated and increasing doses of tuberculin results in both a disappearance of allergy and a beneficial effect upon the disease. This is especially true in certain types of skin and also ocular tuberculosis. They cite the observations of Hamman and Wolman that in a majority of cases treated with tuberculin a change for the better is associated with an increased tolerance of tuberculin. 6. In regard to the assumption that allergic inflammation stimu- lates fibrosis and the encapsulation of a tubercle, they call atten- tion to the fact that connective tissue repair is a nonspecific process and, according to MacCallum, is in no sense a part of in- flammation and may not necessarily follow a violent inflammation. That acute inflammation is not necessary for connective tissue repair is evident to anyone who studies the organization of a thrombus. 7. They call attention to the fact that the tubercle bacillus has little power to cause necrosis in the unsensitized body. After tuberculin allergy develops, it takes but a minute amount of tuberculin to produce extensive necrosis of tissue. In their opinion the killing of tissue within the body cannot be regarded as a defensive process. 8. The authors believe that in reinfection the mechanism of im- munity operates to destroy the tubercle bacillus before the allergic reaction begins. Effect of Administering Vaccine Intravenously. — Additional evidence which seems to support the theory of Rich and his col- SIGNIFICANCE OK ALLERGY 541 h'aiiiK's that tuberculin allcriiy and iiniuunity are not necessarily identical seems to be inherent in certain observations of Clawson (1934) and others. The former reports that animals receiving a heat-killed suspension of B.C.G. vaccine intravenously develop definite immunity to the tubercle bacillus without becoming per- ceptibly allergic to tuberculin as indicated by the Mantoux test. Birkhaug (1933), on the other hand, holds to the opinion that allergy is an important and necessary factor in immunity to tuberculosis. He studied the immunizing property of B.C.G. vac- cine for guinea pigs over a period of two years and reports that intradermal immunization yielded a more desirable and intense allergy as well as a greater percentage of relative immune ani- mals than intraperitoneal immunization. In his opinion B.C.G. is incapable of producing progressive tuberculosis. Experimental and Clinical Studies of Allergy and Immunity in Tuberculosis. — That these apparently' conflicting opinions are not irreconcilable would seem to be apparent from a careful considera- tion of the following experimental studies of Smith (1933), Lurie (1936. 1939), Menkin (1938) and Cannon and Hartley (1938). While the research discussed in the preceding pages of this chapter throws a great deal of light upon allergy and immunity in tuberculosis, there is inherent in them certain artificial and ab- normal conditions with which the student should be made ac- quainted. The factors involved, Avhich may cause one to acquire a misconception of what occurs in naturally acquired tuberculosis, have been clearly set forth in a paper by Theobald Smith (1933). He points out that in natural infection with the tubercle bacillus the entry through the mucous membrane of the respiratory or ali- mentary tract is not accompanied by mechanical injury to the tissue, such as results from hypodermic injection, and the dosage is limited to one or at most a few organisms as contrasted witli relatively massive doses employed in experimental work. Fur- thermore the organisms involved in natural infection have a rate of multiplication and a metabolism that show the effect of a parasitic environment, wherea.s in experimental work the organ- isms have been acclimated to a saprophytic existence in artificial media and possess an accelerated rate of growth. Their meta- bolic products are perhaps essentially different from the organisms that have been growing under parasitic conditions. Some of the 542 IMMUNOLOGY conclusions which he holds as a result of an extensive study of spontaneous bovine tuberculosis and paratuberculosis may be summarized as follows: Summary of Theobald Smith 's Studies of Spontaneous Bovine Tuberculosis. — 1. Whereas the concept of phagocytosis has been regarded as a one-sided affair, in reality one can only postulate that certain bacteria and cell types possess an affinity for each other. The results of such mutual attraction may vary from death of the host to partial or complete suppression of the parasite. In tuberculosis, paratuberculosis, leprosy, glanders, as well as in induced Brucella infections in guinea pigs, the bacteria associated themselves with cells of the connective and adenoid tissue. Their association leads to the development of the epithelioid type of cell. In typhoid and paratyphoid infections the bacteria liave an affinity for endothelial leucocytes, while in diseases due to llickettsia and in mouse septicemia the organisms exhibit a marked affinity for vascular endothelium. 2. In his opinion both allergy and immunity in tuberculosis result from the ''discharge of diffusible products of the living bacilli and the continuous impact of these substances in minute doses upon the forerunners of tlie epithelioid cells throughout tlie system. ' ' 3. He observed that in spontaneous bovine tuberculosis the secondary foci exhibit definite evidence of having developed un- der the influence of an acquired immunity. In contrast with the primary lesions they are smaller, show a lessened tendency to necrosis, and contain giant cells. 4. The primary focus of epithelioid cells observed in spontane- ously acquired bovine tuberculosis is not invaded by neutrophiles as in experimentally induced bovine tuberculosis. The neutro- philes do not seem to be involved in the initial stages of spon- taneous tuberculosis. 5. He attributes a dual role to the focal lesions in tuberculosis. They are places where the bacteria multiply and places where the micro-organisms are being opposed by the host. The focus represents a compromise between the host and parasite. 6. In his opinion it is impossible to visualize all of the factors involved in the inner workings of the tuberculous focus because they are in a state of change. In the primary lesion of spon- SIGNIFICANCE OF ALLERGY 543 taiieous ])oviiie tuberculosis the early development of central necrosis masks the earliest phases. He assumes that the latter consist of the multiplication of the bacteria, the swelling of the cytoplasm of the local tissue cells, and then the development of central necrosis. The diffusion outward of products of metab- olism of the tubercle bacillus leads to the formation of the epi- thelioid cell mantle and connective tissue capsule. 7. He points out that immunity in tuberculosis tends toward a diminishing susceptibility instead of an increased activity on the part of the cells. This results finally in the feeblest reaction, the formation of the giant cells. It is evident that this conclusion of Theobald Smith's is in harmony with the view of Rich and others that immunity and allergy are not necessarily identical. The Immunizing Value of Nonprogressive Primary Lesions. — Lurie (1933), in his experimental studies of contact tuberculosis, seems to have avoided many of the sources of error which have just been discussed. In 1930 he reported that cattle immunized with von Behring's bovovaccine were resistant for several months to the artificial introduction of fatal doses of tubercle bacilli of the bovine type. When, however, such immunized animals were stabled for twelve or more months with tuberculous cattle that were eliminating virulent tubercle bacilli, the immunized animals acquired tuberculosis. Such conditions of exposure to infection ai"e to a certain extent comparable to the environment of a child in contact with tuberculous parents. Lurie also studied contact tuberculosis in guinea pigs. It was evident from these studies that the route of infection depended upon the relative intensity of exposure by the respiratory or alimentary channel. He deter- mined that the reason crowding is so conducive to the spread of tuberculosis is the amount of virulent tubercle bacilli available for contagion. In 1933 Lurie produced localized, nonprogressive infection in rabbits by inoculating them with living tubercle bacilli of the human type. When such rabbits were used as cage mates for normal rabbits, the latter died with contact tuberculosis. In order to ascertain the effect of such localized, nonprogressive tuberculosis upon the resistance of rabbits to contact infection with virulent bovine tubercle bacilli, Lurie exposed these infected 544 IMMUNOLOGY rabbits to cage mates that had been infected with virulent organ- isms. He determined by experiment tliat animals receiving 0.001 to 0.00001 mg. of highly virulent tubercle bacilli of the bovine type develop progressive lesions and eliminate virulent organ- isms. Normal animals v^^ere exposed to contact infection by plac- ing them with cage mates thus infected. By means of a very clever experiment in which a special type of animal cage was used, he was able to show that normal animals and those having nonprogressing lesions acquire infection with virulent bovine tubercle bacilli under conditions in which the opportunity for alimentary infection is eliminated. The i-esults of his experiments may be summarized as follows: Summary of Lurie's Studies on Activi: Immunization.— 1. Of the normal rabbits exposed to cage mates infected with virulent bovine tubercle bacilli, 63.6 per cent contracted the disease within six months and 73 per cent within one year. 2. That the rabbits having a primary nonprogressive lesion gained some immunity from the primary infection is indicated by the fact that only 36.8 per cent of those exposed to cage mates eliminating virulent bovine tubercle bacilli contracted infection within six months and 60 per cent within one year. 3. Since 27 per cent of the normal rabbits and 40 per cent of tlie ones vaccinated with living human type tubercle bacilli escaped infection, it would seem that natural resistance plays a very im- portant role in immunity to tuberculosis. Lurie* points out that vaccination saved only an additional 13 per cent. 4. His results show that "vaccination reduced the incidence, extent, and mortality of the disease, affected the route of infection, changed its pathological character, and retarded its progress. The disease acquired by vaccinated rabbits shared many characteristics with adult type tuberculosis in man. ' ' Lurie's Investigation of the Mechanism of Immunity and the Role of Allergy in Tuberculosis. — Lurie (1936, 1939) has stud- ied the mechanisms of resistance and the role of allergy in normal, tuberculous and vaccinated rabbits and guinea pigs. He intro- duced agar impregnated with tubercle bacilli in the tissues. He found that in normal animals the ])acilli grew unhindered in the ♦Lurie: J. Exper. Med. 58: 305, 1933. SIGNIFICANCE OF ALLERGY 545 aeellular ai>ar at a considerable distance from the phagocytic cells unless the tissue fluids were prevented from reaching them. This was in marked contrast to the inhibition of growth of the bacteria in vaccinated and tuberculous animals. Apparently the fluid from the tissues of normal animals penetrating the agar encouraged growth of the bacteria while the tissue fluid in vaccinated or tuberculous animals penetrated the agar and inhibited growth. This apparently demonstrated that humoral factors play a role in defense against tubercle bacilli. Lurie also suggests that in vivo agglutination, as well as the deposition of a fibrin barrier thrown about the agar mass as a whole, tends to keep the bacteria localized in immune animals. It would appear that extracellular factors may be effective in limiting the spread of infection when small doses are introduced. When larger doses are introduced into vaccinated or tuberculous rabbits, there is a greatly intensified inflammation developed and the factors tending to localize the bacteria are overcome by the increased lymph flow which sweeps the leucocytes and agar par- ticles containing bacteria into the regional lymph nodes more rapidly than in normal animals. This phenomenon has been re- ported by Freund and Angevine (1938) and others. Lurie found that immunity is effective in these vaccinated animals despite the lack of immediate fixation. This immunity is due not only to the humoral factors but to actively phagocytic mononuclear cells. The bacteria that are swept into the regional lymph nodes are fixed and destroyed by the humoral and tissue mechanisms. lAirie found the mononuclear phagocytes much more effective agents in combating infection in vaccinated animals than the neu- trophiles. In fact he states that the mononuclear cells derived from vaccinated or actively tuberculous guinea pigs exhibit a greater in vitro phagocytic capacity for tubercle bacilli and carbon particles than mononuclears from normal animals. Lurie also notes that bacteria are fixed locally more thoroughly in vaccinated or tuberculous guinea pigs than in vaccinated or tuberculous rabbits although the general immunity may not differ. He correlates this difference in local fixation with the differences lie observed in the degree of allergic response and also the kind of fibrin networks laid down. He calls attention to the conclusion of Menkin (1938) that the amount of fixation of bacteria at the 546 IMMUNOLOGY point of infection is to a certain extent in proportion to the de- gree of local tissue injury, e.g., the staphylococcus produces a great deal of local tissue injury and is also fixed more extensively than other organisms producing less local necrosis. Lurie states that the guinea pig develops more allergy to the tubercle bacillus than the rabbit. When tubercle bacilli are introduced into its tissues (agar-bacteria implantation) there is both a greater allergic response and a greater local fixation of bacteria. In the allergic inflammation of the guinea pig the fibrin network is finer, more compact, and tliere is a plugging and oc- clusion of tlie lymphatics. In the rabbit similarly treated, the fibrin network is coarser and the lymphatics remain open. These findings may account for the apparent contradictory conclusions drawn from experimental work mentioned earlier in this chapter. Some of the work on experimental allergy and immunity was done on rabbits and some on guinea pigs. Cannon's and Hartley's Work on Allergy and Immunity'. — Cannon and Hartley rendered rabbits allergic to egg albumen and then injected a mixture of egg albumen and virulent pneumo- cocei. The allergic inflammation did not protect the rabbits from a fatal pneumococcus infection. The organisms "grew profusely in the areas of allergic inflammation and spread evenly through the field of inflammation. While phagocytosis was moderately active, it did not modify the course of infection either in time or degree. ' ' This was in marked contrast to the phenomena observed in rabbits allergic to egg albumen but immune to the pneumococcus. In these rabbits the organisms grew profusely for a time but they became swollen, developed in isolated colonies, many became gram-negative and tended to remain localized. These experiments apparently show that, for the pneumococcus infection in rabbits, allergic in- flammation without the aid of immunity factors such as antibodies cannot be regarded as a defensive mechanism. Discussion. — From a consideration of the results of Smith, Lurie, and Cannon and Hartley presented in the preceding para- graphs the following conclusions seem to be justified : 1. That as the body becomes more immune to the tubercle bacillus there is a diminishing tendency for any visible re- sponse. There is no evidence of allergy where complete im- munity exists. SIGNIFICANCE OF ALLERGY 547 2. That the most important factors in the type of immunity (partial, i.e., not absolute) to the tubercle bacillus resulting from vaccination or infection are mononuclear phagocytes and humoral elements. 3. That in the tuberculin allergic inflanunation in the guinea pig a more efficient mechanical barrier favoring localization is laid down than in the hypersensitive rabbit. 4. That within limits the degree of injury to the tissues in an area of allergic inflammation may affect the amount of local fixation of the infectious agent. Too large a dose of the bacteria may lead to a breakdown in the local mechanism of fixation and permit a more rapid spread to the regional lymph nodes in an allergic animal than in a normal one. 5. Allergic inflanimation unsupported by specific immunity fac- tors fails to prevent the escape of bacteria from the area of allergic inflammation. It would seem from the above discussion that many of the ap- parent contradictory conclusions can be, in a measure, recon- ciled. In this connection a few other observations may be of in- terest. Teale (1935) is of tlie opinion that " hypersensitiveness is due to the state of tlie tissues which cannot deal with the specific antigen even when it is completely saturated with the homologous antibody." Woodruff and Willis (1939) conclude that there is a partial reciprocal relationship between the allergic state of in- fected guinea pigs and the number of bacteria found in the lungs. Corper (1940) has attempted to compare tuberculin al- lergy due to infection with anaphylactic hypersensitiveness to tubercle proteins. Apparently further work is indicated before it can be evaluated. Effect of Primary Lesion in Children Upon Subsequent Infec- tion.— At the present time there is considerable discussion as to the effect of initial or primary infection on subsequent tuber- culous lesions in man. It is quite generally held that primary infection with the tubercle bacillus immunizes the individual and in part at least protects against later infections from exogenous sources. Myers and Harrington (1934) conclude from their study of a very large number of children that five times as many of those with initial positive tuberculin tests fall ill in later years with tuberculosis as do those who on first examination give 548 IMMUNOLOGY negative tests. In their opinion the primarily infected children offer a double liability in that many of them continue to harbor virulent organisms and all of them are allergic. When reinfec- tion occurs the organisms are implanted in allergic tissue. The resulting reaction leads to destructive and progressive changes. Casperis, in discussing the work of Myers and Harrington, sug- gests that the children who show positive tuberculin tests on first examination constitute a group that has greater exposure to tuberculosis than the negative reactors and therefore, it might be expected that more of this group would develop the reinfec- tion type of tuberculosis as a result of i-epeated exposure. Myers states, however, that in his series, contact was broken so far as possible immediately after the skin tests were done. It is difficult to compare these results with the animal experiments of Lurie since the primary infection in the rabbits was with the human type of tubercle bacillus which does not produce progres- sive tuberculosis in rabbits and hence there is no danger of adult tuberculosis arising from the flaring up of old primary foci such as may occur in children. The evidence at hand seems to show that mild ]>rimary tuberculosis confers a relative immunity «is well as definite allergy upon an individual; that excessive ex- posure to virulent tubercle bacilli nuiy break down this inuuTUiity; and that in such cases the allergic inflammation helps in that it assists in mobilizing phagocytic cells but is detrimental in that it plays a definite role in necrosis, extension of the inflammatory processes and general systemic reactions. Different Opinions as to B.C.G. Vaccination. — For a great many years there has l^een a spirited debate over the question of active immunization against tuberculosis. There are at least four dis- tinct views being presented at the present time. One group would not immunize at all but would depend entirely upon measures designed to prevent first infection ; a second group would su})ple- ment such measures by immunizing all tuberculin-negative chil- dren with a killed vaccine; the third group would substitute B.C.G. for the killed vaccine; while the fourth group feels that vaccination should be viewed experimentally for fifteen or twenty >ears before it is generally adopted or rejected. Those who would not immunize at all state that in their opinion immunization is at best a questionable practice, subsequent con- SIGNIFICANCE OF ALLERGY 549 tamiiiatioii may occur and that the administration of the vac- cine produces tuberculin allerj^y in the vaccinated children. This latter they regard as detrimental. The second group is especially concerned over the possible in- crease in virulence of the B.C.G. organisms and the subsequent development of tuberculosis. Petroft' and others have presented evidence supporting this view. The third group, which favors innnunization with an attenuated bovine tubercle bacillus vaccine such as B.C.G., maintains that the vaccine is safe and efificacious. They disregard the development of allergy, in fact many of those favoring this vaccine consider allergy as desirable. The fourth group is made up of conservative individuals who do not desire to experiment upon their private patients. They constitute a very large percentage of group one who are willing to accept vaccination or any other method only after such methods have been tried and found successful over a fairly long period of time. In their opinion adequate data upon which a final conclusion can be based will come only after the present large number of experimentally immunized children are followed for several decades and their ultimate fate determined. Park's Studies on B.C.G. — An investigation of the effect of vac- cination with B.C.G. on children from tuberculous families has been inaugurated in New York by Park, Kereszturi, Camille and Mlshulow (1933). The results of their work may be summarized as follows : 1. They observed no evidence of an increase in virulence of B.C.G, when inoculated into a long series of rabbits and guinea pigs nor from residence in the human body. 2. They immunized 205 tuberculin-negative children by oral and 150 by parenteral vaccination and observed them for five years relative to the phenomenon of allergy and symptoms of clinical tuberculosis. Comparal)le control groups were included in their series. In the orally vaccinated group approximately 1 per cent died from tuberculosis as compared with 3.2 per cent of deaths in the control gi-oup. While no deaths occurred among tliose parenterally vaccinated, it is interesting to note that the death rate was only 1.4 per cent among the tuberculin-negative 550 IMMUNOLOGY controls and 3.2 per cent among the children with positive initial Mantoiix reactions. In regard to the efficiency of oral vaccina- tion, Park states that if, following its administration, the de- velopment of a positive tuberculin test is to be the criterion of its efficiency, then only 20 to 40 per cent of the orally vaccinated children can be regarded as effectively vaccinated. They noted that allergj^ following vaccination does not last, as a rule, longer than two or three years. 3. Park et al. did not find B.C.ri. vaccination to improve the general health nor to protect against other infectious agents as claimed by Calmette. 4. Bogen in discussing the paper presented by Park et al. calLs attention to the value of the tuberculin test in diagnosing active, primary tuberculosis in children. Since B.C.G. immunization leads to the development of tuberculin allergy, it is obvious that the test cannot be used in vaccinated children to diagnose active tuberculosis. He regards this as one objectionable feature of vaccination. 5. Park regards the results they obtained as not conclusive at the present time. The student will find from a perusal of the literature that aside from the work just discussed, the majority of the reports of the use of B.C.G, vaccine represent experiments that are either uncontrolled or only partially controlled. Such are the studies of Wallgren (1934) covering a short series of vac- cinations in Sweden. His conclusions relative to virulence and immunizing value of B.C.G. are in accord with the conclusions of Park and his colleagues. Miller (1940) likewise says that neither B.C.G. nor any other preparation has been proved to be of wide- spread usefulness. The new "Vole tubercle bacillus" vaccine of Wells and Brooke (1940) has been discussed in an earlier chapter. It is apparently superior to the B.C.G. vaccine as an immunizing agent in guinea pigs. Other Bacterial Allergies. — Brucellosis. Reference has been made'earlier in this chapter to the typhoidin, mallein and abortin reactions. The latter is used to a limited ex- tent as an aid in the diagnosis of undulant fever in man although it has not replaced the agglutination test. These reactions are all of the tuberculin type. Schoenholz and Meyer (1927) carried SIGNIFICANCE OF ALLERGY 551 out extensive studies on the purification of abortin. Since a sat- isfactory synthetic medium has not been devised, they proceeded to fractionate coll solutions prepared from whole bacteria by acetic acid precipitation. They report tliat the acid-precipitable fraction of abortin is as active as the original solution. The active principle is destroyed within twenty-four hours by tryptic digestion. Schoenliolz and Meyer could ]n-oducc neither specific increased resistance nor allergy by immunizing guinea pigs with killed suspensions of Br. abortus although the agglutinin titer for the antigen wa.s definitely increased. They conclude that allergy results only when active infection is present. Theobald Smith reports that immunization of cattle with a suspension of killed Br. abortus confers only partial and transient immunity. He finds that a vaccine composed of living organisms is superior to the former. For additional information relative to Brucellosis the student is referred to a few supplementary references and to an excellent monograph by Giltner (1934). Streptococcus Allergies. — In view of the prevalence and fre- quent chronicity of streptococcus infections it is not surprising that many individuals are allergic to the nucleo-protein fraction of scarlet fever streptococci. Dochez and Stevens (1927) carried out extensive studies on streptococcus allergy in guinea pigs and rabbits. They interpret their results as apparently favoring the theory of Bristol (1926) that the rash and clinical symptoms of scarlet fever may be due to the development of allergy to scarlet fever streptococci or their products. Rheumatic Fever. — The etiology of rheumatic fever is not defi- nitely established at the present time. Clawson reports isolating streptococci from a high percentage of blood cultures of rheu- matic fever patients and of producing typical pathological lesions in experimental animals. Herry (1914) and more recently Swift and his colleagues explain the symptoms and clinical course of rheumatic fever upon the basis of streptococcus allergy. An ex- cellent brief summary of this work is given by Zinsser (1931, 1939). Pneumococcus Infection. — McBroom and Schlesinger report that there occurs an early stage of hypersensitiveness to pneumo- coccus nucleoprotein simultaneously with the early stage of re- sistance. 552 IMMUNOLOGY The Shwartzman Reaction.— In 1928 Shwartzman described a new phenomenon of local skin reactivity to certain bacterial cul- tural filtrates. Subsequent studies (1930, 1934) have revealed many interesting facts regarding the reaction. In his original work he reported that if one injects a small amount of a culture filtrate of E. ttfphosa into the skin of a rabbit, there is very little or no local response. If, twenty-four hours later, one administers to the same rabbit a small amount of the filtrate intravenously, one observes a severe hemorrhagic reaction developing at the site where the filtrate was injected into the skin twenty-four hours previously. Shwartzman concludas that the filtrate contains skin preparatory and reacting factors. He has shown that the preparatory effect is not due to trauma, local reticulo-endo- thelial blockade, increased permeability of the capillaries or to inflammation. In Shwartzman 's opinion the vulnerability is due probably to a disturbance of cell function. The susceptibility disappears completely in forty-eight hours. The preparatory and reacting factors are not present in the filtrates of all bacteria nor in crystalline e^^ albumen or horse serum. The preparatory factor in the filtrate of one kind of bacteria, e.g., E. typhosa, can render the skin or organ or a rabbit susceptible to the reacting factor of another bacterial filtrate ; e.g., of the meningococcus. When a potent filtrate is treated with the homologous immune serum, both factors are neutralized in multiple proportions. In this as well as in their synergistic effects they resemble toxins. Because of the short incubation period (twenty-four hours), the inability to transfer susceptibility passively, and the phenomenon of synergism noted above, Shwartzman concludes that his reactions are not to be cla.ssified as anaphylactic in nature. Apitz (1935) has made a rather extensive study of the Shwartz- man reaction and describes a generalized reaction elicited by intra- venous rather than subcutaneous injections comparable to the classical Shwartzman phenomenon. He has also made chemical studies of the Shwartzman-active substances. He finds the active substances are contained in two different chemical fractions of bacterial substances. The so-called "nucleoprotein" fraction is active and is derived from bacteria by autolysis or extraction. There is also an alcohol-precipitable biuret-negative fraction. It SIGNIFICANCE OF ALLERGY 553 is unstable, ])re('ipital)l(' with small volumes of aleohol, and Apitz says it is not the same substance as the type-specific carbohydrates, Stolyhwo (1936) reports that the active Shwartzman substance is concentrated in the urine of typhoid patients. He thinks the active substances may be responsible for severe intoxications in typhoid fever. Frei Test in Lymphogranuloma Inguinale. — DeWolf and Van Cleve (1932) give an excellent discussion of lymphogranuloma inguinale and of the intradermal test suggested by Frei (1925) for its diagnosis. It seems to be established that the disease is venereal and caused by a filtrable organism which according to Tamura (1934, 1935) can be cultivated in vitro. According to DeWolf the antigen for the original Frei test is prepared by making a one to ten dilution of pus obtained from a suppurating gland. Physiological saline is used as a diluent. The prepara- tion is then heated at 60° C. for two hours on one day and one hour on the next day. It is then cultured for sterility, and if no bacterial growth is obtained, the material thus prepared is used as an antigen in 0.1 c.c. amounts for intradermal injection into patients suspected of having the disease. DeWolf and Van Cleve state that cutaneous allergy develops quite early in the disease and persists for a long period after recovery. A positive skin reaction develops at the site of the intradermal injection within twenty-four hours and persists for several days. The reaction appears as an inflammatory papule and resembles a positive tuberculin reaction. The authors regard it as of great diagnostic value. Tamura (1934, 1935) reports the successful cultivation of the virus in a medium consisting of filtered Tyrode's solution and bits of sterile rabbit or guinea pig tissue as suggested by Maitland, Laing and Lythe (1932) in their cultivation of vaccinia virus. Tamura reports that in cultures of the virus in this medium the supernatant fluid becomes cloudy and that it can be heated and used as an antigen in the Frei test and also in immunization ex- periments. Connor, Levin and Ecker (1937) report that the Frei test is specific for lymphogranuloma inguinale and that a positive test has been obtained as long as 39 years after the infection. 554 IMMUNOLOGY Summary and Conclusions. — 1. Evidence is offered which seems to show that tuberculin allergy and immunity are not identical and that the former may be detrimental to tlie host. 2. A summary of Theobald Smith's excellent discussion of ex- perimental tuljerculosis is given. 3. Primary tul)erculosls confers a relative immunity and also allergy upon the individual. There is a difference of opinion as to whether the detrimental effects of the latter outweigh the bene- ficial effects of the former. In any event the primary lesion rep- resents a focus of virulent organisms that may ultimately lead to endogenous reinfection. 4. Relative immunity and allergy are apparently produced by immunizing with suspensions of either killed or living attenuated tubercle bacilli. The route of immunization may determine the degree of allergy that occurs. 5. The work of Park, Kereszturi and Mishulow on the effect of vaccination with B.C.G. on children from tuberculous families is presented. From these studies and those of Lurie it is evident that natural resistance is an important factor in prevention of tuberculosis. They conclude that B.C.G. is safe but confers rela- tively little immunity and considerable allergy when administered to children. Park regards the results as inconclusive. 6. In our opinion, the problem is still in the experimental stage and should be so regarded until more carefully controlled work similar to that of Park et al. is completed. 7. Allergy to streptococcus nucleo-proteins is observed not in- frequently in man. Ando et al. have shown that this is a possible source of error in interpreting the Dick test unless a purified toxin is employed as the indicator antigen. The clinical symptoms and cutaneous manifestations of scarlet fever are regarded by Dochez and Sherman as manifestations of bacterial allergy. There are also a number of investigators who regard rheumatic fever as a manifestation of streptococcus allergy. At the present time the prevailing opinion as to the etiology of scarlet and rheumatic fevers, respectively, is that the former is a toxemic and infectious disease caused by certain hemolytic streptococci, while the etiology of the latter, rheumatic fever, is as yet either undetermined or not generally agreed upon. SIGNIKICANCK OF AI.LERGV 000 8. Shwartzman discovered that the culture filtrates of certain kinds of bacteria, e.g., E. typhosa, EscJi. coli, the meningococci, streptococci and a few other bacteria, contain what he designated as a skin preparatory and reacting factor respectively. If a small amount of such a filtrate is injected into the skin of a rabbit, and twenty-four liours later an intravenous injection of the same or any other filtrate that contains the reacting factor is administered, there develops a severe hemorrhagic reaction at the site of the primary skin injection. In Shwartzman 's opinion the phenomenon is due to a local disturbance of cell function produced by the filtrate. He reports that the skin preparatory and reacting factors of a filtrate may be neutralized by the addi- tion of its homologous immune serum. A few additional observa- tions are reported. 9. Allergy in lymphogranuloma inguinale is mentioned and a skin test as originally described by Frei is described and discussed. The etiological factor is apparently a filtrable virus which Tamura reports having cultured in vitro. According to Tamura the super- natant fluid of positive cultures can be heated and used success- fully in skin testing or in immunization. If his results are con- firmed, a commercial antigen should be available in the near future. References Ando, K., and Ozaki, K.: Studies on the "Toxins" of Hemolytic Strepto- cocci. V. The Dick Test and Allergic Skin Reactions to Strepto- coccus Nucleoproteins, J. Immunol. 18: 267, 1930. Apitz, K. : A Study of the Generalized Shwartzman Phenomenon, J. Im- munol. 29; 2.55, 1935. Studies on the Chemical Nature of Shwartz- man Active Substances, Ibid. 29: 343, 1935. Birkhaug, K. E.: Protection Against Tuberculosis with B. C. G. Vaccine in Guinea Pigs. Am. Rev. Tuberc. 27: 6, 1933. Bohmig, R., and Swift, H. F. : Comparative Histologic Reactions in Cu- taneous Lesions Induced by Streptococci in Rabbits Previously Inoculated Intracutaneously or Intravenously, Arch. Path. 15: 611, 1933. Cannon, P. R., and Hartley, M. A., Jr.: The Failure of Allergic Inflamma- tion to Protect Rabbits Against Infection with Virulent Pneumo- cocci, Am. J. Path. 14: 87, 3938. Cannon, Paul R., and Sullivan, F. L.: Local Immunity and the Local Formation of Antibodies, Am. J. Path. 8: 597, 1932 (Abstract), Clawson, B. J.: Experimental Vaccination with B. C. G., Am. J. Path. 10: 664, 1934 (Abstract), Clawson, B. J.: Studies on the Etiology of Acute Rheumatic Fever, J. Infect. Dis. 36: 444, 1925. Clawson, B, J.: Experimental Subcutaneous Rheumatic Nodules, Am. J, Path. 4: 565, 1928. 556 IMMUNOLOGY Connor, W. H., Levin, E. A., and Ecker, E. E.: Observations on the Frci Test, J. Infect. Dis. 60: 62, 1937. Corper, H. J.: Analysis of the Tubercle Bacillus and Its Natural Products by Immune Allergic and Anaphylactic Tests, J. Infect. Dis. 66: 23, 1940. DeWolf, H. F., and Van Cleve, J. V.: Lymphogranuloma Inguinale, J. A. M. A. 99: 1065, 1932. Dochez, A. E., and Sherman, L.: Some Reactions in Sensitized Guinea Pigs to the Filtrate of Scarlatinal Streptococcus, Proc. Soc. Exper. Biol. & Med. 22: 282, 1925. Dochez, A. R., and Stevens, F. A.: Studies on Biology of Streptococcus; Al- lergic Reactions With Strain From Erysipelas, J. Exper. Med. 46: 487, 1927. Everett, F. R.: The Pathological Anatomy of Pulmonary Tuberculosis in the American Negro and in the Wliite Race, Am. Rev. Tuberc. 27: 411-464, 1933. Feldman, W. H.: A Studv of the Pathogeiiicitv of the Bacillus of Calmette Guerin (B. C. G.), Am. J. Path. 8: 755, 1932. Freund, J., and Angevine, D. M. : The Spread of Tubercle Bacilli in the Bodies of Sensitized and Immunized Animals, J. Immunol. 35: 271, 1938. Garber. C. Z. : Rheumatic Heart Disease Without Valvulitis, Am. J. Path. 9: 443, 1933. Giltner, W. : Brucellosis, a Public Health Problem, Agricultural Experiment Station, Michigan State College Memoir No. 1. Hetherington, H. W. : The Significance of Tuberculous Lesions Found in Adolescent Children in a School Survey, Twenty-Fourth Report of the Henry Phipps Inst., 1932-33, Studies 8. Hughes, J.: Imnmnization Phenomena in Rabbits Vaccinated With Heat- Killed Tubercle Bacilli. A Study of tlie Cutaneous Reactions and the Development of Bacteriotropin, J. Immunol. 25: 103-111, 1933. Lurie, M. B. : A Correlation Between the Histological Changes and tlic Fate of Living Tubercle Bacilli in the Organs of Reinfected Rabbits, J. Exper. Med. 57: 181, 1933. Lurie, M. B.: Experimental Epidemiology of Tuberculosis. The Effect of a Primarv Infection on Contact Tuberculosis in Rabbits, J. Exper, Med. 58: 305, 1933. Lurie, M. B. : On the Mechanism of Immunity in Tuberculosis. The Host- Parasite Relationship Under Conditions of a Localized Agar Focus of Infection and the Generalization of the Disease in Normal and Im- munized Animals, J. Exper. Med. 63: 923, 1936. The Role of Extra- cellular Factors and Local Immunity in the Fixation and Inhibition of Growth of Tubercle Bacilli, Ibid. 69: 555, 1939. The Mobilization of Mononuclear Phagocytes in Normal and Immunized Animals and Their Relative Capacities for Division and Phagocytosis, Ibid. 69: 579, 1939. JtcBroom, J., and Schlesinger, H. : Studies of the Early Stag* of Resistance to Pneumococcic Infection, J. Immunol. 39: 171, 185, 1940. McPhedran, F. M., and Opie, E. L. : Clinical Significance of Latent Pul- monary Tuberculosis, Twenty-Fourth Report of the Henry Phipps Inst., 1932-33, Studies 3. McPhedran, F. M.: Incidence of Tuberculosis in Medical Students, Twenty- Fourth Report of the Henrv Phipps Inst., reprinted from J. Am. Med. Coll. 8: 236, 1933. McPhedran, F. M. : The Occurrence and Progress of Tuberculous Lesions in School Children and Adults, Transactions of the College of Physicians of Philadelphia 54: 98, 1932. SIGNIFICANCE OF .ALLERGY 557 Menkin, V.: Physiol. Eev. 18: liGC), 1938. Cited by Lurie. Miller, J. A.: The Drama of Tuberculosis, J. A. M. A. 115: 1272, 1940. Myers, J. A., and Harrington, F. E. : The Eflfect of Initial Tuberculosis In- fection on Subsequent Tuberculous Lesions, J. A. M. A. 103: 1530, 1934. Opie, E. L. : The Epidemiology of Tuberculosis in Eelation to the Pathological Anatomy and Pathogenesis of the Disease, Twenty-Fourth Eeport of the Henry Phipps Inst., 19;!2-3.''., p. 1. Opie, E. L. : Cellular Reactions of Tuberculosis and Their Relation to Im- munity and Sensitization, Arch. Path. 14: 706, 1932. Park, W. H., Kereszturi, Camille, and Mishulow, Lucy: Effect of Vaccina- tion With B. C. G. on Children From Tuberculous Families, J. A. M. A. 101: 1619, 1933. Petroff, S. A., and Stewart, F. W. : Immunological Studies in Tuberculosis. III. Concerning Allergic Reactions Obtained in Animals Sensitized With Killed Tubercle Bacilli, .L Immunol. 10: 677, 1925. Petroff, S. A., and Stewart, F. W. : Immunological Studies in Tuberculosis. IV. Concerning the Resistance to Infection of Animals Sensitized With Heat-Killed Tubercle Bacilli, J. Immunol. 12: 97, 1926. Rich, A. R.: The Role of Allergy in Tuberculosis, Arch. Int. Med. 43: 691, 1929. Rich, A. R. : Observations on the Relation of Allergy to Immunity, Johns Hopkins Hosp. Bull. 47: 189, 1930. Rich, A. R., and Brown, J. H. : Dissociation of Allergy From Immunity in Pneumococcus Infection, Proc. Soc. Exper. Biol. & Med. 27: 695 and 696, 1929-30. Rich, A. R., Jennings, F. B., Jr., and Downing, L. M. : The Persistence of Immunity After the Abolition of Allergy by Desensitization, Johns Hopkins Hosp. Bull. 53: 172, 1933. Rich, A. R., and McCordock, H. A.: An Enquiry Concerning the Role of Allergy, Immunity and Other Factors of Importance in the Patho- genesis of Human Tuberculosis, Johns Hopkins Hosp. Bull. 44: 273, 1929. Schoenholz, P., and Meyer, K. F. : The Purification of Abortin, J. Infect. Dis. 40: 453, 1927. Sewall, H., De Savitsch, E., and Butler, C. : The Nodules of Experimental Tuberculosis in the Guinea Pig and Their Relations to Immunity, Am. Rev. Tuberc. 26: 1, 1932. Shwartzman, G.: A New Phenomenon of Local Skin Reactivity to B. Tj-phosus Culture Filtrate, Proc. Soc. Exper. Biol. & Med. 25: 560, 1928. Shwartzman, G. : Studies in Bacillus Typhosus Toxic Substances. I. Phenomenon of Local Skin Reactivity to B. Typhosus Culture Filtrate, J. Exper. Med. 48: 247, 1928. Shwartzman, G. : Effect of Immune Sera Upon the Phenomenon of Local Skin Reactivity to B. Typhosus Culture Filtrates, Proc. Soc. Exper. Biol. & Med. 26: 131, 1928. Shwartzman, G.: Studies on Bacillus Tj-phosus Toxic Substances. II. The Effect of Sera Upon the Factors Determining Local Skin Reactivity to Filtrates of Bacillus Typhosus Culture.s, J. Exper. Med. 49: 593, 1929. Shwartzman, G.: Phenomenon of Local Skin Reactivity to Culture Filtrates of Various Microorganisms, Proc. Soc. Exper. Biol. & JMed. 26: 207, 1928. Shwartzman, G.: An Improved Method of Preparing B. Typhosus Toxic Substances Necessary for the Phenomenon of Local Skin Reactivity, Proc. Soc. Exper. Biol. & Med. 26: 843, 1929. Shwartzman, G. : Preparation of Meningococcus Toxic Substances Necessary for Phenomenon of Local Skin Reactivity, J. Infect. Dis. 45: 232, 1929. 558 IMMUNOLOGY Shwartzman, G. : Aninioniuni Sulphate Precipitation of B. Typhosus Toxic Substances in the Phenomenon of Local Skin Eeactivity, J. Infect. Dis. 45: 283, 1929. Shwartzman, G.: Studies on Bacillus Typhosus Toxic Substances. III. The Effect of Sera Upon the Injury Producing Factors of the Phenomenon of Local Skin Eeactivity, J. Exper. Med. 50: 513, 1929. Shwartzman, G. : Therapeutic Antimeningoeoccus Serums : Measurement of Their Neutralizing Potency by Means of the Phenomenon of Local Skin Eeactivity, J. A. M. A. 93: 1965, 1929. Seibert, F. B.: Effect of Sensitization With Tuberculin Protein Upon De- velopment and Course of Experimental Tuberculosis, Proc. Soc. Exper. Biol. & Med. 30: 1274-1276, 1933. Smith, T. : Focal Cell Eeactions in Tuberculosis and Allied Diseases, Johns Hopkins Hosp. Bull. 53: 197, 1933. Stolyhwo, N. : Toxic Substances in Urine and Sweat of Typhoid Fever Patients as Demonstrated by the Shwartzman Phenomenon, J. Immunol, 30: 235, 1936. Swift, H. F.: An Allergic Theory of Eheumatism, J. A. M. A. 90: 906, 1928. Swift, H. F. : Hektoen Lectures, Eheumatic Fever, J. A. M. A. 92: 2071, 1928. Swift, H. F. : Factors Favoring the Onset and Continuation of Eheumatic Fever, Am. Heart J. 6: 625, 1931. Swift, H. F. : Allergy and Eheumatism. See "Eesistance to Infectious Dis- eases" Zinsser, New York, 1931, The Macmillan Co., p. 466. Tamura, J. T. : The Virus of Lymphogranuloma Inguinale. Its Cultivation, Its Antigenic Value as a Vaccine and Also in the Production of an Antiserum, J. Lab. & Clin. Med. 20: 393, 1935. Teale, F. H.: Some Observations in the Eelative Importance of the Eeticulo-Endothelial Tissues and Circulating Antibody in Immunity. II, Hypersensitiveness and Immunity to Foreign Proteins, An Analysis of the Parts Played by the Tissues and Circulating Antibody in These Two States, J. Immunol. 28: 161, 1935. Terplan, K. L. : Anatomical Studies on Primary and Postprimary Tubercu- losis in Wliite Children and Adults, Am. J. Path, 10: 680, 1934. (Abstract.) Walker, T. T., and Hoffman, D. C: The Effect of Testicular Extract on Experimental Tuberculosis in Eabbits. I. Skin Lesions, Am. J. Path, 9: 651, 1933, Wallgren, A.: Value of Calmette Vaccination in Prevention of Tuberculosis in Childhood, J. A. M. A. 103: 1341, 1934. Wells, A. Q., and Brooke, W. S. : The Effect of Vaccination of Guinea Pigs With the Vole Acid-Fast Bacillus on Subsequent Tuberculosis Infection, Brit. J. Exper. Path. 21: 104, 1940. Woodruff, C. E., and Willis, H. S,: Allergy and Desensitization in Experi- mental Tuberculosis, The Effect of Time and Dosage, J. Immunol. 37: 549, 1939, Zinsser, H. : Eesistance to Infectious Diseases, ed. 4, New York, 1931, The Macmillan Co. Zinsser, H., Enders, J, F., and Fothergill, L. D.: Immunology, New York, 1939, The Macmillan Co. CHAPTER XXVIII I-IYPERSENSITIVENESS Human Idiosyncrasies Clinical Allergies. — It is generally recognized, at the present time, that a large nnmber of individuals (variously estimated at from 7 to 15 per cent) are abnormally sensitive to one or more protein or nonprotein substances. In the group of clinical al- lergies one places hay fever, asthma, urticaria, eczema, contact dermatitis, serum disease and drug allergy. Rackemann and others suggest that migraine, angioneurotic edema, erythema multiforme and perhaps epilepsy are manifestations of hyper- sensitiveness. Reactions Occur in Local Shock Organs. — In human idio- syncrasies the reactions tend to be local rather than general. This has led Doerr to introduce the term "shock organs" to indicate the organs or tissues in which evidence of hypersensitiveness ap- pears. Thus in vasomotor rhinitis the ''shock organ" is the mu- cous membrane of the nose, in asthmas it is the lungs, while in eczema, contact dermatitis, etc., it is the skin. Duke and others report numerous cases of allergy in which the "shock organ" is the gastrointestinal tract. Coca calls attention to the fact that not all of the shock organs are affected in every atopic patient. Many hay fever patients do not have asthma and conversely some patients with asthma do not have hay fever or urticaria. Inheritance Factors in Atopy. — Cooke and Vender Veer seem to have established an hereditary factor for the clinical allergies other than serum disease and contact dermatitis (poison ivy, etc.). Their data indicate that the capacity to become allergic is an in- heritable characteristic which follows the Mendelian principles. There is no evidence to indicate that hypersensitiveness to a specific substance is inherited ; instead only the capacity to become allergic appears in the offspring. A parent may suffer from hay fever due to ragweed pollen while the offspring may develop eczema from eating eggs. There is no unanimity of opinion at present as to whether the hereditary character is dominant or recessive. Such 559 560 IMMUNOLOGY forms of hypersensitiveness are designated by Coca as atopy. Zinsser, Enders and Fothergill (1939) and Coca (1931) state that two factors determine the development of atopic hypersensitive- ness ; i.e., contact and the inherited capacity to become sensitized. Reagins Not Demonstrable in All Cases of Allerg:y. — While specific antibodies, which Coca calls reagins, are present in the blood of all asthma and hay fever patients who give positive skin tests, speeific reagins have not been found in those suffering from contact dermatitis, serum disease or drug allergy although there is evidence which suggests that they, too, are mediated by an anti- gen-antibody mechanism. Zinsser (1931) bases his belief that all human idios^mcrasies and anaphylaxis are mediated by similar if not identical mechanisms upon the following facts : The idio- syncrasies are specific, are frequently accompanied by specific skin reactions, are alike regardless of the kind of exciting agent involved, and are often amenable to desensitization. One might add also that in experimentally produced contact dermatitis, in serum disease, and in many cases of drug allergy, the idiosyncrasy develops after an incubation period. The assumption that anti- bodies do not participate in these three types of allergy is based upon negative results where passive transfer has been attempted. We agree with Zinsser that such negative findings do not now constitute adequate reasons for assuming a different mechanism from that demonstrable for most of the atopies and for anaphy- laxis. Loveless (1940) reports the existence of two antibodies in the serum of hay-fever patients. One is the reagin and the other is an antibody that binds and neutralizes the homologous antigen. Passive Transfer of Reagin and the P-K Reaction. — In 1919 Ramirez reported that a patient transfused with blood from a donor sensitive to horse proteins became hjq^erscnsitive to the same exciting agent. Doerr cites a number of examples of passive sensitization following transfusion from allergic donors. It is generally agreed, however, that such cases are extremely rare. In 1921 Prausnitz and Kiistner discovered that the serum of many hypersensitive individuals, when injected intradermally into a nor- mal person, renders the local area injected hypersensitive to the same exciting agent to which the donor is sensitive. Kiistner was sensitive to fish. He demonstrated that his serum contained anti- HYPERSENSITIVENESS 561 bodies or reagins for fish proteins by injecting a small amount of his serum intracutaneously into the forearm of a nonsensitivc person. A few hours later, when the area thus injected was tested with an extract of fish proteins, a positive skin reaction occurred. This phenomenon, which indicates the presence of reagins in serum, is called the Prausnitz-Kiistner or P-K reaction. Coca and Grove state that only about 80 per cent of normal skins are receptive. In 1937 Cowie reported upon a scries of individuals who were clinically sensitive but skin negative to certain food allergies. He stated that the intradermal injection of such a patient's own serum into his skin rendered the injection site sensitive to the allergin. This he called autopassive transfer. Martin (1940) was unable to confirm Cowie 's conclusions. On the contrary he states that the patient's own serum has an inhibitory rather than an enhancing effect. Sensitization Throug-h Alimentary and Respiratory Tracts and Through Placenta, — Walzer (1927) made use of the Prausnitz- Kiistner reaction to ascertain whether the exciting agent is ab- sorbed through the mucous membrane of the intestine. He injected serum from an egg-sensitive patient and from two fish-sensitive patients intracutaneously into the forearms of nonsensitivc in- dividuals. The following morning he fed the recipients egg and fish, respectively, and observed redness and swelling develop at the site of the serum injections. In 1936 Gray and Walzer reported on the absorption of un- digested peanut protein following oral and intraduodenal admin- istration. They have also found (1940) this protein to be absorbed from the rectum. Gay and Chant passively sensitized to ragweed an area in the skin of the forearm of a nonsensitivc individual and after an ade- quate incubation period had elapsed they injected ragweed pollen extract intradermally into the opposite forearm. This caused a positive reaction characterized by erythema, edema, and itching in the area that had been passively sensitized. According to Gay and Chant this reaction was produced by ' ' contralateral injection. ' ' Ulrich (1918) and Eatner and Gruehl (1929) offer evidence which indicates that specific sensitization may develop as a result of absorption through the respiratory mucous membrane. When one considers the amount of exposure to pollen and various finely 562 IMMUNOLOGY divided material in the air, organic and inorganic compounds in food, water and the various substances with which one comes in contact, it is not surprising that allergic disease is common and that the exciting agents are numerous. In those cases in which allergy apparently occurs on the first ingestion of food, Zinsser, Enders and Fothcrgill (1939) suggest that active sensitization may have occurred in utero by the passage of the exciting agent from the mother through the placenta. In fact they state that there is little doubt that both active and passive sensitization of an infant can take place before birth. For some reason, however, the child is not often born hypersensitive. Effect of Heredity on Age of Onset. — Heredity apparently in- fluences the time of onset of allergy. Those with a bilateral in- heritance are all sensitive by the age of forty, 36 per cent being sensitive before the age of five, while hypersensitive individuals with a unilateral and with negative family histories of allergy become sensitive as a rule at relatively later periods of life. Facts About Reagin. — The antibodies or reagins which are responsible for the P-K reaction have been studied extensively. A few of the more important things that have been learned about them may be summarized as follows : 1. When a serum containing reagin is mixed with the specific atopen, the reagin titer is reduced or removed, but the capacity of the exciting agent (atopen) to elicit skin reactions remains unchanged. 2. The reaction between reagin and atopen does not lead to the formation of a visible precipitate. 3. AVhile a very small amount of a serum containing reagin can passively sensitize the human skin, it cannot passively sensi- tize the skin of the lower animals. 4. Reagin is apparently more susceptible to heat than pre- cipitins or protein sensitizers. 5. Many individuals clinically desensitized by the repeated in- jection of extracts of an atopen responsible for hay fever or asthma remain skin sensitive. 6. In some individuals positive skin tests can be obtained re- peatedly in the same area, HYPERSENSITIVENESS 563 7. The injection of an atopen into an atopically sensitive in- dividual may lead to an increase in the reagin content of the blood. 8. A few cases are on record where reagin was detected in the blood of an individual before he became clinically sensitive. 9. Multiple sensitization is quite commonly observed. 10. Among individuals sensitive to food Rowe estimates that only 50 or 60 per cent will give positive skin reactions to the exciting agent. Comparison op Reagin With Antibodies. — It seems to us that these results are not necessarily incompatible with the view that reagin is an antibody. According to the present concept of antigen-antibody union it is a partial filming of dispersed particles of antigen by antibody globulin; therefore, it is conceivable that the toxic property of atopen (antigen) might be retained after partial filming with reagin protein (antibody globulin). In the case of the neutraliza- tion of toxin by antitoxin the properties of the toxin are not destroyed since the antigenic property is unimpaired and the toxic property is demonstrable after dissociation occurs. In regard to the reaction between reagin and atopen not leading to the formation of a visible precipitate, it is of interest to recall that Coca described an antibody which interferes with the ag- glutination of E. typhosa by its specific agglutinin. In toxin- antitoxin neutralization experiments a precipitate is formed only under certain specific conditions. In the case of syphilitic reagin a precipitate is formed when it is brought into contact with a lipoid hapten only under certain conditions. Furthermore, the suppression phenomenon of Landsteiner involves the participation of an antibody in a reaction characterized by the absence of a visible precipitate. It may be that the proper conditions for a precipitin or flocculation test for atopic reagins may at some future time be discovered. In any event, it should be remembered that the phenomena of precipitation and agglutination are secondary and not primary ones in antigen-antibody reactions. Since it has been established that immune serum from one species of animal is not always capable of passivel}' sensitizing another species or, for that matter, every individual of the same species, it 564 IMMUNOLOGY Is not surprising that human serum containing reagin will not passively sensitize the skin of every human or the tissues of the lower animals. The apparent inaetivation of reagin when heated to 56° C. for thirty minutes is not in itself a sufficient reason for omitting reagins from the list of true antibodies. Nuttall states that bac- terial precipitins are inactivated at 58-60° C, whereas other immune precipitins are inactivated at 68-70° C. Sherman found that the apparent destruction of heterohemolysin which occurs frequently when serum containing it is heated to 56° C. for thirty minutes is only an interference or masking phenomenon. When the hemolysin is absorbed at 0° C. from the heated serum, it is found to be uninjured. At the present time Coca is inclined to agree with Zinsser that reagins may be regarded as antibodies. Antibody Responsible for Immunity. — According to Loveless (1940) the antibody responsible for immunity to hay fever is more thermostable than reagin and unites with its homologous antigen. If the results obtained by Loveless meet with confirmation, a defi- nite advance in our knowledge of hay fever will have been achieved. In order to present more clearly certain additional facts about human hypersensitiveness, a few of the clinical allergies will be discussed briefly. For a more comprehensive treatment of the subject the student is referred to the monographs of Duke (1925), Alexander (1928), Coca, Walzer and Thommen (1931), Racke- mann (1931), Zinsser, Enders and Fothergill (1939), Bray (1934), Vaughan (1939). Definition of Hay Fever. — Hay fever is a term that is used quite frequently to designate all forms of vasomotor rhinitis, al- though Rackemann prefers to apply it only to vasomotor rhinitis caused by pollen. It will be used in this chapter in the latter sense. Attacks of pollen hay fever are seasonal and occur only when an adequate concentration of the plant pollen to which the in- dividual is sensitive is present in the air. The symptoms develop when the pollen comes in contact with the mucous membrane of the nose or with the conjunctiva. Since the dates of pollination of plants in most districts of the United States have been determined by botanists and specialists in allergy and are a matter of record, it is obvioiLS that the history of the date of onset and duration of an attack of hay fever is of material value in determining the par- HYPERSENSITIVENESS 565 ticular i)ollcns to be used in skin tests. Since atmospheric condi- tions such as wind, dust and rain affect the amount of pollen in the air, it is not surprising that the patient associates such conditions with the degree of severity of his symptoms. Historical. — The history of hay fever is quite interesting. Duke (1925) states that while cases in which headache, sneezing and itching of the nose were reported during the sixteenth and seventeenth centuries and attributed to the odor of roses, it re- mained for Bostock (1819) to recognize hay fever as a clinical entity. The etiology was not determined until 1856, when Blackley, an English physician, who was subject to seasonal attacks of hay fever and asthma, began his experimental study of the subject and determined that it is caused by pollen. Blackley 's work was not appreciated by clinicians until it was confirmed by Dunbar in 1903. The results of Blackley 's extensive investigation may be partially summarized as follows : Blackley 's Observations. — 1. He tested, on himself, the pollen from approximately one hundred different species of plants and discovered that while he was sensitive to several, he reacted more severel,v to the pollen of rye tluin to other pollens. 2. In his experiments he used both dried and fresh preparations and also extracts of pollen. He employed methods involving inhalation, direct application to the mucous meml)rane of the nose, and instillation into the conjunctiva, and was able to produce liay fever symptoms by all of these procedures. 3. Blackley was the first one to employ skin tests. These he per- formed by rubbing the pollen into a scarified area of the forearm or over the tibia. 4. He made a quantitative study of the amount of pollen present on different days from early spring until August by exposing, for twenty-four hours, glass slides covered with sticky material and noting the kind and number of pollen grains that adhered to the slide. By comparing these results with his own clinical symptoms he found that the latter increased and decreased in severity as the amount of air-borne pollen to which he was sensitive was large or small. He found that the amount of pollen in the air was affected by atmospheric conditions, being considerably decreased after a heavy rain. In his case there was not sufficient pollen in the air prior to June 8 to cause an attack of hay fever. It is now generally 566 IMMUNOLOGY recognized that the June grasses are the principal causes of hay fever in the early summer, while ragweed pollen is the chief offender in August and September. Dunbar Confirmed Blackley's Work. — The confirmation of Blackley's work by Dunbar in 1903 stimulated widespread interest in the subject. The results of subsequent research are discussed at length in the monographs of Rackemann, Coca and others. The most important discoveries are summarized earlier in this chapter. Perennial Vasomotor Rhinitis. — In regard to perennial vaso- motor rhinitis the important facts may be presented by giving a resume of Rackemann 's report on 257 cases studied by him. The majority of these patients were 3'oung people and 73 per cent were women. In only 25 per cent did he find a history of allergy in the family. Sixteen per cent of the 257 individuals presented symp- toms of asthma and 7 per cent eczema along Avith those of vasomotor rliinitis at the time of examination. He was able to identify the ex<'iting agent in only 150 or 58 ])er cent of the 257 patients. Perhaps the reason that sucli a liigh ])erccntagc of his cases ap- peared among young women is that cosmetics arc used so exten- sively by them. He had 44 cases in which tlie exciting agent was orris powder. All of these gave positive skin tests. Other investi- gators have reported cases of vasomotor rhinitis due to rice flour and various other substances used as cosmetics. In Rackemann 's series there were thirteen patients sensitive to emanations from cats, dogs, feathers, etc. In another group he places those sensi- tive to something in house dust. In a few cases of vasomotor rhinitis there was an expression of hypersensitiveness to occupational dust while in others it was due to food allergy. In 72 cases of Rackemann 's series operations were performed to clear up local lesions. Tonsillectomies seemed to benefit two out of sixteen patients while four out of twenty-two were benefited by the drainage of their sinuses. The straightening of the nasal septae of thirty patients resulted in beneficial results to seven. Asthma. — The term "asthma" according to Brown (1917) is used to apply specifically to that type of dyspnea characterized by (1) prolonged and difficult expiration especially if the attack be well developed (in the beginning of a paroxysm, however, botli inspiration and expiration may appear equally difficult) ; (2) a HYPERSENSITIVENESS 567 marked distention — emphysema — of the chest; (3) wheezing and sonorous sounds with both pliases of respiration, but especially with expiration; (4) signs of circulatory disturbance — cyanosis and distention of the blood vessels of the neck and face; (5) secretory changes in the bronciiial mucosa usually producing a considerable quantity of mucous casts, ])lugs, crystals, etc.; and (6) a standing or sitting posture with arms and shoulders l)raced so as to bring the muscles of expiration into best use." Brown also gives an excellent discussion of the early history and theories of asthma. It has apparently been recognized clinically since the time of Hippocrates. Cullen (1788) is said to be the first to suggest ''that asthma might be the result of spasm of muscles of the finer bronchi." According to Rackemann (1931) the location of bronchial obstruction in asthma may be in the larger bronchioles w'hen the interlocking fibers of the bronchial muscles compress their openings; in the terminal bronchioles due to the contraction of true sphincters ; or the bronchi may be, and perhaps always are, obstructed to a greater or less degree by the presence of a tenacious exudate within the lumen of the tube. It is definitely established that the lungs are the "shock organs" in asthma. The exciting agents are similar to those of hay fever and other allergic conditions. Food Allergy. — Food allergy may express itself as vasomotor rhinitis, asthma, urticaria (hives), or eczema. There is a great deal of evidence that food allergy may also manifest itself as a disturbance of the intestinal tract, or perhaps by a number of other clinical symptoms. According to Rowe (1934), positive skin tests are obtained in about 50 per cent of the cases of food allergy and multiple sensitization is quite common. Piness and Miller (1931) claim a much higher percentage of positive skin tests in their series of cases. Vaughan (1930), Ellis (1931) and others have carried out extensive studies on the grouping and classifica- tion of the food allergins. They have proposed a biological classification which is regarded by many as of distinct value in interpreting skin tests and in devising "elimination diets." Vaughan states (1930) that symptoms may be caused by members of a food group to which the patient is skin negative. In his opinion sensitivity to one member of a biological food group warrants careful watching for the development of sensitiveness to 568 IMMUNOLOGY other members of the same group. Many instances of the disap- pearance of food allergy are on record. The value of the botanical classification of plants yielding com- mon food allergies suggested by Vaughan (1930) and Ellis (1931) as a basis for carrying out food testing is definitely questioned by Piness, Miller, Carnahan, Altose and Hawes (1940). In their opinion a botanical classification is an unreliable guide to pre- dicting skin reactivity to a food when reactivity to a botanieally related food is known. Drug" Allergy. — Reports of cases of hypersensitiveness to one or more of the medicinal drugs are quite numerous in the literature. As a rule the principal symptom is some form of an eruption. The latter is usually accompanied by itching. Occasionally there are dyspnea, edema, swelling of the joints and of the lymph nodes. The symptoms appear, in most cases, within a few hours after the drug has been administered. Antibodies are not demonstrable. When hypersensitiveness is once established, the patient as a rule remains sensitive for life. The development of temporary tolerance to quinine in an individual hypersensitive to quinine is reported by Heran and St. Girons (1917). Loveman (1939) lias given an excellent brief discussion of allergic drug eruptions. Landsteiner and his associates (1935, 1936, 1938, 1940) report experimental results indicating that certain simple chemical sub- stances may function as haptens and unite with the body protein to form new antigens, giving rise to symptoms of drug allergy or contact dermatitis, depending upon conditions. Contact Dermatitis. — In contact dermatitis the shock organ is the epidermis. The lesions develop in the allergic individual from surface contact with the exciting agent but do not result from the injection or ingestion of the latter. The results of exten- sive statistical studies indicate that a Mendelian factor is not in- volved in this type of human idiosyncrasy. Contact dermatitis was regarded formerly as due to the action of plant poisons upon the skin and for this reason the nomenclature of dermatitis due to exciting agents in poison ivy, poison oak, etc., is misleading, since the dermatitis is a manifestation of specific hypersensitive- ness to a nonantigenic excitant which is quite often soluble in fat solvents and is not due to a specific toxin. 1 HYPERSENSITIVENESS 569 It was established as early as 1904 by Nestler that this form of hypersensitiveness can be induced by the repeated application of the exciting agent to the skin of a nonsensitive individual. He rendered himself allergic to the juice of primrose leaves. Bloch and Steiner-Wourlisch (1926) confirmed Nestler 's results. Straus (1931) succeeded in sensitizing 48 babies to extracts of poison ivy loaves. In all of these experiments local sensitizing applications caused the entire skin of the individual to become sensitive. Anti- bodies have not been demonstrated by passive transfer. In many individuals a positive skin reaction is obtained only when the excit- ing agent is kept in contact with the skin for a long period of time such as occurs when the ''patch test" is used. The scratch and intradermal tests are always negative except where surface contact occurs in performing the test and the individual is quite sensitive. In many cases of poison ivy, poison oak, pollen dermatitis, etc., an increased tolerance corresponding to desensitization has been pro- duced by the injection of a 1 per cent solution of the exciting agent in sterile almond oil. Divergent results have been reported by numerous investigators. For a more extensive discussion of the subject the student is referred to the studies of Zisserman and Birch (1939), Stratton (1940), and a review of the literature by Goodman and Sulzberger (1940). References to the other papers are given in the supplementary list at the end of this chapter. Substances Responsible for Contact Dermatitis. — Coca (1931) lists a large number of substances that may cause contact dermatitis. Among them he mentioned the leaf, stalk and root of such plants as poison oak, poison ivy, poison sumac, primrose, chrysanthemum, tomato, geranium, lily and a number of others; many chemicaLs such a.s formalin, photogravure ink, hexamethylen- amine, scharlaeh rot and various dyes, etc. ; among the miscel- laneous materials that have been reported as causing contact dermatitis he lists soaps, adhesive plaster, orange peel, cement, furniture polish, feathers, cereals, lanolin and a number of others. In addition to these there are on record numerous cases of contact dermatitis in individuals handling plant pollens. It is interesting to note that in pollen dermatitis the exciting agent is an oil, while in pollen hay fever it is a water-soluble substance. While these 570 IMMUNOLOGY excitants all seem to lack an antigenic property, it is quite possible that they contain specific haptens and it is conceivable that the newer work on haptens, modified antigens and specificity discussed in previous chapters may lead to a better understanding of the phenomenon of contact dermatitis. Physical Factors in Allergy.— Duke (1925, 1932) calls attention lo certain cases of clinical allergy in which the symptoms seem to indicate sensitiveness to heat, cold, or sunlight. He also re- ports cases which he describes as effort sensitiveness. In this connection it is of interest to note that dermographia has long been associated with idiosyncrasy to food, although it is elicited by mechanical streaking of the skin. There is at present a difference of opinion in regard to the "physical allergies." One group of allergists maintains that the symptoms represent a sensitiveness to a physical agent, while another regards the sensitiveness to physical agents as due to physiological disturb- ances associated with hypersensitiveness to organic or inorganic substances. Karady (1939) suggested that a physical stimulus might alter the protein of the blood plasma in such a way as to form an "auto-antigen." Richardson (1940) has been unable to confirm Karady 's work. Attention has also been called to the oc- casional relationship of endocrine dysfunction in skin sensitiveness to physical agents as, for example, the existence of urticaria, ec- zema, etc., in patients suffering from hypothyroidism (Cobb, 1919). For a recent discussion of physical allergy the student is referred to a paper by Swineford (1935). Diagnosis of Allergy. — In the diagnosis of clinical allergy a good history is exceedingly important. When the patient is able to keep an accurate and comprehensive diary for a period that in- cludes sevei'al attacks of allergic symptoms, the diagnosis may be- come quite obvious and definitely verifiable by adequate tests. Among the various other devices used by physicians in attempting to ascertain the source of the exciting agent in any particular case of clinical allergy may be mentioned the following: a. Elimination Tests.- — Suggestion that the patient avoid cer- tain things that the history incriminates. This is the basis for employing elimination diets in the diagnosis of food allergies. HYPERSENSITIVENESS 571 b. Conjunctival Tests. — In cases of pollen hay fever a dilute solution of the pollen under suspicion is dropped into the con- junctival sac. If no reaction occurs within five or ten minutes, the pollen is not incriminated. When a positive reaction occurs, it ap- pears as a diffuse reddening of the conjunctiva accompanied by itching and a watery discharge. The intensity of the reaction can be controlled by dropping into the conjunctival sac either adrenalin (1:5,000) or cocaine (2 per cent). c. Scratch tests are performed on the back, al)domen or arms of the patient. Alexander (1930) demonstrated considerable varia- tion in skin reactions (intradermal) for different regions of the body. The skin of the back and abdomen is apparently preferable to the skin of the forearm. The technique employed by Rackemann in performing scratch tests is described by him as follows : Drops of slightly alkalini7.ed salt solution or Coca's extracting fluid are placed at least one inch apart on the skin. Powdered allergen is I'emoved from its bottle by means of a number 4 (large size) three- cornered, straight surgical needle and stirred into a drop of solvent after thorough mixing. The needle is used to make a short scratch, about 3 mm. long, through the drop of solution. The needle is then wiped thoroughly with gauze soaked in alcohol and used to test another allergen in the next drop of solvent. d. Puncture Test. — In the puncture test the reagin extract is prepared as for the scratch test. Instead of making a scratch through the drop of extract, a few skin punctures are made with the .sharp point of a darning needle. e. Intradermal Test. — The intradermal test is employed by many specialists in allergy. In performing this test one must em- ploy sterile, standardized allergen extracts and preferably a separate, all-glass, accurately graduated tuberculin syringe for each extract. Rackemann employs sterile No. 26 gauge needles and in- jects not to exceed 0.02 c.c. of each allergen extract intradermally as in the Mantoux test. f. Patch Test.— It has been found that in practically all cases of contact dermatitis the exciting agent will give a pasitive skin 7'eaction if it is kept in contact with the skin, whereas negative re- .sults are obtained (juite fre([uently when the scratch or intradermal 572 IMMUNOLOGY method is employed. Ramirez (1933) remarks that both Jadassohn and Block popularized the patch test in Europe Avhile Sulzberger has stimulated interest in the test in America. In conducting the ' ' patch test ' ' one puts a small amount of the material to be tested on an area of healthy skin, moistens it with water if necessary, and covers it with a square of linen and then a square of rubber tissue. These are held in place by a patch of adhesive tape. When posi- tive reactions occur they do so, as a rule, within twenty-four to forty-eight hours. Occasionally a reaction develops twenty-four hours after the material is removed. They appear as an area of redness associated with small vesicles where the material has been in contact with the skin. g. Biological Test. — Since one may be skin positive and clin- ically negative or skin negative and clinically sensitive to an allergen, it is often necessary to ascertain by direct experiment whether the patient is clinically sensitive to an allergen that is under suspicion. This may be accomplished by ingestion of specific foods after fasting where food allergy is under considera- tion; by inducing attacks through exposure to specific inhalants where the latter are under suspicion ; and l)y natural direct contact in ascertaining the cause of contact dermatitis. h. Leucopenic Index. — Vaughan (1934) suggests that in food allergy it is possible to show that the ingestion of the allergen to which the patient is sensitive results in a drop in the leucocyte count (leucopenia) within fifteen minutes and a return to normal after ninety minutes. While Vaughan is apparently of the opinion that the "leucopenic index," as it is called, will prove of value in many cases of food allergy where other methods prove inadequate in identifying the exciting agent, one should remember that this test is not yet beyond the stage of experimental investigation. EosiNOPHiLES AND Allergy. — Aucr (1915) calls attention to the development of an eosinophilia after delayed anaphylactic shock. Davison (1934) lists eosinophilia as one oE the hematological find- ings in allergy. It is generally recognized that eosinophilic cells ak)ng with Charcot-Leyden crystals and Curschmanirs spirals are quite characteristic sputum findings in bronchial asthma. Hansel (1934, 1935) has> carried out an extensive study of the cytology HYPERSKKSITIVENESS 573 of the secretions of the nose and paranasal sinnses in allergy. In his opinion there is invariabl}^ a definite increase in eosinophile cells. These results are of interest when one compares allergy and anaphylaxis, since Schwenke (1912) observed an increase in eosino- phile. cells in the lung tissue of guinea pigs dead of anaphylactic shock. Since eosinophilia is present quite often in chorea, in syphilis and all forms of helminthiasis and in certain other patho- logical conditions, one wonders to what extent it is evidence of allergy. The eosinophile cell is said to be the most sensitive reagent in many bacterial infections, disappearing immediately and reappearing as the first favoral)le symptom. It is evident that tlie presence of an abnormal number of eosinophiles in abnormal secretions is presumptive but not conclusive proof tliat the cause of the symptoms is allergic in nature. Mechanism of the Allergic Response. — At the present time the histamine theory discussed in the chapter on Anaphylaxis is re- garded by many individuals as an adequate explanation of the mcclianism involved in the allergic response. It is quite well established tliat the intradernuil injection of a dilute solution of histamine will cause a wheal similar in appearance and cytology lo that observed when a specific atopen is injected into the skin of a specifically sensitive individual. While we grant that there is a great deal of evidence that the liberation of a toxic substance (K'curs in the shock organ when the exciting agent reaches the latter, nevertheless, we believe that there is a serious objection to calling this substance histamine. To appreciate this objection one must recall that it is at present a well-established fact that the in- jection of a small amount of histamine into the body causes an increase in gastric acidity. In fact, this procedure is used quite often in the diagnosis of true achlorhydria. If histamine is liberated in clinical allergy, it would seem logical to assume that an increase in gastric acidity would occur during an attack. That such does not occur is indicated by the studies of Criep and "Wechsler (1931). They determined the free HCl and total acidity of the gastric juice in forty jiatients and found that the concentra- tion of each varied from zero to normal. In other words there was evidence of hypoacidity rather than hyperacidity. Others have observed an alkalosis in many patients and ketogenic diets 574 IMMUXOLOOY have been suggested. The latter are not recommended by Alex- ander (1928) who has made an extensive study of their value in the treatment of clinical allergy. Abramson, Engel, Lubkin and Ochs (1938) have developed an iontophoretic method of detecting histamine in wheals of the human skin when present in dilutions of 1 :5, 000,000. When this method was applied to wheals produced by ragweed, timothy or ultraviolet light no histamine could be demonstrated. In a review of the literature on asthma and hay fever, Feinberg and Bern- stein state that in their opinion the histamine theory of allergy cannot as yet be accepted. As a corollary to this they feel that the histaminase treatment of allergy is not yet of proved value. In tliis connection the observations of Campbell and Nicoll (1940) are of interest. They report tlie release of an active non-histamine material from sensitized guinea pig lung during in vitro anaphy- laxis. The non-]iistamine substance tliey describe produced definite response in the rat uterus. While their results need confirma- tion, they should stimulate further research relative to the mech- anism of the pliysiological response in hypersensitiveness. Committees on Allergy Clinics.— Within recent years the grow- ing interest in allergy has led to the organization of numerous allergy clinics. The evidence of Avidespread interest in allergy resulted in the appointment of committees by the Society for the Study of Asthma and Allied Conditions and the Association for the Study of Allergy, respectively, to ascertain the minimum re- quirements of equipment and personnel for such clinics. In their joint preliminary report made in 1932 they offer the following suggestions as to the location and equipment of an allergy clinic : Committee Recommendations for Clinic Equipment, Supplies AND Personnel. — ''1. Room in an organized hospital and facilities for bed patients. "2. X-ray facilities. "3. Access to a nose and throat department capable of doing major sinus surgery. "4. Skin test materials, scratch or intradermal (should include intradermal). Facilities for keeping intradermal solutions fresh, particularly after they have been diluted. HYPERSENSITIVENESS 575 "5. Solutions essential — minimum requirements: Common pollen for locality Foods: House dust Wheat Orris root Oats Silk ' Corn Cottonseed Buckwheat Kapok Rice LePage 's glue Rye Danders : l^gg Horse Milk Dog Beer Cat Lamb Cow Pork Rabbit Oyster Sheep Crab Flaxseed One common fish Feathers : Beans Chicken Potatoes Duck Spinach Goose Coconut Cantaloupe Peanut Celery Peas Strawberry Orange Tomato Chicken Mustard Chocolate * ' 6. Syringes and needles. At least four dozen. "7. An adequate record system." In regard to personnel they recommend that anyone who does skin tests should have training and experience that is satisfactory to the Standards Committee. Pollen Extracts.— Noon Pollen Unit. — According to Coca (1934) the first person to apply the principle of specific desensi- tization in hay fever was Noon (1911). He employed as a unit of atopen the amount present in the saline extract from one- millionth of a gram of pollen. Controversy Over Nature of Exciting Agent in Pollen. — Objections have been raised to the unit proposed by Noon, since French observed that the weight of a pollen is not always an ac- curate index of its content of exciting agent. There is at present definite disagreement over the nature of the exciting agent in pollen. Grove and Coca (1925) regard it as nonprotein in nature; Stull, Cooke and Tennant and their associates (1930, 1931, 1932, 576 IMMUNOLOGY 1933) present evidence indicating that the exciting agents in the pollens of ragweed, timothy and certain grasses are proteins, while Black (1932) regards that of ragweed as a carbohydrate. Preparation of Pollen Extract. — To prepare a pollen extract Rackemann (1931) recommends that one gram of pollen be added to 100 c.c. of Coca's extracting fluid, the mixture shaken and allowed to stand at room temperature for three days (it should be shaken several times during this period). It is then filtered first through paper and finally through a Berkefeld N filter. Ac- cording to Rackemann, nitrogen determination shows that such an extract contains approximately 0.20 mg. of nitrogen per cubic centimeter while Coca (1934) says that a 1:100 extract contains about 0.1 mg. of total N per cubic centimeter. The formula sug- gested by Coca for an extracting fluid for pollen and house dust is as follows : NaCl 0.5 per cent NaHCOg 0.275 per cent Phenol 0.4 per cent Carbon dioxide should be bubbled through this fluid until phenolphthalein added to a sample of it remains colorless. The formula for extracting fluids is varied according to the nature of the material to be extracted. These formulas are discussed in de- tail by Coca, Walzer and Thommen. Rackemann suggests that during pollen hay fever season fresh extract should be made every two or three wrecks and kept sealed in the ice box until ready for use. One must be certain of its sterility. Standardizing Pollen Extracts. — Various methods of stand- ardizing extracts have been employed. As previously mentioned, Noon (1911) designated as a unit the amount of exciting agent obtained by extracting one-millionth of a gram of pollen with saline. Complement fixation has been employed, but is no longer recommended. The reaction involves titrating antigenic fractions of pollen against standardized immune serum obtained by im- munizing suitable animals with pollen antigen. Since pollen pos- sesses one or more antigenic fractions distinct from the exciting agent of pollen hay fever, it is obvious that complement fixation is a measure of antigenic fractions whose relationship to the exciting HYPERSENSITIVENESS 577 ai>eiit is unknown. Olliers hnvv sui>i>ostcd standarclizinji>" it by skin tests on allersi'ic individuals, l)ul Ihis method lias not met with favor. Cooke and Stull (1933) standardize pollen extracts on the basis of their protein nitrogen content, while others regard total nitrogen as a better criterion. Coca (1933) offers numerous objections to the method of Cooke and Stull. He suggests (1934) a new definition of the Noon pollen iniit and recommends that the Noon unit be "the quantity of pollen extract which contains 0.00001 milligram of total nitrogen." Coca says that this represents ap])roximately the total N present in the exti^act of one-millionth of a gram of ])ollen. Character of an Allergic Skin Reaction. — Positive skin reac- tions to atopens are characterized by the formation, within a few minutes after the test is performed, of a wheal one or more centimeters in diameter. The reaction consists of a reddening of the skin, which is called the flare, and the development of a central blister showing one or more pseudopods. Recording Skin Reactions.- — In recording skin reactions some individuals describe them as + to + + + + depending upon the size of the wheal, number of pseudopods and the size of the area of erythema. According to Berkoff (1933) others record reactions as slight, moderate, marked, or very marked. Not infrequently the reactions are descril)ed by measurements. Berkoff suggests that a sheet of a modified celluloid called ''absolite"' or ''plas- tocele" 9 by 16 cm., be ruled oft' in 1 cm. squares and that 4 eye holes 1, 1.5, 2 and 3 cm. in diameter, respectively, be made and used for measuring the size of the skin reaction. These holes repre- sent reactions of + to + + + +, respectively, and the results are so recorded. Treatment of Allergy. — Two types of treatment apply to all forms of allergy except perhaps serum disease and these are avoid- ance of the exciting agent and desensitization, respectively. Ex- perience has shown that it is not always possible to employ either of these forms of therai)y and that palliative measures have to be used. The latter vary with the type of clinical allergy encountered. The promising reports of Bloom (1938) on the use of potassium chloride and of Ertl (1939) on the use of histamina.se in the treat- ment of allergy have not met with general confirmation. Spain, 578 IMMUNOLOGY Westeott and Gaillard (1940) report that 12 out of 15 patients with liay fever obtained no relief from symptoms when treated with potassium chloride. Feinberg and Bernstein state that a healthy skepticism should be maintained relative to the use of histaminase and of histamine in the treatment of allergy until more well-planned and carefully controlled series of cases are reported. Correct Breathing. — Brown (1925) states that in the past morphine has been the "sheet anchor" in the treatment of asthma. He recommends that asthma patients be taught to breathe slowly and quietly. In his opinion asthmatic attacks may be induced by prolonged, forced expiration and acute attacks may be relieved if the patient will let the air out of his lungs slowly and without force. Adrenalin and Ephedrine.— It is quite customary to treat asthmatic attacks by the injection of adrenalin or by the use of some drug that will relax the ])ronchial musculature. Ephedrine has been used quite extensively in controlling attacks of hay fever. It should be rememl)ered that the continued use of ephedrine and adrenalin to shrink the mucous membrane is undesirable, since the mucous membi-ane ultimately becomes chronically congested. Diathermy. — Diathermj- has been used in the treatment of hay fever and asthma with variable success. This and other methods of fever therapy are discussed by Feinberg, Osborne and Afremow (1931), Leopold and Stewart (1931) and by Miller and Piness (1931). For a more comprehensive discussion of therapy in allergy the student is referred to Rackemann (1931) or one of the other monographs mentioned at the beginning of this chapter. References Abramson, H. A., Engel, M., Lubkin, V., and Ochs, L. : Eeveised loutoplioresis of Histamine From Human Skin; Its Bearing on the Histamine Theory of Allergic Wheal, Proc. Soc. Exper. Biol. & Med. 38: 65, 1938. Alexander, H. L., and MeConnell, F. S. : The Variability of Skin Eeaction.s in Allergy, J. Allergy 2: 23, 1930. Alexander, H. L. : Bronchial Asthma: Its Diagnosis and Treatment, Phila- delphia, 1928, Lea & Febiger. Alexander, H, L. : The Treatment of Hay Fever by Specialists in the United States and Canada, J. Allergy 4: 169, 1933. (Report of an inquiry.) Alexander, H. L.: The Basal Metabolic Rate in Allergic Disease, J. Allergy 2: 507, 1931. Auer, J.: The Functional Analysis of Anaphylaxis. Chap. II of Forch- heimer's Therapeusis of Internal Diseases, New York, 1915, D. Apple- ton-Century Co. 5: p. 39. Balyeat, R. M.: Allergic Eczema, J. Allergj' 1: 516, 1930. HYPERSENSITIVENESS 579 Tice, 41. Becker, F. E., and Black, W. C. : Investigations of the Mechanism of the Passive' Transfer of Skin Sensitization, J. Allergy 2; 405, 1931. Bell, S. D.: Transfer of Skin-Sensitizing Antibodies (Abstract), J. Allergy 2: 398 1931. Berkoflf,' H. S.: A Standard for Reading Skin Tests, J. Allergy 4: 227, 1933. Black, J. *H.: A Soluble Specific Carbohydrate of Ragweed Pollen, J. Allergy 2: 161, 1931. Black, J. H.: The Pollen Allergen, J. Allergy 3: 1, 1931. Bloch, B., and Steiner-Wourlisch, A.: (1926.) Cited by Coca m Ti Practice of Medicine, Hagerstown, Md., 1927, W. F. Prior Co. 1: p. 1' Bordet, P.: L'AUergie Non Specifique, Ann. Inst. Pasteur 56: 325, 1936. Bordet, P.: Le Phenomene de Shwartzman, Ann. Inst. Pasteur 57: 357, 1936. Bostock, J.: Case of Periodical Affection of the Eyes and Chest, Med. Chir. Tr., London 10: 161, 1819. Cited by Duke, 1925. Bowman, K. L. : On the ' ' Protein Unit ' ' Standardization of Pollen Extracts Proposed by Cooke and StuU, J. Allergy 5: 341, 1934. Bray, G. W.: Recent Advances in Allergy, ed. 2, Philadelphia, 1934, The Blakiston Co. Bray, G. W.: The Hereditary Factor in Hypersensitiveness, Anaphylaxis and AUergy, J. Allergy 2: 205, 1931. Brown, A., Milford, E. L., and Coca, A. F.: Studies on Contact Dermatitis. I. The Nature and Etiology of Pollen Dermatitis, J. Allergy 2: 301, 1931. Brown, O. H. : Asthma. Presenting an Exposition of the Nonpassive Ex- piration Theory, St. Louis, 1917, The C. V. Mosby Co. Brunner, M. : Active Sensitization in Human Beings, J. Allergy 5: 257, 1934, Campbell, D. H., and Nicoll, P. A.: Studies on in Vitro Anaphylaxis and Release of Active Non-histamine Material From Sensitized Guinea Pig Lung, J. Immunol. 39: 103, 1940. Carey, T. N., and Gav, L. N.: Skin Reactions in Infants, J. Allergy 5: 488, 1934. Caulfeild, A. H. W., and LaRush, F.: Local Passive Transfer to a Fre- quently Recovered Strain of Staphylococcus Hemolyticus. Case re- port, J. Allergy 2: 372, 1931. Caulfeild, A. H. W.: Some Comments Upon Antigens and the Practice of Allergy, J. Allergy 5: 1, 1933. Cobb, I. G. : Organs of Internal Secretion: Their Diseases and Therapeutic Application, New York, 1919, William Wood & Co., p. 70. Coca, A. F. : Hypersensitiveness, Practice of Medicine, Tice 1: Hagerstown, Md., 1931,' W. F. Prior Co., pp. 107-168. Coca, A. F., Walzer, M., and Thommen, A. A.: Asthma and Hay Fever— In Theory and Practice, Springfield, HI., 1931, Charles C Thomas Co. Coca, A. F. : On the Plan of Standardization of Pollen Extracts Proposed by Cooke and Stull, J. Allergy 4: 354, 1933. Coca, A. F.: A New Definition of the Noon Pollen Unit, J. Allergy 5: 345, 1934. Cohen, M. B., Rudolph, J. A., Wassermann, P., and Rogoff, J. M.: Studies on the Relation of the Adrenal Glands to Allergic Phenomena. I. The Output of Epinephrine During Anaphylactic Shock in Dogs, J. Allergy 5: 221, 1934. Colmes, A.: Serial Skin Tests as a Guide in the Treatment of Ha}' Fever. Preliminary Report, J. Allergy 3: 449, 1932. Colmes, A.: Notes on the Treatment of Hay Fever, J. Allergy 4: 98, 1933. Committee on Survey of Allergv Clinics and Standardization of Materials. Report, J. Allergy 3: 526,'l931. Cooke R. A., and Stull, A.: The Preparation and Standardization of 'PoUen Extracts for the Treatment of Hay Fever, J. Allergy 4: 87, 1933. 580 IMMUNOLOGY Cooke, E. A., and Vander Veer, A., Jr.: Human Sensitization, J. Immunol. 1: 301, 1916. Cowie, D. M.: Autopassive Transfer in Allergy, Ann. Int. Med. 11: 949, 1937. Criep, L. H., and Wechsler, L.: Studies in Urticaria: The Influence of Metabolic Factors, J. Allergy 2: 479, 1931. Davison, W. C. : See, The Compleat Pediatrician, 1934, Duke University Press, Sec. 66c. Duke, W. W. : Allergy, Asthma, Hay Fever, Urticaria and Allied Manifesta- tions of Eeaction, St. Louis, 1925, The C. V. Mosby Co. Duke, W. W. : Clinical Manifestations of Heat and Effort Sensitiveness and Cold Sensitiveness, J. Allergy 3: 257, 1932. Dunbar, W. P.: The Present Status of Our Knowledge of Hay Fever, J. Hyg. 13: 105, 1913. Ellis, E. v.: A Eational Grouping of the Food Allergens, J. Allergy 2: 246, 1931. Farmer, L.: Xonspecific "Desensitization " Through Histamine, J. Immunol. 36: 3^ 1939. Farmer, L.: Experiments on Histamine — E«fractoriness; II. Nonspecific "Desensitization" Through Oral Application of Histamine, J. Immunol. 37: 321, 1939. Feinberg, S. IM., and Bernstein, T.: Asthma and Hay Fever, J. Allergy 11: 281, 1940. Feinberg, S. M.: Eeview of Hay Fever Literature of 1929, J. Allergy 1: 313, 1930. Feinberg, S. M., Osborne, S. L., and Af remow, M. L. : Fever by Diathermy in the Treatment of Allergic Disease, J. Allergy 2: 414, 1931. Fisher, A. M., and Beck, J. P.: Death in Asthma. Eeport of a Case With Autopsy, J. Allergy 2: 149, 1931. Goodman, J., and Sulzberger, M. B. : Allergy in Dermatology, J. Allergy 11: 407, 1940. Gray, I., and Walzer, M. : Studies in Absorption of Undigested Proteins in Human Beings. VII. The Absorption of Unaltered Protein Introduced by Duodenal Tube Into the Abnormal Gastro-Intestinal Tract, Am. J. Digest. Dis. 5: 345, 1938. Gray, I., and Walzer, M. : Studies in Absorption of LTndigested Protein in Human Beings. VIII. Absorption From the Eectum and a Compara- tive Study of Absorption Following Oral, Duodenal, and Eectal Ad- ministrations, J. Allergy 11: 245, 1940. Gray, I., and Walzer, M. : Studies in Absorption of Undigested Protein in Human Beings. VI. The Absorption of Unaltered Protein From the Abnormal Gastro-Intestinal Tract, Am. J. Digest. Dis. & Nut. 3: 403, 1936. Hansel, F. K. : Observations on the Cytology of the Secretions in Allergy of the Nose and Paranasal Sinuses, J. Allergy 5: 357, 1934. Harkavy, J.: Vascular Allergy in the Pathogenesis of Bronchial Asthma With Eecurrent Pulmonary Infiltrations and Eosinophilic Poly- serosites, J. Allergy 11: 622, 1940. Heran, J., and St. Girons, F. : (1917.) Cited by Coca in Tice, Practice of Medicine, Hagerstown, Md., 1927, W. F. Prior Co. 1: p. 159. Huber, H. L. : A Critical Analysis of the Information Obtained From Hay Fever Sufferers, J. Allergy 2: 48, 1930. Hurwitz, S. H.: Hav Fever — A Sketch of Its Early History, J. Allergy 1: 245, 1930. Karady, S.: J. Immunol. 37: 457, 1939. Cited by Eichardson (1940). Kern, E. H., and Teller, Ida: Basal Metabolism and Blood Calcium Studies in Asthma and Allergic Eczema, J. Allergy 2: 488, 1931. Landsteiuer, K., and Jacobs, J.: Studies on the Sensitization of Animals With Simple Chemical Compounds, J. Exper. Med. 61: 643, 1935. HYPERSENSITIVENESS 581 Laudsteiner, K.: Serological and Allergic Eeactions With Simple Chem- ical Compounds, New England .7. Med. 215: 1199, 1936. Landsteiner, K., and Di Somma, A. A.: Studies on the Sensitization of Animals With Simple Chemical Compounds. V. Sensitization to Diazomcthane and Mustard Oil. .J. Kxper. Med. 68: 50.'), lO.'lS. Landsteiner, K., Rostenberg, A., and Sulzberger, M. B.: Individual Dif- ferences in Susceptil)ility to Eczeraatous Sensitization With Simple Chemical Substanc<;s, J. Invest. Dermat. 2: 1'."). 1939. Landsteiner, K., and Chase, M. W.: Studies on the Sensitization of An- imals With Simple Chemical Compounds. VI. Experiments on the Sensitization of Guinea Pigs to Poison Ivy, J. Exper. Med. 69: 767, 1939. Landsteiner, K., and Chase, M. W. : Studies on the Sensitization of An- imals With Simple Chemical Compounds. A"II. Skin Sensitization by Intraperitoneal Injections, J. Exper. Med. 71: 237, 1940. Leopold, S. S., and Stewart, S. G.: The Effect of Fever, Either Accidentally Incurred or Artifieiallv Produced in Bronchial Asthma, J. Allergy 2: 425, 1931. Loveless, Mary H.: Immunological Studies of Pollinosis. I. The Presence of Two Antibodies Related to the Same Pollen Antigen in the Serum of Treated Hay-Fever Patients, J. Immunol. 38: 25, 1940. Loveman, A. B. : Allergic Drug Eruptions, J. Allergy 11: 48, 1939. Martin, S. : The Fallibility of the Autopassive Transfer Test (Cowie), J. Allergy 11: 26G, 1940. Michael, P. P., and Rowe, A. H. : Pathology of Two Fatal Cases of Bronchial Asthma, J. Allergy 6: 150, 1935. Miller, H., and Piness, G. : Hypertliermia Induced by High Frequency Electric Current in the Tieatment of Intractable Asthma, J. Allergy 2: 436, 1931. Nelson, T.: Constitution and Allergic Manifestations. I. Age-Sex Incidence of Allergic Conditions. Preliminary Report, J. Allergy 5: 124, 1934. Newell, J. M.: Attempts to Detect a Reaction Between Human Allergic Serum and Its Antigen, J. Allergy 11: 35, 1939. Piness, G., and Miller, H.: Skin Tests in 1,589 Cases of Allergic Disease, With Criticism Concerning Elimination Diets, J. Allergy 4: 18, 1932. Piness, G., Miller, H., Carnahan, H. D., Altose, A. R., and Hawes, R. C: Relationships Between Foods as Shown by the Skin Test in 1,000 Chil- dren, J. Allergy 11: 251, 1940. Pinkel, J. H., and Balyeat, R. M. : The Localization and Specificity of Cellular Sensitization, J. Allergy 3: 567, 1932. Pottenger, F. M. : Similarities and Differences in Bacterial and Non- bacterial Allergy, J. Allergy 1: 235, 1930. Prausnitz, C, and Kiistner: (1921.) Cited by Rackemann, Clinical Allergy, New York, 1931, The Macmillan Co., p. 68. Rackemann, F. M., and Colmes, A. : Studies in Asthma. V. The Clinical Characteristics of Hypersensitiveness, J. Allergy 1: 2, 1929. Rackemann, F. M.: The Nature of Allergy, .J. Allergy 1: 536, 1930. Rackemann, F. M., and Simon, F. A.: Technic of Intracutaneous Tests and Results of Routine Tests in Normal Persons, J. Allergy 6: 184, 1935. Rackemann, F. M., Simon, F. A., Simon, M. G., and Scully, M. A.: Further Observations on the Nature of Allergy, J. Allergy 4*: 498, 1933. Rackemann, F. M.: Clinical Allergy — Asthma and Hay Fever. Mechanism and Treatment, New York, 1931, The Macmillan Co. Rackemann, F. iL: A Review of the Phases of Allergy, .7. Allergy 6: 17, 1934. Ramirez, M. A.: (1919.) Cited by Zinsser, Resistance to Infectious Dis- eases, New York, 1931, The Macmillan Co., p. 439. 582 IMMUNOLOGY Ramirez, M. k., and Eller, J. J.: The "Patch Test" in "Contact Dermatitis" (Dermatitis Venenata), J. Allergy 1: 489, 1930. Ratner, B.: Rabbit Hair Asthma in Children, Am. J. Dis. Child. 24: 346, 1922. Ratner, B., Jackson, H. C, and Gruehl, H. L. : Respiratory Anaphylaxis, Am. J. Dis. Child. 34: 23, 1927. Ratner and Gruehl, H. L. : Cited by Zinsser, Resistance to Infectious Diseases, New York, 1931, Tlie Macmillan Co., p. 413. Richardson, E. H., Jr.: Physical Allergy and "Auto-Antigens," Proc. Soc. Exper. Biol. & Med. 45: 787, 1940. Rowe, A. H. : An Evaluation of Skin Reactions in Food Sensitive Patients, J. Allergy 5: 135, 1934. Rowe, A. H.: Food Allergy and Pollen Hay Fever, J. Allergy 1: 531, 1930. Sherman, W. B., Stull, A., and Cooke, R. A. : Serologic Changes in Hay Fever Cases Treated Over a Period of Years, J. Allergy 11: 225, 1940. Simon, F. A., and Rackemann, F. M. : The Development of Hypersensitiveness in Man. I. Following Intradermal Injection of the Antigen, J. Allergy 5: 439, 1934. II. Absorption of the Antigen Through the Na.sal Mucous Membrane, Ibid. 5: 451, 1934. Smyth, F. S., and Bain, K. : A Study of the Allergic Skin-Test Substance, J. Allergy 2: 177, 1931. Smyth, F. S., and Bain, K. : Studies in Passive Transfer, J. Allergy 2: 181, 1931. Smyth, F. S., and Bain, K. : Enteral Absorption of the Antigen and the Apparent Failure of Antigen Secretion in Human Milk, J. Allergy 2: 282, 1931. Smyth, F. S., and Bain. K. : The Direct Skin Test in Allergy, J. Allergy 2: 316, 1931. Spain, W. C, and Newell, J. M. : On the Reagin Content of Blister Fluid, J. Allergy 5: 331, 1934. Spangler, R. H.: Eosinophilia in Syphilis (Abstract), J. Allergy 6: 200, 1935. Sterling, A. T.: Status Asthmaticus, J. Allergy 6: 189, 1935. Stratton, E. K.: Poison Oak and Poison Ivy, J. Allergy 11: 591, 1940. Straus, H. W. : Artificial Sensitization of Infants to Poison Ivy, J. Allergy 2: 137, 1931. Straus, H. W.: Cited by Coca in Tice, Practice of Medicine, Hagerstown, Md., 1927, W. F. Prior Co. Stull, A., Cooke, R. A., and Chobot, R. : Cited by Stull, Cooke, and Tennant, J. Allergy 4: 455, 1933. Stull, A., Cooke, R. A., and Tennant, J.: The Allergen Content of Pollen Extracts. Its Determination and Its Deterioration, J. Allergy 4: 455, 1933. Stull, A., and Hampton, S. F. : A Study of the Antigenicity of Proteoses, J. Immunol. 41: 143, 1941. Sulzberger, M. B., Spain, W. C, Sammis, F., and Shahou, H. I. : Studies in Hypersensitiveness in Certain Dermatoses. I. Neurodermatitis (Dis- seminated Type), J. Allergy 3: 423, 1932. Sulzberger, M. B., and Kerr, P. S.: Trichophytin Hypersensitivene.ss of Urticarial Type, With Circulating Antibodies and Passive Transference, J. Allergy 2: 11, 1930. Swineford, O.: Physical Allergy, J. Allergy 6: 175, 1935. Templeton, H. J.: Trichophytin — Its Use According to Allergic Principles, J. Allergy 5: 521, 1934. Ulrich, H. L.: Cited by Ratner, Jackson, and Gruehl, 1927. Unger, L., and Moore, M. B.: Studies on Pollen and Pollen Extracts. IX. A New Extracting Solution, J. Allergy 4: 92, 1933. Vaughan, W. T.: The Future of Allergy, J. Allergy 11: 588, 1940. Vaughan, W. T.: Practice of Allergy, St. Louis, 1939, The C. V. Mosbv Co, HYPERSENSITIVENESS 583 ■\^augluui, W. T.: Further Studies in the I^eucopenic Index in Food Allergy, J. Allergy 6: 78, 1934. ^'aughan, W. T.: Food Allergens. I. A Genetic (Classification, With Eesults of Group Testing, J. Allergy 1: .385, 1930. Vaughan, W. T. : Food Allergens". 111. The T.eucopenic Index. Preliminary Report, J. Allergy 5: 601, 1934. Walzer, M.: Cited by Eackemanii, Clinical Allergy, New York, 1931, The Macmillan Co., p. 70. Waters, I.: Elimination Diets for tlie Diagnosis and Treatment of Food Allergy, J. Allergy 2: 22.5, 1931. Westcott. F." H.. and Spain, W. C. : Sedimentation of Eed Blood Cells in Allergic Diseases, J. Allergy 4: 370, 1933. Wilnier, H. B., and Cobe, H. M. : A^accine Therapy: The Uses and Misuses, J. Allergy 4: 414, 1933. Wilson, R. A.: Heat Sensitiveness, Report of a Case, J. Allergy 2: 499, 1931. Zinsser, H., Enders, J. F., and Fothergill, L. D. : Immunology, Resistance to Infectious Diseases, New York, 1939, The Macmillan Co. Zisserman, L. : Susceptibility to Poison Ivv Dermatitis, J. Allergy 11: 600, 1940. Zisserman, L., and Birch, L.: Prophylaxis and Treatment of Poison Ivy Dermatitis With an Extract of" Rhus Toxicodendron, J. Allergy 10: 596, 1939. Supplementary References Albert, M., and Walzer, M.: Contact-Reactions in Atopy. I. Contact- Reactions to Silkworm in Atopic Subjects, J, Immunol. 38: 201, 1940. Barksdale, E. E.: Cutaneous Manifestation From Tobacco, J. A. M. A. 115: 672, 1940. Coulson, E. J., Spies, J. R., and Stevens, H.: The Immunochemistry of Allergens. I. Anaphylactogenic Properties of a Proteic Component of Cottonseed, J. Immunol. 41: 375, 1941. Fanburg, S. J.: Dermatitis Following the Wearing of Nylon Stockings, J. A. M. A. 115: 354, 1940. Feinberg, S. M., Foran, F. L., Lichtenstein, M. R., Padnos, E., Rappaport, B. Z., Sheldon, J., and Zeller, M.r Oral Pollen Therapy in Ragweed Pollinosis: A Cooperative Study, J. A. M. A. 115: 23, 1940. Figley. K. D.: Karaya Gum Hypersensitivity, J. A. M. A. 114: 747, 1940. Goldberg, L. C: Histaminase "in the Treatment of Allergic Dermatoses, J. A. M. A. 115: 429, 1940. Keener, E. L.: Histaminase in the Treatment of Hay Fever, J. A. M. A. "114: 2448, 1940. Pear.son, E. F. : Clinical Desensitization to Wheat by Use of an Acetylcholine Derivative, Ann. Int. Med. 13: 2241, 1940. Prickman, L. E., Lillie, H. I., Roth, G. M., and Fleming, R. G.: Results of the Use of Extract of the Intestinal Mucosa in the Treatment of Vaso- motor Rhinitis, Ann. Int. Med. 13: 2235, 1940. Rubin, S. S., Aaronson, A. L., Kaplan, M. A., and Feinberg, S. M.: Potas- sium Salts in the Treatment of Pollinosis: A Clinical Evaluation, J. A. M. A. 114: 2359, 1940. Seideman, R. M.: Cutaneous Reactivity of Guinea Pigs to Gum-arabic, J, Immunol. 38: 237, 1940. Sherman, W. B.: Changes in Serological Reactions and Tissue-Sensitivity in Hay-Fever Patients During the Early Months of Treatment, .7. Immunol. 40: 289, 1941. APPENDIX COLLOIDS Crystalloids and Colloids. — In the older literature, sul)stances were divided into crystalloids and colloids. At the present time, it is thought that the term ''colloid" should be applied to the state of the substance and that so-called crystalloids may be prepared in the colloidal state. We now speak of substances being in true solu- tion and in the colloidal state. True Solution. — In the case of true solutions, one is dealing with a solvent such as water and a dissolved substance, as e.g., sodium chloride, glucose, etc. When sodium chloride goes into solution in water, it tends to ionize, i.e., the molecule breaks up into sodium and chloride ions, while glucose goes into solution in the molecular state. Because of this difference, the sodium chloride tends to diffuse through a membrane more rapidly than glucose, although both are diffusible. It is estimated that the size of a molecule of sodium chloride is 5.6 x 10"^ cm. while that of glucose is 14.7 x 10~* cm. It is obvious that the glucose molecule is considerably larger than that of sodium chloride and that it is also of a different quality. Colloidal State. — If sodium chloride or glucose could be present in the water not in the io7iic or molecular state but in particles or aggregates ranging in size from 0.00001 em. to 0.0000001 cm., it would be present in the colloidal state. The water would be called the "dispersion medium" and the small particulate substance dispersed in it would be called the "dispersed phase." Homogeneous and Heterogeneous Systems. — In nature we find that such su])stances as the serum globulins and albumins and the cell protoplasm are found in the colloidal state. True solutions are also spoken of as "homogeneous systems" in which there are no apparent physical surfaces of discontinuity, while colloids are "heterogeneous systems" consisting of two or more phases sepa- rated from each other by surfaces of discontinuity. Alexander cites Zsigmondy's observation that perhaps this basis of differentia- tion between homogeneous and heterogeneous systems is more 584 AI'PENDIX 585 apparent than real and is probably due to tlie limitation of our means of observation. In any system there is a tendency for equilibrium to be established and this is of general importance in science. The Tyndall Effect. — Another difference between a homogene- ous system and a heterogeneous one containing very small dis- persed particles can be demonstrated ])y passing a 1)eam of liglit through them and making observations from a position at right angles to the beam of light. A true solution, which is an homoge- neous system, should be clear, wherea.s the colloidal solution, which is an heterogeneous system, would appear turbid. Tlii.s phenom- enon is well illustrated by passing a beam of sunlight through a very small opaning into a darkened room. An observer within the room can see the beam of light extending across the room owing to the presence of dust particles in the air. The light is scattered and part of it is completely polarized. The beam of light from a searchlight is also a good example of the scattering and polarizing of light by fine particles in suspension. This phenomenon, in- volving polarization, was first descrilicd by Faraday (1857) and later by Tyndall in 1869. It is usually called the Tyndall Effect. Colloids are usually divided into two classes, suspensoids and emulsoids. Suspensoids and Emulsoids. — Suspensoids. — In these the dis- persed phase is merely suspended in the dispersion medium and does not change the viscosity of the latter. Emulsoids. — Here the dispersion medium and dispersed phase enter into such intimate relationship with each other that the viscosity is changed. Transferability of Emulsoid to Suspensoid. — It is iiossible to convert an emulsoid into a suspensoid, as, e.g., when 50 per cent ammonium chloride is added to blood serum, which is a complex emulsoid, sufficient water is witlidrawn from the .serum globulin, present as a dispersed phase, to change it from an emulsoid to a suspensoid, and hence we say it is salted out. Chemical vs. Colloid Reactions. — It is important to note that material in the colloidal state exhibits properties quite different from those when in true solution. When substances in true solu- tions react with each other, they do so according to known chem- 586 APPENDIX ical laws which give results that are predictable. When material is in the colloidal state, reactions do not follow the laws of mass action, etc., but are dependent largely upon the amount of surface exposed to the dispersion medium per gram of dispersed material (i.e., specific surface) and to kinetic activity of the dispersed par- ticles. The nature of the material dispersed, as well as of the dis- persion medium, is also an important factor. Specific Surface. — The simplest example of the meaning of this term would be to take material having a density the same as water, i.e., at a standard temperature one cubic centimeter weighs one gram. In the form of a cube, the surface area of one cube would be 6 sq. cm. If this were divided into 1,000 cubes, each 1 mm. on the side, the total surface would be 60 sq. cm. If the original cube were divided into 1,000,000,000,000,000 cubes, eacli being 0.0001 mm. (0.1 micron) on the side, the total surface would be increased to 6,000 sq. cm. This represents what is commonly regarded as the upper limit of size of particles forming colloids. If the original cube were divided into cubes each having a length of side corre- sponding to the minimum of those in colloids, i.e., 1.0 millimicron or 0.001 micron, the total surface would be increased to 14.83 acres. In this case the specific surface woukl be per gram of dispersed material. Surface Tension.— Wells has defined surface tension as ''the force with which a fluid is striving to reduce its free surface to a minimum." At the surface of contact of a liquid with air there is an interfacial tension and Avitliin a colloid there exist interfacial tensions where the dispersion medium is in contact with the surface of the dispersed material. This can, perhaps, be understood by visualizing not a colloid but a drop of fat added to hot water. It appears as a flattened sphere floating on the surface because the attraction of the fat molecules for each other is greater than the attraction of the surface water molecules for the fat. In this case, part of the fat is exposed to air and part to Avater. The force or tangential pull that maintains the surface of the oil droplet ex- posed to air is its surface tension in respect to air, while the force that maintains the integrity of the surface in contact with water is its surface tension with respect to water. Thus the student should be able to appreciate that at the surfaces of contact between two substances there exist interfacial tensions. Ordinarilv the sur- APPENDIX 587 face tension of a medium is measured against air. In the colloidal state each colloidal particle is exposed at its surface to the disper- sion medium and there exists at the surface a tangential force which maintains the integrity of the surface and attempts to re- duce the surface area to a minimum and this is the interfaeial tension. Electrical Phenomena and Surface Potentials. — The terms ''membrane potential" and "critical potential" have, during re- cent years, crept into immunological papers having to do with ag- glutination, precipitation and complement fixation. It is hoped that the following simple explanations may liel]) the student in his reading of current literature. + Fig. 24. Fig. 24. — Negatively charged particle surrounded by single layer of positive charges, theory of Helmholtz. Fig. 25. — Negatively charged particle surrounded by diffuse atmosphere of positively charged particles, theoiT suggested by Gouy. Electrical Double Layer, Helmholtz. — In colloidal solutions the dispersed particles are conceived of as being somewhat like a condenser in that at the surface there is a double layer of opposite electrical charges. One layer is within the wall of the particle and the layer of opposite charges is at a molecular distance away but in the dispersion medium. This donl)le layer conception was origi- nated by Helmlioltz. More recently the outer layer is conceived of as forming a diffuse atmosphere rather than a single layer. These two concepts are illustrated in Figs. 24 and 25 which show the particle to be negatively cliarged and surrounded with a simple or diffuse atmosphere of positive charges. 588 APPENDIX Me]\ibrane Potential. — It will be observed that the outside layer has the same total electric charge as the inside layer although op- posite in kind. There is then a difference in potential between those two layers. The stability of colloids runs parallel with the drop in the potential in the double layer. This drop in potential is due to changes in the outside layer. It is now known that this layer is more complex than has been indicated here and that one portion only of the total potential difference is important in colloid stability. Effect of Cations on Colloids. — It has long been known that free hydrogen ions in the dispersion medium profoundly affect the stabilit.y of colloids. When their concentration is sufficient to bring al)out comi)lete neutralization of charge, tlie colloid becomes very unstable and is readily precipitated. This is called the isoelectric point. This is in accordance with the observation tliat colloidal particles liaving like charges repel each other. Critical Potential. — The point in the drop in potential where the colloidal particles barely repel each other is called the critical potential, a fall below which indicates the loss of the property of repulsion. This discussion applies more nearly to suspensoids than to emulsoids. In the latter group of colloids the dispersion medium enters into intimate relationship with the dispersed material. Factors Governing' Stability of Emulsoids.— It is thought that there are at least two factors involved in maintaining the dispersed state of emulsoids. One factor is the charge and potential and the other is the so-called film of water around each particle. Removal or reduction in amount of this water fihn favors precipitation. If an adequate water film is maintained, a drop in potential may not lead to precipitation. This will be more fully discussed under Cohesion and Precipitation. Migration of Charged Particles. — Cataphoresis. — As is evident from the preceding discussion, one of the important properties of colloids is that the dispersed particles are electrically charged with respect to their surroundings. When an electric current is passed through a colloid, any negatively charged particles will travel to- ward the positive pole, or anode, while positively charged particles will travel toward the cathode or negative pole. This migration phenomenon is called cataphoresis. If the dispersed particles have APPENDIX 589 lost their charge, they will not travel to either pole. This state of affairs exists at the "isoelectric" point where neutralization of charge has occurred. By cataphoretic experiments the nature of the charge of various colloids has been determined. Adsorption. — Adsorption and Surface Affinities. — It is gen- erally known that finely powdered charcoal will remove colored sub- stances that are present in a solution. The intensity of color of certain dye solutions is markedly reduced by charcoal. In each case, the material removed has attached itself to the surface of the finely dispersed particles of charcoal. This plienomenon is called adsorption and is a characteristic of all matter in the colloidal state. Positive Adsorption. — The term positive adsorption means that there has occurred an increase in concentration of some substance in the boundary layer of the dispersed substance and dispersion medium. Surface Concentration of Dissolved Substance. — Willard Gibbs called attention to a principle that is perha])s fundamental to adsorption, and that is, that when a substance is dissolved in a liquid and lowers its surface tension, it appears in greater con- centration at the surface. It is now generally thought that the way the surface molecules are oriented is responsible for many of the properties of adsor])tion. Surface Wetting. — It is readily appreciated that when oil or fat is dispersed in water the water is unable to wet the greasy surface of the dispersed fat droplets. On the other hand, if oil droplets can be filmed with proteins, the surfaces may readily be made wet with water. Devaux Experiment. — Alexander cites an experiment of De- vaux which seems to explain the phenomenon of surface wetting. Devaux floated a drop of molten fatty acid on the surface of some hot water and allowed it to cool. The upper surface was in contact with the air, while the lower was adjacent to the Avater. He found that after he dried the lower surface he coiild wet it with water but that he could not wet the upper surface with water. Alexander says that apparently the "hydrophile" or "friendly-to-water" ends of the fatty acid molecules were turned toward the surface that cooled in contact with the water, while the "hydrophobe" or 590 APPENDIX "greasy" ends were turned outward in the surface which had cooled in contact with the air. Molecular Orientation. Harkins' Theory. — This surface orientation of molecules is part of Harkins ' orientation theory. He would say that on the surface wet l)y water, the molecules were oriented at right angles to the surface with a polar group attached to a short carbon chain and that this polar group attacted the water so strongly that it tended to drag the hydrocarbon chain out into the water. This affinity for water was responsible for the wetting. On the side exposed to air, the other end of the chain was outside and it had no affinity for water, hence, the surface Air uOjj616 6ll6lloL(5 Water Fig. 26. — Orientation of molecules of an alcohol (or an organic acid) at the surface of its aqueous solution. From "Atoms, Ions, Salts and Sui-faces," by William D. Harkins in Neicer Knotoledge of Bacteriology and Immunology edited by E. O. Jordan and I. S. Falk. Reprinted by permission of the University of Cliicagd Press. could not be wetted. Langmuir has studied this plienomenon of molecular orientation and arrived at the same conclusions as Har- kins. This surface orientation of molecules is graphically illus- trated ])y Harkins. (Fig. 26.) Attraction of Colloids for Each Other. — Cohesion, Adhesion and Precipitation. — Graham noted that colloids adhere to each other with great tenacity. Within a colloid there are powerful residual forces that hold adjacent molecules together. At the surface there are unsatisfied fields of force that make it possible for the dispersed particles to stick together if the forces liolding them APPENDIX 591 apart are not sufficient. Every colloid is a heterogeneous system ill which there is a continual offort loward establishing equilibrium. Fundamental Problem of Colloid Chemistry. — Kruyt says that llie fujulniuciital ]U'o])lein of colloid chemistry is to ascertain the reason for the inability of the surface tension to unite the particles. In the case of suspensoids it seems that the surface charge and dif- ference in potential are fundamental factors in keeping the ]iar- ticles apart, while the orientation of surface molecules is funda- mental in determining the cohesive or adhesive forces. In emulsoids, conditions are a little more complex and a third factor perhaps ranfe first in importance. Attention has previously been called to the difference between emulsoids and suspensoids. In emulsoids, the dispersion medium forms an intimate relationship with the dispersed material. Since the emulsoids encountered in immunity are made up of water as the dispersion medium and protein the dispersed substance or phase, the water is in intimate relationship with the dispersed particles of protein. It is con- sidered that each particle has an intimate w^ater envelope around it. Factors in Emulsoid Stability. — The factors involved are the particle and its surface, the water layer and the electrical double layer. Anything that reduces this water layer to a certain critical amount increases the cohesive properties. The lowering of the potential to the critical point favors precipitation and also cohesion, and as mentioned above the nature of the material of the surfaces is an additional factor. The water envelope is of sufficient importance to prevent precipitation even when the critical potential is reached. This is illustrated by Kruyt (Fig. 27). Precipitation by Electrolytes. — It will be seen from Fig. 27 that if the charged emulsoid is dehydrated, it becomes a charged sus- pensoid that can be precipitated by electrolytes neutralizing the charges. If the charge on the emulsoid is removed by elec- trolytes, it remains an emulsoid. but without a charge, and can be precipitated by dehydrating it with alcohol. Thus it will be seen that the neutralization of charge causes ])recipitation or coagulation of suspensoids but not of emulsoids. Macleod says that ''a (juantity of electrolyte which is capable of producing complete precipitation when added all at once to sus- pensoids will be ineffective when added in small quantities. This 592 APPENDIX phenomenon, which is exhibited when toxins and antitoxins are mixed together, is probably due to the fact that precipitation de- pends on inequality and irregular distribution of electric charges, a condition which becomes established when the electrolyte is sud- denly added, but not so when it is gradually added." This phe- nomenon is of considerable importance in immunology. Charged .suipfinsoid dehydration b>j alcohol ptecipitatincj particle \jnchar$ei emulsoid Fig-. 27. — Factors involved in dispersion and precipitation of colloids. (After Kruyt and Van Klooster, Colloids, John Wiley and Sons, 1930.) Protective Action of Colloids. — From the preceding discussion it can be seen that emulsoids are much more resistant to the precipitating action of electrolytes than suspensoids. This is due to their water layer. If the dispersed particles of a suspensoid like colloidal gold, which is quite sensitive to the precipitating power of electrolytes, can be made to adsorb a film of a stable emulsoid around each particle, then the suspensoid will be pro- APPENDIX 593 tected from the precipitating action of electrolytes. It has ac- quired the stability of the adsorbed emiilsoid. Autoprotection. — Autoprotection, double and ])lural protec- tions have been observed. As an example of autoprotection, Alexander mentions that in pure iron "one allotrope, y - iron, seems to be adsorbed by a - iron." Effect of Speed of Mixing*. — When oppositely charged col- loids are mixed, it is quite possible for flocculation to occur as a result of the neutralization of charges. If, however, an excess of a positively charged emulsoid is added to a negatively charged sus- pensoid, the emulsoid may be adsorbed before precipitation occurs. If a small amount of the emulsoid instead of an excess is added, or if the emulsoid is added very slowly, precipitation may occur be- fore the protection is established. This shows that relative propor- tions and speed of mixing profoundly affect colloidal reactions. This is commonly given as the explanation of the zoning phe- nomena seen in many immunological reactions. It can also be shown experimentally that when small amounts of the protective colloid added bring the other colloid to the isoelectric point, precipitation commonly occurs. Solubility of Precipitates. — In regard to protection and protec- tors, Alexander* says, "although some salts (citrates, sulpho- cyanates) may act as protectors, protection is generally accom- plished by adding a reversible or emulsoid colloid to an irreversible one, which thereupon acquires reversible properties, that is, it be- comes insensitive to electrolytes, redissolves after desiccation (at any temperature that does not render the protector insoluble) and passes through ultrafilters that would otherwise hold it back." References Alexander, Jerome: Colloid Chemistry, New York, 1929, D. Van Nostrand Co. Harkins, W. D. : Atoms, Ions, Salts, and Surfaces, in Jordan and Falk, Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, University of Chicago Press, p. 136. Kruyt, H. E.: Colloids, Translated by H. S. Van Klooster, New York, 1930, John Wiley and Sons. Macleod, J. J. E.: Physiology and Biochemistry in Modern Medicine, ed. 6, St. Louis, 1930, The C. V. Mosby Co. Wells, H. G.: Chemical Patholog\^, Philadelphia, 1920, AV. B. Saunders Co. Wells, H. G. : Chemical Aspects of Immunity, New York, 1925, Chemical Catalogue Co. ♦Alexander, Jerome: Colloid Chemistry, D. Van No.strand Company, Inc. Quoted by permission. AUTHORS INDEX Aarouson, 583 Abderhalden, 327, 487 Abel, 298 Abels, 33, 44 Abramson, 574, 578 Adelsberger, 153, 156, 185, 190, 195 Afremow, 578 Airila, 479 Albert, 583 Alexander, 564, 571, 574, 578, 584, 593 Allen, 243, 270 Almon, .127 Almquist, 43 Altemeier, 138 Altose, 568 Aiiiarol, Do, 35, 44 Anderson, E. J. M., 106, 122 Anderson, G. W., 300 Anderson, J. F., 201, 258, 261, 269, 273, 471, 473, 498 Anderson, K., 74, 133, 141 Anderson, R. J., 353, 356, 367 Ando, 41, 44, 273, 277, 555 Andrewes, 384, 389 Andrews, A. H., 139, 140 Andrus, 494, 498 Angevine, 142, 545, 556 Apitz, 552, 555 Arkwright, 37, 42, 44 Arloing, 470, 502 Armstrong, 395 Arueth, 54, 61, 66 Aronson, 511, 513, 515, 521-523, 526, 529, 530, 534, 535 Arrhenius, 251, 252, 269 Arthus, 471, 479, 498, 502 Aschoff, 79, 88, 97, 128 Ascoli, 228 Auchard, 302 Auer, 473. 477-479, 498, 501, 572, 578 Autenberg, 191 Avery, 195, 206, 271, 284-287, 298, 300, 354, 356, 359-363, 369, 372, 395, 398, 474, 498, 502 Avinery, 65 Aycock, 139, 140, 291, 298 B Babes, 529 Baer, 94, 98 Baerthlein, 37, 42, 44 Baier, 233, 236 Bail, 39, 44 Bain, 582 Baker, 363, 449, 464, 467 Baldwin, 524, 525, 534 Bally, 473, 474, 479, 491, 497, 408 Balyeat, 578, 581 Bancroft, 234, 236 Banzhaf, 255, 259, 262, 269, 298 Barber, 36, 41, 44 Barenberg, 290, 298 Barksdale, 583 Barr, 263, 264, 270 Bauer, 40, 427 Baughman, 243, 270 Baumgartel, 353 Baumgartner, 102, 105, 122, 125 Bawden, 39, 44 Bayne-Jones, 31, 33, 43, 46, 111, 127, 230, 236, 248, 251, 269, 278, 279, 281, 296, 364, 373 Bay-Sehmith, 102 Beach, 146, 155 Seattle, 284, 303 Beatty, 400 Bechold, 213, 219 Beck, 580 Becker, 579 Behring, 33, 44, 242, 254, 256-258, 261, 269, 293, 470, 502 Beintema, 464, 467 Belding, 104, 122, 432, 438 Bell, 146, 155, 579 Bendersky, 65 Beres, 172, 190 Bergeim, 287 Berger, 322, 329 Berk, 468 Berkoflf, 577, 579 Bernal, 40, 44 Bernstein, 497, 574, 578, 580 Berthelsen, 273, 274 Besredka, 477 Best, 494, 498 Bezi 274 Biedl, 477, 480, 498 Bien, 384 Bigler, 263, 269 Birch, 569, 583 Birger, 483, 500 Birkhaug, 269, 282, 541, 555 Birnbaum, 78, 85 595 596 AUTHORS INDEX 405, 188, 161, 217, 403- 348, Black, 576, 579 Blackfan, 292 Blackley, 565 Blake, 77, 78, 85, 277, 278, 298, 303, 395, 398 Bliss, 287, 298, 304 Bloch, 569, 579 Bloom, 89, 97, 577 Bloomfield, 71, 85 Boak, 387, 398 Body, 500 Boerner, 159, 166, 388, 396, 399, 417 Bohmig, 555 Boivin, 34, 44, 115, 122 Bonanto, 274 Bond, 150, 155, 161, 166, 175, 192, 435, 442, 469 Bordet, 42, 44, 145, 148, 152, 154, 163, 166, 208-212, 214, 215, 219, 251-253, 269, 350, 401, 405, 417, 423-425, 529, 579 Borries, 40 Bostock, 565, 579 Bouffard, 88, 97 Bowman, 310, 316, 579 Boyd, 190, 198, 200, 207, 345 376 Boynton, 443, 520, 537 Bradford, 116 Bradshaw, 309, 314 Brady, 283 Brahdy, 298 Brand, 157, 166 Brandes, 481, 501 Branham, 353, 368 Braun, 246, 478 Bray, 564, 579 Brett, 511, 536 Brewer, 438 Brill, 500 Bristol, 551 Britton, 104, 122 Brodie, 298, 312, 314, 481, 498 Bronfeiibrenner, 427, 442, 476, 477, 481, 489, 493, 498 Brooke, 550, 558 Brooks, 159, KiC) Brown, A., 579 Brown, C, 363 Brown, C. P., 417, 439 Brown, G., 309, 310, 314 Brown, J. H., 282, 285, 366, 398, 557 Brown, O. H., 566, 567, 578 Brown, R., 258, 285, 303, 356, 357, 359, 368, 373 Browne, 494, 499, 502 Browning, 146, 156, 427 Bruck, 403, 404, 423, 425, 426, 442, 444, 465, 467, 469 Bruetsch, 420, 442 Brunner, 579 Bucca, 284, 303, 400 Buchanan, 190, 213, 219 Buclmer, 144, 154, 157, 160, 166, 197, 198 Bull, 153, 154, 241, 269, 298 Bullock, 96, 98 Bullowa, 286, 298, 307, 314 Bunnell, 154, 156, 399 Bunney, 274, 275, 537 Bunting, 54, 69, 93, 94, 98 Burchard, 261 Burmeister, 150 Burn, 107, 127 Burnett, 112, 122 Buslmell, 391, 398 Butler, 557 Butt, 499 Butterworth, 433, 442 Buttle, 287, 304 Buxton, 134, 140 Cabot, 81 Calder, 398 Calmette, 230, 237, 248, 269, 540 Cameron, 255, 269 Campbell, 127, 537, 574, 579 Cannon, A. B., 434, 438 Cannon, P. E., 94, 98, 128, 129, 132, 133, 135, 136, 140, 201, 205, 229, 237, 480, 498, 538, 539, 541, 546, 555 Canuteson, 150, 155, 435, 442, 469 Carey, 288, 304, 579 Carnahan, 568, 581 Carpenter, B. R., 195, 206 Carpenter, C. M., 387, 398 Casals, 105, 122, 537 Casper, 350, 372 Castaneda, 111, 398 Castellani, 350, 368 Caulfeild, 579 Cavazuti, 179, 189 Cecil, 77, 78, 85, 298 Cesari, 248, 272 Chandler, 146, 155 Chant, 561 Chapin, 96, 98 Charlton, 279, 302 Charrin, 208 Chase, 197, 205, 213, 220, 354, 364, 368, 473, 500, 503, 509, 534, 581 Ghesney, 420, 433, 434, 438 Chickering, 395 Chilcote, 312, 315, 491, 500 AUTHORS INDEX 597 Chiu, 5o7 Chu, 291, 301 Citron, 425, 42(5, 438 Clark, A. R., 129, 131, 140 Clark, H., 150, 155 Clark, J., 359, 373 Clark, L. T., 508, 530 Clawson, 541, 551, 555 Cobb, 570, 579 Coca, 140, 150, 168, 174, 179, 188, 190, 241, 243, 270, 309, 311, 314, 442, 473, 474, 479, 480, 484, 485, 498, 559, 560, 563, 564, 569, 575- 577, 579 Coggeshall, 78, 86 Coghill, 309, 310, 314, 353, 368, 370 Cohen, 301, 537, 579 Cohn, 41, 42, 44, 164, 165, 167, 532, 533 Cole, 285, 298, 323, 330, 395 Colebrook, 287, 304 Colmes, 579 Conn, 87, 98 Connor, 553, 556 Conroy, 292 Cook, 325, 330 Cooke, 559, 575, 577, 579, 580, 582 Cooper, F. B., 287, 288 Cooper, G., 284, 299, 304, 394, 398 Coriell, 102, 137, 140, 147, 154 Corper, 532, 533, 534, 547, 556 Coryllos, 78, 85 Cotier, 284, 303, 400 Coulson, 583 Coulter, 214, 219 Courmont, 470 Coventry, 252, 253, 270 Cowie, 96, 98, 561, 580 Cox, 105, 124 Craig, 403, 404, 417, 438 Craigie, 38, 44 Creighton, 309, 314 Criep, 497, 573, 580 Crile, 190 Crocker, 283 Crowe, 303 Cruikshank, 427 Culbertson, 105, 122 Cullen, 567 Cunningham, 54, 57, 67, 68, 89, 98 Curnen, 358, 369 D Dakin, 326 Dale, 297, 312, 314, 322, 329, 478, 487, 489, 493, 498 Danysz, 230, 248, 270 Daranyi, 39, 44 Davidsohn, 311, 314 Davies, 194, 206, 468 Davis, 79, 85 Davison, 572, 580 Dean, 96, 98, 161, 166, 229-232, 242, 261, 270, 313, 314 Debains, 248, 272 DeCastello, 168, 188 De Eds, 488 Deibert, 174, 188 Deissler, 449, 464, 467 DeKruif, 37, 44, 20S, 214-217, 221, 469, 482, 500 Delves, 199, 206 DeNecker, 104, 125 Denison, 420, 422, 438 Derew, 190 Dernby, 245, 270 Devaux, 589 DeWolf, 553, 556 Dick, 35. 44, 160, 201, 241, 270, 276- 278, 299 Dienes, 353, 368, 526-529, 531, 534 Dimitrijevic-Speth, 242, 271 Dineur, 208, 220 Dingle, 146, 155, 364, 368 Di Somma, 503, 581 Doan, 54, 57, 60, 68, 69, 142, 536 Dochez, 241, 270, 284, 285, 298, 299, 354, 395, 398, 551, 556 Doerr, 153, 155, 197, 322, 323, 329, 330, 470, 486, 489, 492, 498, 502, 559, 560 Domagk, 287 Dombrowsky, 462, 467 Donath, 149, 155 Dorset, 505, 532, 534 Douglas, 96, 101 Dowxiey, 54, 89 Downing, 557 Downs, 149, 155, 233, 237, 278, 292, 299, 322, 329, 379, 388, 398, 415, 417, 483, 484, 486, 498, 501 Dragstedt, 494, 498, 499 Dreyer, 398, 511 Drinker, 481 Dubos, 286, 287, 299, 300, 306, 308, 314 Duclaux, 209, 220 Dudley, 354, 355, 370 Duke, 564, 565, 570, 580 Dulaney, 325, 329, 385, 398, 403 Dulitskiy, 291, 299 Dunbar, 566, 580 von Dungern, 172, 174-177, 188, 311, 323, 329, 403, 418 Dunham, 422 Durham, 208, 220, 222, 237, 319, 320, 329, 350, 379, 399 Duryea, 289, 299 598 AUTHORS INDEX E Eagle, 165, 1(3G, 199, 200, 20G, 213, 220, 434, 435, 438, 443, 448, 465, 467, 468 Eaton, 33, 45, 200, 206, 245, 247, 270 Ebaugh, 420, 442 Eberson, 299 Ecker, 104, 157, 167, 465, 468, 499, 553, 556 Eddy, 106, 124 Edmunds, 481, 499 Edwards, J. C, 397, 399 Edwards, M., 299, 398 Eggstein, 487 Ehrlich, 54, 87, 88, 98, 152, 155, 157, 163, 166, 197, 198, 247, 250, 251, 258, 270, 403, 404, 417, 471 Ehrmann, 434 Eisenberg, 225 Eisenbrey, 322, 330, 480, 501 Elias, 468 Ellis, 567, 568, 580 Elmore, 324, 329 Elvidge, 298 Embleton, 163, 164, 167, 418 Emmerich, 143 Enderlein, 43 Enders, 33, 46, 161, 167, 199, 207, 278, 292, 309-311, 313, 316, 317, 331, 353, 356, 369, 373, 433, 443, 483-487, 502. 530, 537, 560, 562, 564, 583 Engel, 574, 578 Engle, 104, 124, 301 Epstein. 172, 188, 433, 438, 461, 468 Ertl, 577 Etinger-Tulczynska, 107, 124 Eusterman, 106, 123 Evans, 262, 271 Everett, 556 i]wins, 287 Eyer, 498 Eyre, 285 F Eabricius-Hansen, 173 Fahey, 195 Falgairolle, 184, 188 Falk, 39. 45, 299 Fanburg, 583 Faraday, 585 Farmer^ 494, 499, 580 Faucett, 38, 45 Fehleisen, 282 Feierabend, 152, 155, 242, 270 Feinberg, 497, 499, 574, 578, 580, 583 Feldman, 556 Felix, 121, 123, 351, 383, 384, 390, 399 Fell, 309, 310, 314 Felton, 286, 299, 358 Fenger, 508, 518, 519, 535 Fenn, 97, 98 Ferguson, 175 Ferrata, 157, 166 Ferry, 299, 300 Fex, 191 Figlev, 583 Fildes, 427 Finkelstein, 137, 141, 146, 157 Finland, 105, 123, 126, 284, 286, 300, 303, 358, 369 von Fish, 222 Fisher, 580 Fitzgerald, 270, 433, 434, 439, 457 Fleischner, 524-526, 535 Fleisher, 310, 314, 322 Fleming, 147, 255, 271, 272 Fletcher, 400 Flexner, 289, 291, 300, 470, 502 Flick, 439 Flosdorf, 158, 166 Foa, 285 Foerster, 287 FoUenby, 300 Foran, 583 Ford, 34, 45 Fornet, 228, 237 Forssman, 153, 155 Fosbinder. 287 Foshay, 298. 300 Fothei-gill, 33, 146, 155, 161, 167, 199, 207, 278, 292, 309-311, 313, 316, 317, 331, 353, 364, 368, 373, 483- 486, 502, 530, 537, 560, 562, 564, 583 Fox, 153, 155 Fracastoro, 419 Francis, 115, 123, 359, 372, 397, 399 Frankel, 261, 270 Eraser, 106, 122 Freedlander, 129, 140 Frei, 553 Freund, 132, 140, 217, 218, 274, 353, 368, 499, 537, 545, 556 Fried, 516, 536 Friedberger, 477, 483, 489, 499 Friedemann, 213, 220, 475 Friedenreich, 186, 188 Fuller, 366, 369 Funk, 513, 518, 519, 535 Furth, 41, 45, 325, 329 Futagi, 102, 126 G Gabritschewsky, 241, 270 Gage, 300 Gager, 427, 442 AUTHORS INDEX 599 Gahiinger, 484, 499 Gaillard, 578 Gallagher, 111, 123 Garber, 556 Gardner, 365, 371, 384, 399 Garrod, 52, 77, 78, 89, 93 Gaspari, 255, 272, 274 Gasul, 280, 302 Gate, 465, 468 Gay, F. P., 33, 92, 98, 99, 128, 129, 131, 140, 152, 154, 161, 166, 168, 188, 283, 284, 382, 399, 415, 417, 499, 502 Gay, L. N., 579 Gebauer-Fulnegg, 498 Gebhardt, 302 Gelmo, 287 Gengou, 161, 166, 401, 403, 404, 417, 423-425 Gennerich, 432 Georgi, 444, 445, 465, 469 Gerlough, 274 Gibbard, 255, 2(i9 Gibson, 398 Gilbert, 438 Gill, 264 Gillespie, 284, 299, 398 Gilman, 287, 288, 304 Giltner, 551, 556 Githens, 274 Glenn, 515, 536 Glenny, 243, 254, 261-264, 270 Glover, 516, 536 Glynn, 191 Goebel, 287, 356, 359-363, 369, 370, 372, 395, 502 Goldberg, 583 Goldmann, 88, 99 Goodman, 287, 288, 304, 569, 580 Goodner, 78, 85, 115, 123, 174, 188, 190, 197, 199, 206, 286, 300, 303, 306, 308, 314, 315, 322, 324, 329, 395, 474, 483, 489, 499 Goodpasture. 29, 43-45, 74, 79, 85, 128, 133, 134, 141 Gordon, 157, 167, 300 Gorter, 190 Gortner, 40 Gotschlich, 43, 45 Gottlieb, 233, 237, 245, 271 Gottschall, 537 Gouy, 587 Grabar, 309, 314 Gradwohl, 388, 399 Graham, 264, 270, 590 Gramenitski, 159, 166 Gratia, 40 Gray, 287, 561, 580 Green, A. A., 195, 206 Green, R. G., 40, 262 Greenberg, 300 Greenwood, 50 Gregory, 303 Grendel, 190 Griffith, 365 Gross, 287, 288, 304 Grove, 483, 487, 502, 575 Gruber, 208, 209, 220, 222, 237, 319, 320, 329, 350, 379, 399 Gruehl, 561, 582 Griinbaiim, 208, 220 Gundel, 52 Gunn, 78, 85, 139, 141 Gutherie, 179, 181, 188 H Hadaek, 281 Haden, 54, 61, 66, 68 Hadfield, 52, 77, 89, 93 Hadley, 42 Hagen, 274 von Halban, 190 Hall, 295, 302 Halvorson, 262, 271 Hamilton, 288, 304 Hamman, 540 Hampton, 525, 582 Handel, 285, 301 Hankins, 160, 166 Hanks, 101, 529 Hansel, 572, 580 Hanson, 309, 315 Hanzlick, 305, 315, 484, 488, 499 Harkavy, 580 Harkins, 218, 590, 593 Harrington, 520, 537, 547, 548 Harris, 282, 308, 315 Harrison, 261, 262, 270 Harten, 309-311, 315 Hartley, G. A., 300 Hartley, M. A., Jr., 541, 546, 555 Hartley, P., 133, 140, 322, 329 Hartman, 104, 123 Hartoch, 477, 499 Haughey, 502 Havens, 31, 46, 264 Hawes, 568 Heath, 512 Heathman, 324, 329, 387, 399, 403, 418, 423, 432, 439 Hecht, 427 Heffron, 300, 306, 315 Heide, 403 Heidelberger, 195, 197-199, 206, 212, 213, 220, 234, 237, 287, 302, 353- 356, 358, 359, 361, 362, 369, 395, 399, 531, 535, 536 600 AUTHORS INDEX lleiubeckor, 103, 123 Hektoen, 90, 99, 159, 1(36, 108, 188, 222, 228, 233, 237, 239, 278, 300, 322, 323, 325, 329, 330 Helmholtz, 587 Henderson, 300 Henley, 532, 534 Henry, 300 Heran, 568, 580 Hericourt, 471, 503 Herry, 551 Hershey, 365, 370 Hertzler, 138, 141 Hess, 292 Hetheringtou, 538, 556 Hidaka, 445, 467 Higgenbotham, 423, 432, 439 Higgins, 88, 89, 93, 99 Hill, 87, 104, 106 Hinton, 463, 468 Hintze, 153, 155 Hirsch, 143, 155, 233, 237 Hirschfeld, 172, 173, 175-177, 188, 323, 329 Hirszfeld, 107, 123 Hoagland, 397, 399 Hodes, 50, 53, 108, 109, 127 Hoelsclier, 365, 372 Hoffman, 391, 399, 558 Hoffmann, 419, 442 Hofmeier, 246 Hogan, 435, 438 Holmes, 43 Holden, 139, 141 Holm, 303 Holmes, Oliver Wendell, 81 Holt, 481, 499 Holtman, 156 Hooker, 153, 155, 178, 188, 198, 200, 207, 213, 220, 300, 308, 311, 315, 345-348, 376 Hoppe, 275 Horsfall, 112, 115, 123, 196, 197, 199, 200, 286, 300, 306, 308, 314, 315, 403 499 Hosepian, 312, 315, 491, 500 Hotchkiss, 301, 370 Howe, 193, 207 Howell, 94, 99, 481, 497 Howitt, 301 Hoyne, 305 Hiibener, 258 Huber, 580 Huck, 179, 181, 188 Huddleson, 365, 370, 388 Huff, 142 Hughes, 52, 109, 123, 550 Hunt, 69 Huntoon, 508, 513, 518, 519, 535 llurwitz, 580 Ilurxthal, 301 Hutc-liinson, 309, 315 Ichida, 190 Irvine-Jones, 103, 123 Irwin, 107, 109, 123, 146, 155, 156 Ivanic, 242, 271 Ivy, 79, 85 J Jackson, 582 Jacobs, 344, 348, 340, 472, 500, 503, 580 Jacobstahl, 444, 445, 408 Jacoby, 159, 167 Jaffe,*87, 89, 99, 104, 124 Jamieson, 311, 315 Jamuni, 139, 141 Jancso, 94, 99 Jansky, 168-171, 188 Janssou, 285 Jantzen, 468 Jenkins, 190 Jenner, 110, 124 Jennings, 557 Jobling, 325, 330 Johlin, 274 Johnson, A. J., 191 Johnson, C, 278 Johnson, G. E., 300 Johnson, T. B., 353, 370 Johnson, T. L., 252, 271 Jones, A. E., 191 Jones, H. P., 149, 155 Jones, H. W., 439 Jones, L. E., 310, 314, 315 Jones, N, C, 138 Jordan, E. O., 41, 45, 114, 300 Jordan, H. E., 501 Jordan, K., 520, 530 Jorgensen, 399 Jovanovic, 242, 271 Joyner, 305, 371 .Judd, 301 Julianelle, 302, 306, 370 Jungeblut, 104, 124, 153, 155, 301 K Kabat, 200, 206 Kabler, 312, 315, 482, 497, 499 Kahn, 149, 150, 155, 421, 435, 439, 445, 457, 468 Kane, 271 Kaplan, 583 Karady, 494, 499, 502, 570, 580 Kareliitz, 291, 301 Karsner, 307, 315, 322, 330, 499, 502 AUTHORS INDEX 601 Kast, 441 Kauft'inan, L'^ii Kauffniaiin, :]8, 45, 2S4. ;;ul Kaufmann, 282 Keeney, 583 Kemp, 150, 155, 4;54, 4;!9, 457, 4(17, 469 Kempf, oil, ;!15 Kempner, 241, 271 Kendall, 29, 35, 43, 45, 198, 199, 20(;, 212, 213, 220, 234, 237, 358, 3(59 Kennedy, 169, 191 Kenny, 287 Kereszturi, 536, 557 Kerr, 582 Kessel, 399 Kessler, 105, 122 Kesten, 325, 330 Kettle, 296, 301 Kilduffe, 431, 432, 439 Kimball, 443 King-, 105, 124 Kirkbride, 274, 278, 301 Kitasato, 33, 44, 241, 242, 256, 258. 269, 271, 293 Klausner, 444, 468 Klebs, 242, 271 Klein, 179, 188 Klemperer, 285 Klenge, 490 Kligler, 241, 272 Kline, 421, 441, 445, 457, 461, 46S, 469 Klopstock, 355 Kliiver, 142 Koch, 40-42, 45, 353, 470, 502, 504, 510, 524, 529, 535 Koerber, 149, 155 Kolk, 303 Kolmer, 158, 167, 202-205, 226, 284, 288, 301, 388, 396, 399, 404-406, 412, 415, 417, 421, 426, 427, 439- 441 Kopeloff, 485, 503 Kosofsky, 192 Kozelkai 191 Kramar, 361, 370 Kraus, 208, 220, 222, 223, 237, 320, 323, 330, 477, 480, 498, 538, 539 Krikorian, 385, 400 Kritehevskv, 483, 500 Kroo, 94, 99 Krumwiede, 274 Kruyt, 591, 593 Kuhn, 107 Kurotchkin, 474, 502 Kusclmarjew, 488, 500 Kustner, 150, 156, 560, 561, 581 Kuttner, 110, 124 Lacayo, 419. 443 Lackman, 121, 124 Lackmann, 38, 45 Laidlaw, 39, 40, 45, 354, 355, 37U Laing, 553 Lambert, 107, 124 Lampl, 344, 349 Lan, 399 Lancefield, 365, 366, 370, 371, 39(>j 399 Landsteiner, 41, 45, 121, 148, 149, 153, 155, 168-172, 174, 175, 181, 189, 191, 194, 195, 207, 213, 220, 225, 234, 237, 291, 317, 320, 323, 325, 327, 330, 332-335, 339-341, 343-345, 348-350, 359, 364, 371, 376- 378, 389, 399, 420, 426, 434, 441, 472, 473, 500, 503, 509, 525, 534, 568, 580, 581 La Roche, 420 Lar.sen, 253, 271, 432, 442 Larson, 262, 271 La Rush, 579 Lattes, 179, 184, 189 Lawrence, 65, 66 Leasure, 102 Leathes, 469 Leblanc, 222, 322 Leclef, 96 Lederer, 178, 190, 192 Ledingham, 143, 147, 155 Leggett. 535 Leishraann, 96, 99 Lembcke, 442 Lemetayer, 275 Lenarsky, 298 Lennette, 403 Leonard, A. B., 135, 141 Leonard, A. E., 135, 141 Leonard, B. F., 274 Leonard, G. F., 303 Leopold, 578, 581 Leslie, 365, 371 L'Esperance, 442 Levaditi, 291, 420. 42(), 434, 442 Levin, 384, 399, 553, 556 Levine, 153, 155, 181, 192. 301, 323, 330, 466, 468, 469 Levinson, 303, 423, 441, 488 Lewis, J. H., 355 Lewis, J. M., 290, 298 Lewis, P. A., 31, 46, 104, 108, 124, 291, 300, 322, 478, 492, 493, 498, 511, 526, 531, 535, 536 Libby, R. L., 207, 365, 371 Lichtenstein, 583 Liofmann, 99. 1()4, l(i5, 1(57 602 AUTHORS INDEX Lilly, 318 Limper, 301 Lindau, 78, 85 Linden, 52 Linton, 128, 141 Lister, 143 Litarezek, 362 Little, 207 Littman, 4(58 Locke, 83, 86, 143, 155, 274 Lockwood, 288, 305 Loeb, 214, 220, 317, 318, 330 Loeffler, 242, 244, 261, 271 Loescheke, 78, 86 Loewenstein, 261, 263 Loewenthal, 526 Logan, 395 Long, 41, 353, 355, 371, 504, 505, 510, 512, 514, 515, 521-524, 535 Longcope, 271, 311, 315, 487, 500 Lord, 30(i, 315 Lott, 287 Lottermoser, 214 Love, 287 Loveless, 560, 564, 581 Lovenian, 568, 581 Lowell, 286, 300 Lubkin, 574, 578 Lucke, 97, 100 Lukens, 159, 160 Lurie, 541, 543-546, 548, 556 r^ynch, 443 Lytlie, 553 M MacCallum, 540 Mackenzie, 149, 150, 155 Mackie, T. J., 137, 141, 146, 156 Mackie, T. T., 106, 124 MacLeod, C. M., 307, 308, 315 Macleod, J. J. R., 591, 593 MacMaster, 281 Madsen, 251, 252, 261, 271, 384, 399 Magendie, 470, 503 Main, 274 Maitland, 553 Maizels, 284 Major, 288, 305, 487, 500 Malcolm, 275, 301 Mallery, 95, 99 Mallory, 244, 245, 271, 526, 527, 534 Maltaner, 418 Mann, 88, 89, 93, 99 Mantoux, 516 Manwaring, 153, 159, 167, 197, 206, 312, 315, 476, 480, 481, 489-491, 500 Marchand, 96, 99 Marcus, 468 Marie, 426, 442 Mariette, 508, 518, 519, 535 Marine, 104, 124 Marino, 500 Marrack, 195, 199, 206, 353, 357, 371 Marshall, 201, 205, 287, 480 Martin, L., 244, 271 Martin, S., 561, 581 Maschmann, 354, 535 Massol, 230, 237, 248, 269 Masucci, 506, 535 Matson, 174, 189 Matsunami, 439, 440 Maver, 246, 253, 271 Maximow, 54, 57, 68, 100, 128, 188 Mayer, 353, 372 Mazzini, 466, 469 McAlpine, 506, 535 McBroom, 551, 556 McCann, 282, 301 McCartney, 241, 272 McClaskey, 298 McConnell, 578 McCordock, 518, 536, 539, 540, 557 McCoy, 294, 301 McCullough, 487 McCuteheon, 95, 97, 99, 100 McDermott, 468 McDonald, 420, 422, 432, 438 McHenry, 494, 498 Mcintosh, 39, 45, 427 McJunkin, 525 McKenzie, 427 McKliann, 195, 206, 291, 301 McKinstry, 164, 166 McLeod, 33 McPhedran, 535, 538, 556 Meade, 494, 498, 499 Meader, 301 Medlar, 69 Mehlman, 301 Meier, 469 Meinieke, 444, 445, 461, 469 Mellon, 43, 287, 288, 304 Menkin, 57, 66-68, 95, 100, 142, 541, 545, 557 Mennes, 96, 100 Menzel, 354, 355, 369, 531, 535, 536 Mesrobenu, 34, 44 Messer, 236 Metchnikoff, 87, 100, 160 Meyer, H. H., 245, 271 Meyer, J. A., 524-526, 535, 550, 551, 557 Meyer, K., 104, 124, 153, 301 Meyers, 214 Michael, 581 Michaelis, 444, 469 AUTHORS INDEX 603 Migula, 42, 45 Milford, 579 Milian, 432 Miller, C. P., Jr., 175, 176, 191 Miller, F, A., 274 Miller, F. E., 536 Miller, H., 567, 568, 578, 581 Miller, J. A., 550, 557 Miller, P., 246 Miller, R. H., 294, 301 Miller, W. R., 137, 140, 147, 154 Millice, 300 Mills, 106, 124 Mishulow, 274, 536, 557 Mita, 483, 489, 499 Mitchell, 292, 419, 442 Mitra, 364 Mohr, 443 Molitch, 301 Molomut, 104 Moloney, 251, 255, 262, 271 Monreas, 108 Moon, 66 xMoore, J. E., 443, 467, 469 Moore, M. B., 582 M0rch, 284, 301 Moreschi, 161, 167 Morgan, H. J., 230, 237 Morgan, I. M., 38, 45, 105, 121, 124 Morgan, W. T. J., 153, 364, 371 Morgenroth, 158, 222, 237, 417 Moriwaki, 102, 126 Morton, 274 Moser, 241, 271 Moskey, 532, 534 Mosley, 274 Moss, 168-171, 189, 191 Mott, 325, 330 Mudd, 38, 40, 45, 73, 95, 100, 121, 124, 158, 166, 199, 206, 211, 217, 218, 220 Mueller, E. F., 60, 68 Mueller, J, H., 225, 246, 325, 330, 355, 362, 371 Muir, 96, 100, 146, 156 Miiller, 228, 237, 426, 441 Mulsow, 391, 399 Munday, 506, 513, 536 Murdick, 274, 301 Murphee, 264, 270 Myers, H. R., 274 Mvers, J. A., 520, 535, 537, 547, 548, 557 N Nageli, 42 Neal, 301 Neckermann, 135, 136, 140 Neff, 76, 86, 280, 301 Neill, 41, 46, 255, 271, 272, 274, 275 Neisser, 41, 46, 213, 220, 425, 426, 434, 442, 444, 469 Nelson, E., 262, 271 Nelson, H., 494, 501 Nelson, T., 581 Nestler, 569 Neubauer, 468, 469 Neufeld, 96, 100, 107, 124, 285, 301, 320, 330, 395 Nevin, 301 Newell, 299, 300, 581 Newman, 350, 367 Neymann, 427, 442 Nicole, 433, 442 NicoU, 537, 574, 579 Nicolle, 248, 272, 472, 500 Nigg, 102, 125, 174, 187, 189, 190, 191, 253, 271, 389, 399, 432, 442 Noguchi, 404, 418, 420, 427, 442 Nolf, 100, 160, 167 Nonne, 423 Noon, 575, 576 Northrop, 208, 214-217, 220, 22], 257, 272, 419 Norton, 300 Nourse, 279, 302 Novy, 482, 500 Nungester, 78, 85 Nuttall, 144, 156, 223-225, 228, 230, 238, 320, 330, 564 O Obermayer, 225, 238, 332, 333, 345, 349 O'Brien, 302 Ochs, 574 Ohlmacker, 503 Okell, 513, 536 O'Leary, 433, 442 Olitsky, L., 65 Olitsky, P. K., 105, 124, 241, 272 Olmstead, 284, 301 Olsen, 420, 442 O'Neill, 487 Opie, 57, 68, 69, 87, 100, 230, 238, 480, 487, 490, 500, 523, 536, 538, 556, 557 Oram, 140, 301 Orr, 296, 301 Osborne, 324, 578, 580 Osier, 421 Ottenberg, 168, 172, 177, 190, 191 Otto, 471, 503 Ouvang, 537 Ozaki, 41, 44, 555 Padnos, 583 Panghorn, 356, 371 604 AUTHORS INDEX Papaeostas, 465, 468 Pappenheim, 54 Pappenheinier, 257, 272 Paifentjev, 309, 310, 315, 316 Parish, 513, 536 Park, 113, 124, 255-258, 261-263, 280, 281, 284, 290, 292, 301, 306-308, 315, 418, 536, 549 Parker, F., Jr., 482, 501 Parker, G., 164, 166 Parker, J. I., 482, 501 Parker, J. T., 194, 241, 272, 354, 373 Parr, 191 Pasteur, 112, 350 Paterson, 488 Paul, F., 461, 468 Paul, J. R., 154, 156 Paul, J. S., 399 Paul, S. B., 433, 438 Pauli, 213, 221 Pauzat, 465, 469 Payne, 285 Pearce, 322, 330, 480, 501 Pearse, 516, 536 Pedersen, 354, 355, 509, 536 Peizer, 299, 398 Pennell, 365, 370 Perkin, 87, 100 Peterson, 292, 295, 302 Petroff, 59, 69, 536, 539, 557 Pettersson, 147, 156 Pettit, 38, 45. 121, 124 Pfeiflfer, H., 317, 322, 330 PfeiflPer, R., 144, 156, 208 Phillips, 287 Pick, 153, 155, 225, 238, 247, 272, 320, 328, 330, 332, 333, 345, 349, 350, 354, 371 Pickels, 40 Pillemer, 157, 167 Piness, 567, 568, 578. 581 Piney, 69 Pinkel, 581 • Pinner, 355 Piper, 307, 316 Pirie, 39, 44 von Pirquet, 310, 311, 515, 530 Pitt, 121, 123, 350 Pittman, 364, 371 Plaut, 426, 442 Plummer, 298 Plunkett, 537 Polayes, 178, 190, 192 Pons, 69 Pope, 257, 262, 272, 309, 315 Porges, 469 Portier, 470, 471, 480, 488, 503 272, 302, 356, 261, 341, Pottenger, 503, 581 Potzl, 426, 441 Poutas, 534 Powell, 153, 156, 302, 311, 315 Povvis, 213, 221 Prausnitz, 150, 156, 560, 561, 581 Predtechensky, 294, 302 Price, 405, 418 Prickett, 107, 126 Prickman, 583 Pritchett, 241, 269, 295, 298 Proca, 529 Provitsky, 272, 275 R Rac'kemann, 311, 315, 559, 566, 567, 576, 578, 581, 582 Raistrick, 34, 46, 391 Raiziss, 418 Rake, 290, 302, 366, 372 Ramirez, 560, 572, 581, 582 Ramon, 200, 201, 247-250, 264, 272, 275, 294 Ramsdell, 316 Ranson, 501 Ranvier, 88 Rappaport, 583 Ratcliflfe, 466, 469 Rathmell, 439 Ratner, 110, 124, 561, 582 Ravenel, 292 Ravitch, 303 Ravfield, 384 Reagh, 383, 399 Redmond, 142 Reed, 353, 371 Reeves, 302 Regan, 292 Reichel, 87, 100 Reichell, 508, 536 Reimann, 31, 46 Rein, 469 Reiner, 275 Renaux, 250 Reznikoff, (i9 Rhoads, 280, 302 Rice, C. E., 353, 371 Rice, J. P., 419, 443 Rich, 133, 141, 518, 526, 531, 536, 539, 540, 557 Richardson, E. H., 570, 582 Richardson, L. V., 271, 274, 275 Richet. 243, 470, 471, 480, 488, 503 Rife, 174. 190 Rigdon, 274 Riley, 302 Rimpau, 96, 100 Rischkaw, 39, 46 AUTHORS INDEX 605 Ritter, o02 Rivers, 40, 46, 47, 52, 00, (iS, 111. 125, 139, 141 Robertson, D. H., 78, 80 Robertson, H. E., 523, 530 Robertson, M., 241, 273, 295, 297, 302 Robertson, R. C, 38, 46 Robinson, E. S., 257, 272, 275 Robinson, G. C, 480, 498, 501 Roby, 421, 422, 442 Roemer, 511 Roger, 208, 294, 301 Rogoff, 104, 125, 120, 579 Romer, 254, 255, 273 Rons, 284 Rosenau, 471, 473, 49S, 503 Rosenow, 302 Rosenstein, 398 Rosenthal, 460, 469 Rosling, 107, 125 Rostenberg, 503, 581 Rothberg, 191 Rous, 501 Roux, 32, 46, 241, 242, 244, 246, 273 Rowe, 567, 581, 582 Rubin, E. H., 536 Rubin, S. S., 583 Rule, 301, 440 Ruska, 40 Russ, 486, 498, 502 Russell, 243, 270 S Sabin, A. B., 105, 124, 395, 399 Sabin, F. R., 54, 57, 59, 68, 91, 93, 100, 142, 524, 530 Sachs, 152, 417, 426, 435, 444, 445, 464, 405, 409 Salazar de Sanza, 291, 302 Salkowski, 325, 330 Salle, 34, 46 Sames, 254, 273 Sammis, 582 Sams, 442 Sanderson, 275 Sanford, 61, OS Sarles, 142 Sarrabat, 87, 100 Sauer, 116, 125 de Savitsch, 557 Sawin, 192 Scabia, 285 Scammon, 103, 125 Schaeffer, 324, 330 Schamberg, J. F., 433, 439, 442 Schamberg, J. T., 418 Schaudinn, 419, 442 van (ler Scheer, 191, 195, 200, 234, 237, 349, 434, 441 Scherp, 300, 372 Schick, 255, 273, 291, 301, 310, 311, 530 Schiemann, 356, 372 Schiff, 153, 156, 185, 190, 195 Schilling, 54, 01-08 Schlesinger, 551, 550 Schmidt, 251, 273 Schmith, 102, 284, 301 Schoeflfel, 305 Schoenheit, 526, 534 Schoenholz, 550, 551, 557 Schott, 108, 125 Schottmiiller, 283 Schrader, 174, 189 Schroder, 261, 263, 272 Schroeder, 511, 53() Schubert, 152, 155, 270, 292 Schucht, 426 Schulhof, 323, 329 Schultz, E. W., 279, 302 Schultz, W. H., 478, 481, 489, 493, 501 Schur, 223, 238 Schiitze, A., 159, 167, 222 Schiitze, H., 179, 190 Schwenke, 573 Scott, J. P., 39, 46 Scott, W. M., 104, 125, 470, 501 Seegal, 301, 302 Seibert, F. B., 41, 353-355, 371, 372, 505-509, 513, 515, 521, 525, 530, 531, 535, 530, 558 Seibert, W. W., 530 Seideman, 583 Seifter, 157, 107 Selye, 502 Serota, 259, 272 Sewall, 557 Shaffer, 290, 302, 305 Shahon, 582 Shattock, 184, 190 Shaw, A. F. B., 09 Shaw, E. B., 301, 524-520, 535 Sheldon, 583 Shepard, 434, 457 Sherman, L., 241, 270, 550 Sherman, W. B., 564, 582, 583 Sherwood, 101, 102, 125, 137, 140, 147, 150, 154, 159, 160, 166, 167, 302, 312, 315, 324, 330, 391, 399, 403, 418, 435, 442, 469, 479, 481- 484, 480, 491, 494, 497, 499, 501, 502 Shiblev, 214-216, 221, 365, 372 Shope,' 31, 46 Shrigley, 146, 156 606 AUTHORS INDEX Shwartzman, 552, 557, 55S Sides, 302 Siebenmann, 275 Sievers, 366, 372 Silva, 476, 501 Silvette, 104, 122 Simmons, 115, 125, 153 Simon, 581, 582 Simonds, 481, 501 Simpson, 179, 190 Slater, 520, 536 Slavin, 116 Sleeswijlv 96, 101 Smeall, 395 Smedley-MaeLean, 356, 372 Smillie, 302 Smith, C, 160, 167 Smith, L. W., 191, 303 Smith, T., 47, 52, 74, 109, 125, 258, 273, 383, 391, 399, 400, 471, 502, 503, 541, 542, 551, 558 Smyth, 582 Sneath, 294, 302 Snyder, L. H., 107, 126 Snyder, L. N., 168, 172, 173, 175, 177-179, 190 Sohma, 323, 330 Solomon, 468 Sondern, 61 Soo Hoo, 198, 206 Sordelli, 353, 372 Sorenson, 344 Soule, 34. 46 Southard, 499, 502 Spain, 483. 487, 502, 582, 583 Spangler, 582 Spiegel, 302 Spies, 583 Spring, 286, 300 Stadnichenko, 537 Standenath, 490, 502 Stankovic, 42(5, 441 Stanley, 39, 46 Starin, 328, 331 Stiiubli, 400 Steele, 300 Steinecke, 324, 331 Steiner-Wourlisch, 569 Steinhardt, 477 Stephenson, 303 Sterling, 582 Stern, 427, 464, 469 Stevens, F. A., 551, 556 Stevens, H., 583 Stevens, J. W., 351, 372 Stevens, E. E'., 303 Stevenson, 283, 287 Stewart, C. A., 520, 521, 537 Stewart, F. W., 540, 557 Stewart, G. N., 104, 126 Stewart, R. L., 385 Stewart, S. G., 578, 581 St. Girons, 568, 580 Stillman, 77, 86, 303 Stockton, 499 Stoland, 312, 315, 479, 481, 491, 494, 501, 502 Stolyhwo, 553, 558 Stone, 363 Stookey, 278, 292, 299 Stovall, 127 Strandskov, 191 Stratton, 569, 582 Straus, 582 Streng, 152, 154 Streukens, 520, 537 Stuart, C. A., 192, 363 Stuart, G., 385, 400 Stull, 525, 575, 577, 579, 582 Sudmersin, 261, 270 Sugg, 255, 271, 272, 274, 275 Sullivan, E. R., 72, 75, 86 Sullivan, F. L., 94, 98, 135, 136, 140, 555 Sulzberger, 569, 572, 580, 582 Supplement No. 9, 442 Supplement No. 11, 442 Surgeon General, 275 Sutliff, 105, 123, 126, 194, 206, 284, 300, 303 Svedberg, 537 Sweanv, 537 Swift." 427, 555, 558 Swineford, 570, 582 Swingle, 104, 126 Szent-Gyorgyi, 106, 126 Takaki, 294 Taliaferro, 135, 140, 142, 152, 156, 252, 273 Tamiya, 355 Taraura, 553, 558 Tanigiechi, 153, 156 Taylor, 255, 271, 273 Tchistovit(^h. 208, 221. 224, 238, 320, 321 Teale, 128, 137, 142, 547, 558 Templeton, 582 Ten Broeck, 400 Tennant, 575 Terplan, 523, 524, 537, 558 Terrell, 78, 86 Thelander, 300 Thiele, 163-165, 167, 418 Thonmaen, 564, 576, 579 Thompson, J. E. M., 305 Thompson, R., 397, 399, 400 AUTHORS liNDEX 607 Thompsou, W. R., 418 Thomsen, 191 Tillett, 115, 123, 359, 372, 474, 498, 502 Tiselius, 354, 355, 509, 536 Tobias, 432, 442 Todd, 61, 68, 191, 241, 273 Toenniessen, 361, 372 Tomesik, 325, 330, 474, 502 Tompkins, 89, 98 Toomey, 129, 140, 279, 303 Topley, 34, 46, 50, 52, 101, 108, 121, 126, 198, 363, 373, 391, 405, 418 Toyoda, 102, 120 Trask, 277, 278, 303 Trist, 439, 440, 441 Tschernogubow, 427, 442 Tuft, 307, 308, 311, 316, 537 TuUoch, 213, 221 Turgenseu, 111 Turner, 284 Tyndall, 585 U Uhlenhutli, H., 323, 331 Uhlenhuth, P., 222, 223, 238 Ulrich, 356, 561, 582 Unger, L. J., 191, 582 VanCleve, 552, 556 Vander Veer, 559, 580 Varley, 274 Vaughan, V. C, 503 Vaughan, W. T., 307, 316, 564, 567, 568, 572, 582, 583 Veintemillas, 111, 126 Virchow, 54 Volk, 263 Vollum, 511 W Waddington, 262 Wadsworth, 275, 278, 279, 282, 303, 356, 359, 373, 405, 418 Wagner, 439 Walker, E. W. A., 398, 421 Walker, T. T., 558 Wallace, 262 Wallgren, 550, 558 Walsh, 132, 133, 140 Walter, 284, 287, 303, 394, 400 Walters, 60, 68 Walzer, 308, 309-311, 315, 561, 564, 576, 579, 580, 583 Ward, 487 Warner, 308, 316 Warnshuis, 303 Warthin, 105, 126 Washbourne, 2S5 Wassermann, A., 222, 223, 241, 273, 294, 403-405, 423, 425, 426, 442, 469 Wassermann, P., 579 Wasson, 288, 304 Waters, 583 Watson, 512 Webb, 537 Webster, J. R., 94, 98 Webster, L. T., 50, 52, 53, 105-109, 122, 126, 127 Weehsler, 497, 573, 580 Weech, 292 Weed, 274 Weil, A. J., 202, 310, 316 Weil, E., 383, 387, 390, 399, 400 Weil, E., 486, 488, 489, 492, 502 Weiskotten, 69 Weiss, 69 Weissbecker, 280 Weld, 251, 262, 271 Welker, 222, 237, 239, 322, 330 Weller, 420, 442 Wells, A. Q., 550, 558 Wells, D. J., 264, 273 Wells, H. G., 245, 246, 252, 253, 273, 317, 322-324, 326-328, 331, 344, 348, 349, 355, 373, 435, 442, 485, 487- 489, 492, 502, 586, 593 Wells, J, E., 103, 127, 152, 156, 242, 273 Wels, 403 Werigo, 136 Werner, 263, 269 W^ernicke, 258, 273 Wertheimer, 487 West, E. J., 303 West, E., 101, 160, 167 Westcott, 578, 583 Western, 96, 98 Wheeler, K. M., 192 Wheeler, M. AV., 275, 278, 282, 301, 303 Wherry, 74 Whitby, 287 White, B., 274, 275, 301 White, H. J., 508, 518, 519 White, P. B., 304, 373, 379, 384, 400 White, E. G., 191 AMiite, W., 274 Whitehead, 157, 167 Whitney, 132, 140 Whittaker, 284 Widal, 208, 209, 221, 222, 238, 350, 379, 391 Wieghard, 366- 370 608 AUTHORS INDEX Wiener, 175, 17«, 19(l-l!i'J Wikle, 385 Wilbur, 106, 123 Wilcox, 494, 498 Williams, A. W., 257, 272, 282, 302, 306, 307, 315 Williams, H. U., 419, 443 Williams, J. W\, 70, 86 Willis, 539, 547, 558 Wibner, 583 Wilson, G. >S., 50, 52, 61, 108, 126 Wilson, J. L., 536 Wilson, R. A., 583 Winn, 59, (59 Winternitz, 76, 86 Winters, 305 Wiseman, 536 Witebsky, 355, 403, 464 Witt, 179, 191 Wolff-Eisner, 516 Wolman, 540 Wolpert, 111, 123 Wong, 537 Woodbury, 312, 315, 481. 502 Woodruff, 547, 558 Woolpert, 84 Wormall, 157, 167, 344, 345, 349 Wright, 281 Wright, A. E., 96, 101 Wulff, 535 Wvman, 104, 122 Yagle, 440, 441 Yaguda, 69 Yamakami, 192 Yamanouchi, 420 Yersin, 32, 46, 241, 242, 244, 246, 273 Yoffey, 72, 75, 86 Yol, 275 Young, 469 Yu, 38, 46 Z Zacks, 534 Zeller, 583 Zetterberg, 366, 372 Zla, 398 Zimmerman, 363 Zingher, 259, 272, 281, 290, Zinsser, 31, 33, 43, 46, 101, 127, 161, 165, 167, 168, 194, 198, 199, 203, 207, 243, 261, 273, 278, 279, 296, 303, 309-311, 313, 326, 331, 350, 353-356, 433-435, 442, 483-487, 489, 526, 529-531, 537, 539, 560, 562, 564, 583 Zisserman, 569, 583 Zozaya, 218, 221, 329, 331, Zsigmondv, 584 302 111, 121, 190, 192, 235, 238, 281, 292, 316, 317, 373, 418, 502, 524- 551, 558, 359, SUB,JECT INDEX Ablastin, nature of, 152 Abortin, fractions of, 551 reaction, necessary factors for, 525 use of, in suspected ])rucellosis, 387 Abscess, retropharyngeal, 73 Acetylated proteins, preparation of, 334 Acid-fast bacteria, antigenic com- parisons of, 355 factors in, 531 Acidosis, infection and, 73 Active immunity, 110 bacterial vaccines and, 113 diphtheria prevention and, 258, 259 examples of, 257 tuberculosis in rabbits and, 544 immunization, Park's work with T.A.T., 258 poliomyelitis and, 292 programs for, 117, 264 Active-passive immunity, Eanion's results and, 264 Acute infectious mononucleosis, heterophile antibodies in, 392 Adenoids, lymphatic drainage, 75 Adhesions, value of, 138 Adrenal gland, resistance and, 103, 104 Adrenals, cortex function, 103 Adsorption, 589 antigen-antibody mechanisms and, 210, 214 effect of dilution on, 211 phenomenon, Bordet's example of, 253 two concepts of, 162 Willard Gibbs' principle, 589 Aerobic spore-forming bacteria, an- tigenic study of, 366, 367 Agar, cause of errors due to, 353 Age, anatomical changes in, 105 resistance and, 105 Agglutination, adsorption of anti- body and, 214 Bordet's definition of, 210 experiments on, 210 theorv of, 200 Agglutination — Oont M Buchanan's explanation, 213 cataphoresis experiments and, 213 critical potentials and, 213 denaturing of protein in, 213 early investigations of, 208 effect of charge on, 213 of salt on membrane potential and, 211, 213-215 errors due to extraneous material, 353 extent of surface coating for, 217 Gruber's theory of, 208 Heidelberger's theory of, 200 history of use in diagnosis, 379 importance of cellular surfaces and, 211, 217, 218 law of multiple proportions and, 211, 212 limitation of, for identication, 351 mirror reaction, 393 Northrop and de Kruif's explana- tion, 214 or precipitation, use of, in identi- fying bacteria, 392-397 Pauli's explanation of, 213 period of discovery of, 208 pH and, 214 reaction, value of, in cholera, 392 in glanders, 392 in S. dvsenteriae infection, 392 scope of investigations, 211, 379 Shibley's summary c>.' Northrop 's work, 214-216 subsequent lines of research, 211 test, undulant fever and, 387 use of formalized suspensions in, 209 Agglutinin response, variation in, 384, 385 titer, allergy and, 385 Agglutinins, brucellosis infection and, 387 carriers and, 383 cold, 184 cross reactions, S. gallinarum and E. typhosa, 391 demonstration of O and H, 381 during infection and vaccination, 386 (effect of dilution on absorption of, 311 609 610 SUBJECT INDEX Agglutinins, effect of — Cont M of fractional addition of anti- gen on, 212 fine flocculating, 384, o8() flagellar, somatic, and labile, 383- 385 Landsteiner's postulation of, 168 loose-flocculating, 386 measurement of, 201 mechanism of, 208-221 natural and immune, 118 normal and diagnostic titers of, 383 partially immune animals and, .'188. ■ 389 substances inciting granular, 391 time of appearance and maxinuim titer of, 385 variation in production, 385 in time of appearance, 382, 383 in titer following vaccination, 384 Agglutinogens, comparison of human and monkey blood for, 176 human red cells and, 168-187 Landsteiner's postulation of, 168 Aggressins, definition of, 39 Alcohol-water ratio, flocculation tests and, 451 Alexin, nature of, 144 Algae, biological and antigenic stud- ies on, 324, 325 immunological studies of, 324 Alimentary tract, sensitization through, 561 Aliphatic side chains, 336 Alkalosis, definition of, 84 Allergy, adrenalin and ephedrine in, 578 age of onset in, 562 agglutinin titer in allergic indi- viduals, 385 alimentary sensitization, 561 and immunity, Cannon's and Hart- ley 's studies in, 54() antibodies in, 560, 564 antibody and immunity, 544 asthma and, 566 auto-antigens in, 570 autopassive transfer in, 561 bacterial, 550 biological tests in, 572 brucellosis and, 550 character skin reactions, 577 clinic equipment for, 574 Coca's extracting fluid, 576 conjunctival tests in, 571 contact dermatitis. 568 Allergy — Cont 'd contralateral injection and, 561 diagnosis of, 570-573 diathermy in, 578 drugs producing, 568 duration in IjTnphogranuloma in- guinale, 553 elimination tests in, 570 endocrine dysfunction in, 570 eosinophiles in, 572 extracting fluids for, 576 food, 567 gastric acidity in, 573 histamine and histaminase in, 578 liistory of hay fever, 565 immunity and, 133 incubation period and, 560 inheritance factors in, 559 intradermal tests in, 571 leucopenic index in, 572 mechanism of, 573 necessary factors foi' tuberculin, 524-526 patch tests in, 571 paths of sensitization in, 561 perennial vasomotor rhinitis, 566 physical factors in, 570 P-K reaction in, 560 placental sensitization, 561, 562 pollen extracts and, 575 units, 575 possible explanation of drug, 378 potassium chloride in, 577 preparation of pollen extract, 576 puncture tests in, 571 reagins in, 150, 560 respiratory sensitization, 561 scratch tests in, 571 shock organs in, 559 significance of, in infections, 538- 558 standardizing pollen extract, streptococcal, 551 summary of conclusion in culosis, 554 symptoms due to food, 567 treatment of, 577 tuberculosis and, 504-558 Alpha lysins, 144, 146 Alum toxoids, preparation and use of, 263 xVmanita phalloides, toxin of, 34 Amboceptor, definition of, 146 effect of concentration of, on sensi- tization, 163 hemolytic unit, 202 Ameba, biological and antigenic re- lationship, 324 57(; tubei SUBJECT INDEX 611 American Indians, scarlet fever among, 102 Amines, role of, in infection, 74 Amino acids, acid and basic groups of, 328 possible number of compounds from, 327 removal of acid properties of, 341 of basic properties of, 342 Anaerobes, invasive power of, in gas gangrene, 297 Anaerobic spore-forming bacteria, toxins of, 296" Anamnestic reaction, 120 Anaphylactic sensitizers, definition of, 151 shock, effect of excess of liapten on, 474 ratio of antigen to antibody in, 474 Anaphylactoid reactions, 306, 307, 489 Anaphylaxis, acute shock in guinea pig, 477 and tuberculin allergy, possible ex- planation of dissimilari- ties, 526, 527 antitryptic index and, 477 Arthus phenomenon, 471, 480 bacterial systemic vs. skin sensi- tivity, 525 Bally 's blanching reaction in rab- bits and, 479 canine, 480, 481 carbohydrate haptens and, 474 chickens and, 484 criteria of, 485, 486 crop reaction in, 484 Dale phenomenon in, 478 desensitization, 476 with histamine, 494 discovery of, 470 duration of sensitization, 474 early studies of, 470 effect of endothelial blockades on, 490 of trypan blue on, 490 embryonic chick and, 484 evidence favoring H-substance, 493 feline, 481 frogs and, 483 further discussion of physical the-^ ory of, 494 guinea pig, 477 histaminase in, 494 histamine theory of, 493-497 Anaphylaxis — Cont 'd H-substance and, 493-497 incubation period in active, 473 liver and, 490 man and, 485 Manwaring's tlieory of, 490 mechanisms of reaction, 492 metabolic changes in shock, 487 monkeys and, 484 nature of, 488 of sensitizing antibody in, 486 passive sensitization, 472, 475 permeability change in, 491 protection of drugs in, 477 protracted, in guinea pig, 478 rabbits and, 479, 480 rats and, 482 refractory state, 476 reversed, 480, 484 Richet and Portier's work, 471 role of trypsin in, 476 sensitizing dose in, 473 substance, 472 serum sickness and, 312-314 shocking dose of antigen, 474 )Simond's work on, 492 simple chemical compounds and, 472, 473 site of reaction, 489 specificity of, 487 studies of Rosenau and Anderson, 471 symptoms in dogs, 470 Theobald Smitli phenomenon, 471 theories of, 492-493 tissues involved, 490 turtles and, 483, 484 washed precipitates and, 488 Anatomical factors, 70 Anatoxin, history of, 247 Anergy, 434 Anilin dyes, history of use, 87 use of, in hematology, 87, 88 Animal parasites, immunity mech- anisms against, 135 tissue resistance to Cysticercus pisiformis, 135 proteins, nature of, 321, 322 reagins, 150, 151 Animals, agglutinogens in, 174 bleeding of, 227 effect of toxin on, 242, 243 inoculation • of, according to Dean, 226 to Kolmer, 226 lipoid differences in antibodies of, 196 612 SUBJECT INDEX Aiitliiux bucteria, lelatiou of colony t>-pe to virulence of, 38 Anthropoid apes, blood group fac- tors in, 176 Antiaggressins, 152 measurement of, 202 Antianaphylaxis, 477 Antibacterial sera, causes of failure of, 283 Antibodies, ablastin, 152 absorption of, 210, 214 age and, 105 alpha lysins, 144, 146 anaphylactic sensitizers, 151 animal species and molecular weights, 197 antiaggressins, 152 antipolysaccharide, 196 antitoxins, 151 atoxyl-azo antigens and, 198 blood groups and, 105 Buchner's theory of, 197 cephalin in, 196 cold agglutinins, 184 comparison of, with reagin, 563 concentration of pneumococcal, 285, 286 diagnosis, importance of, in, 379- 400 difference in animal response to polysaccharide Type I, 197 differences between rabbit and horse, 197 effect of dilution on adsorption of. 211 of temperature on isohemagglu- tinins, 181, 183, 184 experimental streptococcus infec- tion and, 129 explanation of, 194 Heidelberger 's method of separa- tion of, 197 hemolysins, 148, 149 heterophile, 153, 154, 156, 185 importance of, in diagnosis, 379- 400 irregular hemagglutination and, 183 isopliile, 187 lecithin in, 196 lipoid differences in, 196 mammary secretions and, 109, 258 Manwaring's theory of, 197,' 198 measurement of agglutinins, 201 of antiaggressins, 202 of bacteriotropins, 202 Antibodies, measurement of — Cont'd of hemolysins, 202 of precipitins, 202 methods of measuring, 200, 204 natural, 137, 148 and immune, 143-156 resistance and, 109 nature of, 193 and origin of, 195, 196 of natural, 193 of sensitizing, 486 normal in chick embryos, 147 in kittens, 147 in young chicks, 147 rabbits, 147 opsonins and bacteriotropins, 152 origin of, 197, 198 peptic-digest method for, 309, 310 placental transfer of, 109, 561, 562 pneumococcal in infants, 194 precipitins, 151 relation of amount produced to antigen injected, 198 reticulo-endothelial system and, 193, 194 serum fractions and, 195-197 fractions containing, 196 side chain theory of, 197, 198 skin tests for, 397, 398 strychnine and, 348 substance of known constitution and, 332 variation during infection, 415 in titer of, 414 water-soluble ones, 197 Antibody fractions, Goodner and Horsf all's work on, 197 response, variation in animals to Type I polysaccharide, 115 Antibody-globulins, 195, 196 denaturing of, 213 Eagle's explanation, 199 Anticomplementary substance, 159, 160 titration protocol of bacterial anti- gen, 407 Antigen and antibody, reasons for co- existence of, 232 coexistence of, in the blood, 311, 313 An^gen-antibody mechanism, Heidel- berger and Kendall's the- ory of, 212, 213 ratio, anaphylaxis and, 474 reactions, colloidal phenomena and, 253 factors in, 162 SUBJECT INDEX 613 Antigen-antibody, reactions — Oont M lipids and, 196 theories of agglutination, 208, 209 union, Marrack's hy]:)othesis of, I!)p mechanism of ^^ 199, 200 Antigenic fractions, work on, .'io.';, 354 homogeneity, species showing, ;')51 relationship, Mallei and Whitmorii, 392 tj'phosus and enteritidis, 384, 391 and gallinarum, 391 titration, protocol of bacterial, 408 Antigens, acetylated proteins as, 334 acid-fast bacteria and. 354-356, 531 agar in, 353 alcohol-soluble ones, 355 altered by iodine, 333 animal proteins as, 321, 322 antibody adsorption by, 210, 214 anticomplementary titration of bac- terial, 406, 409 bacterial, for complement fixation, 405 body response to, 94 Brucella, carbohydrate fractions and, 365 carbohydrate-lipid fractions of E. typhosa, 391 cholesterinized, 427 complexity of bacterial, 350 components and antibodies, 218 of bacterial, 218 conjugated, of Landsteiner, 335 cross reactions of altered, 343 defatted, 405 definition of, 193, 374 dosage used in animal inoculation, 226 early work on, 350 on conjugated, 332 on fractions of, 353 on modified, 332 effect of acid on, 326 of coagulation on, 326 of enzymes on, 327 of formaldehyde on, 121 of racemization on, 326 esterification of, 343 explanation of group reactions of, 345, 346 factors in diazotizing, etc., 329, 330 finished lipoidal for flocculation tests, 451 Forssman, 153, 323 Antigens — Cont 'd fungi and, 325 H, O, and Vi, 383, 384 haptens and, 332 hemolytic titration of bacterial, 405, 406 heterophile, 153, 154, 156, 185 history of flocculation tests, 444 of Wassermann, 426, 427 importance of aromatic amino acids in, 328 of digestibility of, 327 of dosage in testing, 359 of physical and chemical changes ' in, 326 of surface factors in, 195 keratin, 323 Kolmer's, 429 labile, 121, 383, 384 and virulence, 38 Lanceiield 's streptococcal, 366 Landsteiner 's views on specificitv of, 195 lens, 322 linkage with salt-forming groups and, 341 living attenuated, 350 major and minor, 319 methyl ation of, 343 milk, 323 miscellaneous factors affecting li- poidal, 451 modified and conjugated, 332-349 mosaic nature of, 195 of cellular ones, 121 pattern in red cells, 185 mucin, 323 multiplicity of bacterial, 319 nature of, for flocculation tests, 447-449 of H. influenza, 364 non-protein, 359 and protein, 321 non-species specific, 322, 323 O, H, and 0 of bacteria, 218 optical activity and, 326 original Wassermann, 425 preparation of H and O, 384 Price 's bacterial, 405 properties of, 195, 196, 321 of protein, 374 proteoses as, 525 protocols of Kahn titration, 454, 455 ratio of components in lipoidal, 450 recent work on iodized, 344 red cell haptens and, 185 614 SUBJECT INDEX Antigens — Cont 'd requirements for bacterial comple- ment fixation, 405 role of alcohol in lipoidal, 449 of electrolytes in lipoidal, 449 of synergistic substances in li- poidal, 447, 448 of water in lipoidal, 448. 449 Shigella dysenteriae, 364 species specific, 320, 322 specific reactions of altered, 343 specificity factors in cellular, 321 soluble, 321 standardization of for Kahn test, 453 standardizing suspensions, 380 steroisomers and, 339 suggested mosaic structure of, 378 synergistic substances used in li- poidal, 447, 448 T.A.T. as, 260 thyroglobulin, 323 titration of Kolmer, 429, 430 toxoids as, 261, 263 tuberculins and, 525 use of, in Widal, 379, 380 Vi and inagglutinability, 351 Vi and virulence, 38 Wadsworth bacterial extract, 405 Zozaya's work on, 218 Antimeningococcus serum, use of, 254 Antistreptococeus serum, in septice- mia, 250 Antitoxins, definition of, 35, 151 dosage of, 257 Ehrlich's measurement of, 247 flocculation of purified, 250 mammary secretions and, 258 measurement of scarlet fever, 201 methods of administration of, 257 new unit of, 200 old unit of, 200 peptic-digest methods for, 309, 310 prophylactic dose of, 258 reference to improved methods of preparation, 257 to preparation of, for tetanus, 294 scarlatinal requirements for, 280 serum reactions and, 307 standardizing of, 200, 201 therapeutic dose of scarlatinal, 280 unit of tetanus, 200, 201 use of antistreptoeoccal for blanch- ing test, 279 in gas gangrene, 297 in tetanus, 293 Antitryptic index, importance of, in anaphylaxis, 477 Apes, agglutinogens in higher, 175 Appendicitis, leucocyte count in, 61 routes of infection in, 80 Armies, typhoid in, 115 Armstrong virus, 293 Arneth and Schilling counts, 61 Aromatic amino acids, importance of, 328 Arsauilic acid antigens, 376, 377 Arsonic haptens, employment of, 335 protein compounds, 336 Arthus phenomenon, 471, 480 Aspiration pneumonia, 77 Association, specific microbic, 31 Asthma, definition of, 566 history of, 567 sputum findings in, 572 Atopen, injection, effect, 563 reagin, effect, 563 Atopic hypersensitiveness, mechanism of, 573 reagins, discovery of, 150 Atopy, definition of, 559, 560 inlieritance factors in, 559 serum reaction and, 307 Autoagglutinins, 183 Autopassive transfer, controversy over, 561 Autoprotection, 593 Autopsies, incidence of tuberculosis found, 522 Auxilysin, definition of, 153 x\.zo dyes, diazotization and, 338 B Bacillus anthracis, haptens in, 367 mesentericus, haptens in, 367 whitmori, disease caused by, 392 Bacteremia, definition of, 30 Bacteria, chemical composition of, 40 classification using serology, 351, 352 effect of environment on, 41 habituation to various conditions, 41 heterophile antigen in, 153 identification by agglutination or precipitation, 392, 397 removal of, from blood stream, 135- 137 theories as to effect of environment on size, 43 types of, 393 Bacterial allergies, brucellosis and other, 550, 551 SUBJECT INDEX 615 Bacterial — Cont 'd anaphylaxis, systemic vs. skin sen- sitivity, 525 antigens, attenuated, 350 complexity of, 325, .350 killed suspensions as, 350 specificity and, 350, 373 used in classification, 351-353 association in gaseous gangrene, .'!4 carboliydrate, discovery of, 41 characteristics, early theorie.s of^ 42 complement fixation, antigenic preparation for, 417 antigens used by Wassermann in, 424 basis of, 401-418 correlation of with clinical findings, 413 discussion of, 413, 415 quantitative and qualitative tests, 413 significance of tubes in, 409- 412 summary of recommendations, 415-417 three methods available, 409 dissociation, 37 Bordet's description of, 42 surfaces, studies of, 217 toxins, names of, 241 nature and formation of, 245 types, 320, 321 vaccines, use of, 114 Bactericidal action, mechanism of normal, 146 Bacteriolysis, visible phenomena of, 163 Bacterio-precipitins, effect of heat on, 224 Bacteriotropins, 152 nature of, 96 titration of, 202 Bacterium lepisepticum, de Kruif 's work and, 37 Bail's aggressins, nature of, 39 Bally 's blanching reaction, anaphy- lactic shock in rabbits and, 479 Barley, antigens in, 324 B. C. G., nature of, 112 vaccination, different opinion.s of, 548-550 Park's views on, 550 Petroff 's views of, 549 vaccine, value of, 113 Behavior of cells, Mudd 's woik on. 218 Bernstein, triple allelomorph theory of, 178, 179, 188 Beta lysins, definition of, 147 Biological and antigenic specificity, 317-331 relationships, precipitin tests and, 223 tests, 572 Blanching reaction, rabbit anapliy- laxis and, 479 Blastomyces dermatitis, antigenic properties of, 325 Blood agglutinogens, incidence of N factor in Eskimos, 173 banks, suggestions for, 284 cells, 56, 90 origin of, 92 cold agglutinins in, 184 composition of, 143 count, discussion of white cells in, 60, 67 groups, 168-192 classifications of, 169-171 based upon agglutinogen con- tent of, 169, 170 comparison of human factors with monkey agglutino- gens, 176 discovery of, 168 distribution of agglutinogens among lower animals, 174- 176 frequency in different popula- tions, 173, 174 genetic formulas for, 177 importance of temperature in ag- glutination, 181 inheritance of factors, 172 irregularity of agglutination in, 181 M, N, and P factors and, 181, 182 medico-legal application of, 176, 177 racial distribution of, 173, 174 subgroups in, 179-181 time of establishment of, 172 nature and distribution of haptens in, 185 rouleaux formation in, 184 stream, removal of bacteria from, 135-137 supply, importance of, 80 Boil, nature of ' ' core, ' ' 55 Bone marrow, indication of activity of. 54 616 SUBJECT INDEX Bordet-Gengou technique, use of, in serology of syphilis, 4:2'.', 424 Wassermann 's modification of. 424 Bordet's criticism of theories of ag glutination, 209 definition of agglutination, 210 two-phase theory, 209 Botulism, cause of, 33 etiology of, 298 Brodie reaction, cause of, 312 Brucella abortus, antigenic faetois in, 365 Brucellosis, agglutinins for P. tularensis, 388 allergy in, 525, 551, 552 laboratory procedures in, 387 reference to Huddleson's technique in, 388 significance of antibodies in, 387, 388 skin allergy in, 387 specific agglutinins in, 387 strains of organism in, 387 vaccination in cattle and, 551 Bruck's nitric acid reaction, syph- ilitic serum and, 465 Buccal inflammations, protection against, 70 Buchanan, review of agglutination, 213 Buchner, antibody theory of, 197 Buffer mechanisms, importance of, 83 C "C" substance, nature of, 359 Canine anaphylaxis, 480, 481 Manwaring's work on, 491 Capsule, virulence and, 37 Carbohydrate, specific bacterial, 41 Carbohydrate-lipoid complexes, anti- genic ones, 391 endotoxins and, 34 from E. typhosa, 121 O and Vi antigens of E. typhosa and, 363 red cell haptens and, 186 Carbohydrates, yeasts and, 325 Carriers, agglutinins in, 383 definition of, 48 types of pneumococci in, 522 Casein, non-species specificity of, 323 Cataphoresis, 588 effect of antibody adsorption on, 213 Catheter cystitis, factors in, 81 Cations, effect on colloids, 588 Cats, Brodie reaction in, 312 Cell count, effect of intraspinal se- rum on, 289 sensitization, Bordet 's and Ehi - lich's views of, 163 Thiele and Embleton's observa- tions of, 163 surfaces, molecular orientation and, 218 studies of, 217 Cells, blood, 56 clasmatocytes, 58 giant-formation of, 57, 58 lymphocytes, 58 monocytes, 57, 58 origin of blood, 92 reticulum, 89, 90 supra-vital preparation of, 58 Cellular antigens, Durham's concep- tion of, 319 types and, 320, 321 theory, origin of, 87 Cephalin, complement and, 158 Cerebrospinal lues, serology of, 423 Chancre, description of "Hunteri- an," 419 Charcot-Leyden crystals, asthma and, 572 Charge, depressing effect of electro- lyte on, 215 effect of sensitization on, 215, 216 Chemical reactions, colloidal and, 585 Chemistry of bacteria, acid-fast group, 355, 356 Chemotaxis, 95 fertilization and, 317, 318 Chemotherapy, Br. abortus and, 288 CI. oedematiens and, 288 CI. welchii and, 288 E. typhosa and, 288 endocarditis and, 288 H. influenzae and, 288 history of, 287 Lancefield 's group D and, 288 meningococcal and gonococcal in- fection and, 288 pneumococcus infection and, 288 rheumatic fever and, 288 S. paratyphi B and, 288 staphylococcus infection and, 288 virus disease and, 288 Chickens, anaphylaxis in, 484 Children, tuberculin test in, 520, 521 Cholecystitis, factors in, 79, 80 Cholera, agglutination reaction in Asiatic, 392 SUBJECT INDEX 617 Cholesterol, use of, in the Wasser- mann, 427 Chorio-allantoic membrane, experi- mental infection of, 133, 134 Clironaxia, anaphylaxis and, 312 (jhronic inflammation, tubercle forma- tion, 57 Cilia, effect of nasal medication on, 1 oo Xoo importance of, 72 Clarification reaction, 4(i2 Clasmatocytes, 58 Sabin's views on, 91 streptococcus infection and, 128 Classification, blood groups and, 169- 171 Climate, effect of, on imnmnity mech- anisms, 50 Clinical allergies, 559-578 diagnosis of, 570 allergy, adrenalin and ephedrine, 578 antibodies in, 560-564 asthma, 566 atopy, 559, 560 character skin reactions, 577 contact dermatitis, 568 correct breathing and, 578 diathermy in, 578 drugs producing, 568 endocrines and, 570 equipment for clinic, 574 foods producing, 567 gastric acidity in, 573 hay fever, 564 heat, cold, and effort, 570 heredity, age of onset of, 562 history of hay fever, 565 importance of history in, 570 incubation period, 560 leucopenic index in, 572 mechanism of, 573 patch tests, 571 paths of sensitization, 501 perennial vasomotor rhinitis, 56() P-K reaction in, 560 preparation of pollen extract, 576 shock organs in, 559 treatment of, 577 Clinics, allergy, committees on, 574 (Uostridium botulinum, types and tox- ins of, 394 chauvei, aggressins of, 39 oedematiens, effect of. on tissues, 297 Clostridium — Cont 'd septicum, effect of, on tissue, 297 tetani, types of, 394 welchii, effect on tissues, 297 Coca's extracting fluid, 576 Cohesive force, agglutination and, 214-216 measurement of, 216 Cold abscess, 57 agglutinins, 184 vaccines, value of, 114 ''oli, sugar-lipoid complexes of, 363 Collodion particles, Loeb's adsorp- tion experiments with, 214 Colloid chemistry, fundamental prob- lem of, 591 definition of, 584 Colloidal state, 584 Colloids, 584-593 attraction of, 590 autoprotection, 593 cataphoresis and, 588 classes of, 585 critical potential of, 588 effect of cations in, 588 of speed of mixing, 593 factors in dispersion, 592 protective action of, 592 Colonies, rough and smooth, 37 Colony variation, Arkwright 's studies on, 37 Colostrum, antibodies in, 258 Combined tetanus and diphtheria tox- oids, use of, 263 Complement, 157-167 bacteriolytic vs. hemolytic, 146, 147 Boerner and Lukin's method of preservation, 159 comparison with enzymes, 157 definition of, 146, 157 desirable qualities of, 160 Ecker's studies on, 158 effect of ammonia on, 157 of concentration of, on absorp- tion, 163, 164 of heat on, 159 of inorganic and organic com- pounds on, 159, 160 of prolonged shaking on, 159 of temperature on action of, 165 of zvmin on, 157 exact unit of, 204, 416 fixation, antigen requirements for, 405 antigenic unit in bacterial, 417 antigens for gonococcal, 405 application of, 402 618 SUBJECT INDEX Conipleinent fixation — Cont 'd bacterial antigens used in, -iO'j basis of bacterial, 401-418 by-products of lysis and, 164 development of modern tech- nique, 4on diluents used in, 405 discussion of bacterial, 418-415 Eagle's studies, 165 effect of kind of lipoid in, 196 final volume in, 416 glassware in, 416 gonococcal infection and, 413 history of, 401 inactivation of sera for, 417 Kolmer's recommendations, 415- 417 mechanisms of, 165, 435 methods available, 409 modifications of original, 404, 405 necessary controls in, 409-412 nonspecific reactions in, 149, 4.".4 original technique, 401. 402 precipitates and, l(il protocol of Bordet, 402 of simplified bacterial, 410 quantitative and qualitative tests, 413 reagents and factors involved, 403 reporting results, 413 results due to antibodies, 412 specific, 161 syphilis and, 419 time and temperature of incuba- tion, 416 tuberculosis and, 413 fourth component of, 157 Kolmer's full unit of, 204, 416 method of titrating, 203, 204 nature of, 157, 158 of end-piece and fourth com- ponent, 158 nonspecific adsorption of, 162 opsonification and, 96 origin of, 160 phospliatide fraction of, 158 preservation of, 158, 159, 416 reversibility of reaction, 159 role of, 145 specific fixation of, 161 time of fixation, 161, 162 unit of, 203, 204 various animals and, 160 Concentration of antihodv, historv of, 285 Conglutinins, discovery of, 152 Conjugated antigens, 335 Landsteiner 's, 378 studies of Averv and Goebel, 360, 361 proteins, antigenicity of, 322 Conjunctival tests, 571 Constancy of characters, theory of, 41, 42 Contact dermatitis, causes of, 568, 569 Contagious disease, definition of, 32 Contralateral injection, skin allergy and, 561 Controls, complement fixation and, 412 Convalescent and immune sera, 27()- 305 serum, blanching test and, 279 danger of intraspinal, in polio- myelitis, 292 measles and, 290 mumps and, 292 poliomyelitis and, 292 scarlet fever and, 279, 280 variola and, 292 virus disease and, 289 Corynebacterium diphtheriae, aviru- lent toxicogenic strains of, 244 hypersensitivity to toxins of, 41 identification of, 393, 394 invasive powers of, 152, 242 toxin of, 245 Coughing reflex, value of, 71 Cow's milk, precipitins for, 222 Criteria of anaphylaxis, 485, 486 Critical potentials, agglutination and, 213 Cross reactions, pneumococcus Type II and Friedlander 's ba- cillus, 154 Tvpe XIV and liuman EEC, 358 rickettsia and proteus 0X19, 154 tularemia and brucellosis, 390 Croton tiglium toxin, 35 Cryo-ehem process, preservation of complement by, 158 Crystalline lens, non-species specific- ity of protein of, 322, 325 Crystalloids, 584 Customs, effect of, in freedom from disease, 50 Cysteine, diazotization of, 338 Cysticercus pisiformis, tissue resist- ance to, 135 Cystitis, factors in, 81 SUBJECT INDEX 619 D l);ilo plienoiuenon, bacterial liajjtcns and, 48(i description of, 478, 479 l)aiij>er area, location of, 72 Danysz phenomenon, Bordet 's expla- nation of, 212, 253 description of, 212 Defatted antigens, for complement fixation, 405 Degeneration phenomena, definition of, 41 Dermal pneumonia, gravitation of fluid in, 78 Desensitization, criterion of, 47G explanation of, 495 histamine and, 494 Determinative bacteriology, use of serology in, 392-397 Detoxified toxin, history of, 2(51 immunization with, 116 Devaux's experiment, 589 Developmental anatomy, natural im- munity and, 103 Diabetes, infection and, 73 Diagnosis, importance of antibodies in, 379-400 Diagnostic tests, tuberculin and, 515 Diapedesis, definition of, 72 Diathermy, in hay fever and asth- ma, 578 Diathesis, constitutional in tubercu- losis, 108 Diazotization, aliphatic side chains and, 336 cysteine and, 338 method of, 336-338 Dick test, dose of toxin for, 279 reading of, 279 significance of, 227 toxin valency and, 278 use of, 119 Diet, effect on agglutinins, 106 Diluents, Kolmer's recommendation, 416 Diphtheria, active and passive im- munity to, 256-259 active-passive immunity, 264 antitoxin, Ehrlich 's units of, 200 causes of death in, 245 etiologic agent of, 242 immunity, results of retesting, 265 Schick test and, 255 immunization. New York City Board of Health, 264, 265 results of, 260, 261, 263 natural immunity in, 102, 103 Dijihtheria — ('out M nature of, 34 Park's studies of, 255, 256, 259 susceptibility, T.A.T. and, 256 symptoms and pathology of, 244, 245 toxin, discovery of, 242 Ehrlich 's measurement of, 200 hypersensitivity to, 255 source and nature of, 245 synthetic media and, 246 variation in potency, 246 use of antitoxin in, 257 variation in susceptibles, 255 Direct extension, infection and, 77 Disease, definition of infectious and contagious, 32 human, 102 toxemic, examples of, 32, 33 Dispersion, factors in, 592 Dissemination, routes of, 74 Dissociation, bacterial, 42, 43 Dogs, anaphylaxis in, 470 Dosage, importance of, in determin- ing antigenicity, 359 Dressings, hot, wet, value of, 83 Drug allergy, 568 duration of, 568 haptens and, 568 possible explanation, 378 Ducts, parotid, 70 Dyes, use of, in hematology, 87, 88 Dvsenterv, agglutination reaction in, 392 E Eagle fiocculation test, 465, 466 Ear, lymphatic drainage of, 75 Eberthella typhosa, antigenic fac- tors in, 363 uniformity of, 394 carbohydrate fractions in 0 and Vi antigens, 115, 121, 363. 391 Craigie's bacteriophage for in- agglutinable strains, 38 relationship to S. galliuarum, 391 Vi antigen of, 38, 121 Edema, definition of, 74 Edematogenic substances, nature and source of, 74 Effort sensitiveness, 570 Egg, antigenic components of, 323, 324 phenomenon of fertilization of, 318 Egg white arsanilic antigen, 345-348 620 SUBJECT INDEX Elirlich, coutributious of, 88 criticism of Bordet's work, 211 toxins and, 247, 248 units of diphtheria antitoxin of, 200 Electrocapillary mechanism, role of, 73 Electrolytes, emulsoids and, 591 role in agglutination, 210 Electron microscope, use of, 40 Elimination, importance of, 82 tests, 460, 571 Embryo, antibodies in, 147 Empyema, cause of, 77 Emulsoids, electrolytes and, 591, 592 stability factors, 591, 592 Endameba histolytica, antibodies for, 324 Endemic disease, definition of, 48 Endocrines, natural immunity and, 103, 104 Endotoxins, antigenic property of, 34 carbohydrate-lipid complexes of, 34 nature of, 33, 34 yhiga dysentery bacteria and, 34 Enol form, racemization and, 327 Enolization, definition of, 327 Enteritidis infection, hereditary factors, 108 Euvirouinent, effect of, on bacteria, 41 factors not involving imiminitv, 48 relation of size of bacteria, 43 Environmental conditions, effect on immunity mechanisms, 50 Enzymes, effect of, on antigens, 327 treatment of antisera with, 309, 310 of pneumoeoccus infection with, 286 Eosinophiles in allergy, 572 Epidemic disease, definition of, 48 Epidemiological factors, importance in measles serum therapv, 291 Epidemiology, experimental studies in, 50-52 Equipment for allergy clinics, 574 Erysipelas, definition and cause of, 281 types of lesions in, 281, 282 use of scarlet fever antitoxin in, 282 Escherichia coli, antigenic factors in, 362 mutation of, 41 Eskimos, diphtheria among, 102, 103 Euglena, antigenic studies of, 325 Euglobulins, Pi, Pj,, Pm fractions of, 195 Exact unit of complement, 204 Exogenous infection, definition of, 81 Exotoxins, nature of, 33, 245 Shiga dysentery bacteria and, 34 Experimental infections, 128-142 Altemeier and Jones on perito- nitis, 138 Buxton's studies, 134 Cannon's work, 129-131, 135 Freedlander and Toomey's work, 129 Gay's work, 128, 131 Goodpasture and Anderson 's work, 133 Hertzler's work on peritonitis, 138 immunity mechanisms in, 128- 142 Leonard's work, 135 Lurie's work, 544-546 Taliaferro and Cannon's work, 135 Teale's work, 138 Extra agglutinin 1, 183 Extracting fluids, allergens and, 576 Exudate, peritoneal, supra-vital stain of, 58 F Factors, chemical and physical, in cell sensitization, 162 of safety, mechanical, 82 Fascial planes, importance of, 77 Fatigue, effect on resistance, 83 Feline anaphylaxis, 481 Felix-Weil phenomenon, nature of, 377, 389 Fertilization, phenomena of, 318 specificity and, 317 Fever, importance of, 83 Fibrinogen, specificity of tissue, 322 Film hypothesis, antigen-antibody, 199 Fitness, Locke 's indices of, 83 Fixation of bacteria, 130, 545, 546 phenomenon, in inflammation, 130, 545, 546 Flagella, agglutination and, 209 Flavobacterium orchitidis, source of, 392 SUBJECT INDEX 621 Flexner dysentery bacteria, ciulolox in of, 34 studies on venoms, 35 Flocculatiou tests, antigens in, 447 Brack's, 465 citochol reaction, 464, 465 iloubtful value of hypersensitive tests, 466 Eagle's, 465 forinol reaction, 465 Hinton's, 463, 464 history of, 444 Kahn's, 452, 453 Kline's, 457-461 Mazzini's, 466 Meinicke's, 461 non-specific reactions, 149, 4.'14 principle of, 445, 446 role of ingredients in, 446 Eosenthal's, 466 Sachs-Georgi 's, 465 simplicity of method, 445 status of, 445 syphilitic reagin and, 444-468 Vernes, 465 Fluctuating variability, 42 Flushing mechanism^ respiratory tract and, 71 Focal infection, location and impor tance of, 31 Food allergy, incidence of skin re- actors in, 563, 567 symptoms of, 567 Formaldehyde, effect of, on protein, 344 Formalin, use in bacterial suspen- sions, 209 Formalized proteins, antigenic prop- erty of, 344 rabbit serum, antigenic propertv of, 344 Formol reaction, 465 titration, Sorenson's, 344 Forssman antigen, nature of, 153, 323 relation of, to group A factor, 154 Francis skin test, use of, in pneu- monia, 397 Frei test, lymphogranuloma and, 553 Freundlich's equation, value of, 165 FriedlJinder 's bacillus, cross rela- tionship with Type II pneumococcus, 362 polvsaccharide hapten of, 361- 363 types of Julianelle, 362, 363 Frogs, anaphylaxis in, 483 Full unit of complement, 204 Fungi, antigens in, 325 Fuiuncle, description of, 55 G Gangrene, etiology of gaseous, 294- 296 gaseous, cause of, 34 effect of hemolytic streptococci in, 34 Gaseous gangrene, cause of, 34 antitoxin in, 297 bacteria in, 294-296 chemotherapy in, 288 contaminating bacteria and, 295 effect of toxins on heart and adrenals in, 297 factors affecting spore germina- tion, 295 removal of devitalized tissue in. 295 summary of important factors in, 296 tissue reactions to different an- erobes, 297 tissues affected in, 296 Gastric acidity, allergy and, 497 Gay's summary of cell origin, 92 Gelatin arsanilic acid antigen, 345- 348 Gelding, 496 General paralysis, malaria and di- athermy in, 428 Genetic formulas, blood groups and, 177 Genitourinary tract, infection of, 81 Giant cells, description and formation of, 57, 59 Gibbs, Willard, views on adsorption, 589 Glanders, agglutination reaction in, 392 Globulin, comparison of horse and rabbit, 358 Globulin synthesis, antibody forma- tion and disturbance of, 198 Gonococcal infection, complement fix- ation in, 413 Gouy, theory of, 587 Gramicidin, source and action of, 287 Granular agglutinins, substances in- citing, 391 Group A factor, Forssman 's antigen and, 154 622 SUBJECT INDEX Guinea pig, acute anaphylactic shock in, 477, 478 protracted shock in, 478 staphylococcus infection in, 129 Gunn's views on tolerance, 139 H H antigen, preparation of, 384 Haptens, adsorption of carbohydrate on carbon particles, 359 anaphylactic shock with, 474 bacterial, 354 conjugated antigens and, 377 cross reactions and, 377 definition of, 193, 194 discovery of, 194 distribution of group A substance in body, 18(i Forssman's antigen and, 153, 154, 185 importance of, 234 measurement of, bv precipitin test, 232 nature and distribution in red cells, 185 non-protein carriers* and, 218 polypeptide nature of one, 367 polysaccharide of Friedlander 's bacillus, 361-363 proteosis as, 525 red cell, 185 role of, 376 synthetic, of Goebel and Avery, 359 Harkins' theory, 590 Hay fever, antibodies in, 560, 564 " Blackley's tests, 565 controversy over exciting agents, 575 definition of, 564 Dunbar's work, 566 history of, 565 treatment of, 577 Heat, value in infection, 83 Heidelberger and Kendall, theory of, 199 Helmlioltz's double layer, 587 Hemagglutination, irregularity in, 181 pH and, 214 Hemagglutinins, hetero-, 148 immune, 148 Landsteiner 's discovery of, 148 Hematogenous extension, infection and, 76 Hemogram, example of, 64 Schilling, 61 Hemolysins, Bordet's studies of 148 hetero-, 148 immune, 148 iso-, 149 measurement of, 202-204 mechanism of, 148 preservation of, 416 unit of, 416 Hemolysis, definition of, 148 phvsicoehemical interpretation of, 164 Hemolytic streptococci, Lancefield 's studies on, 365, 366 titration, protocol of bacterial an- tigen, 40(5 Hemophilus influenzae, soluble specif- ic substance of, 364 pertussis, Bordet's rough and smooth cultures of, 43 phases of, 365 Saner 's vaccine, 116, 365 Hen egg, antigenic components of, 324 Hepatization, gray, in pneumonia, 55 red, in pneumonia, 55 Heredity, immunity and, 107, 108 Heterogeneity, species showing anti- genic, 351 within a culture, 42 Heterohemagglutinins, 148 Heterohemolysins, 148 Heterophile antibodies, acute infec- tious mononucleosis and, 154, 392 antigen, acquisition of, by bac- teria, 150 cross reactions and, 154 distribution of, 153, 154 nature of, 153 antigens and, 153, 154, 156 Hinton 's test, description of, 463, 464 glycerated indicator solution for, 463 Hirschfeld 's studies, blood groups, 173 Histaminase, discovery of, 494 tuberculin allergy and, 532 Histamine, allergy and, 573, 574 anaphylaxis and, 493-497 effect on leucocyte count of, 66, 67 reaction and Bier's spotting, 49() role of, in allergy, 573, 574 summary of physiological studies on, 496, 497 theory, further discussion of, 495 Histidindiazoarsanilic acid, modified antigens and, 345 SUBJECT INDEX 623 Histiocytes, function of, 9:> Hormones, specificity and, .'^IS Horse serum, comparison willi rabbit, 358 permeability cliangcs fi'oni, 491 Host cells, young plasma cells, 44 factors, resistance and, ?,9 Host-parasite relationships, 47, 51, 74, 133, 134, 542 the carrier state and, 51, 52 various diseases and, 542 Human diseases, 102 idiosjmcrasies, 559-583 gastric acidity in, 497 Humoral theory, 143 Hunterian chancre, description of, 419 Hydration of tissue, effect on bac- teria, 74 Hyperemia, in inflammation, 54, 57 Hvpersensitiveness, anaphylaxis, 470- 503 Brucella carbohydrate and, 365 development of, 59 diphtheria toxin and, 41, 255 serum reactions and, 308 significance of, in infection, 538, 558 Hypertonic salt, antibody fractions and, 197 Hypothesis, Dineur's, of agglutina- tion, 208 filming of antibody, 199 Landsteiner 's, on reciprocal rela- tionships, 170 physiocliemical, of lysis, 164 Welch's, 37 I linnuiue precipitates, mechanism in- volved, 199, 200 nature of, 233 obtaining, 227 ral)bit vs. horse, 197 sera, 227, 276, 305 serum, effect of, on scarlet fever, 277 liorse vs. rabbit, 358 inoculation of animals for, 226 measurement of antibodies in, 201 obtaining of, 227 preparation and prerequisites of, 225-227 use of, in tularemia, 298 Immune-globulins, 195, 196 nature of Piv fraction, 19() use in measles, 118, 291 Imnuinitv, absence of antibodies and, 110 acquired, 110 active and passive, 256, 257 active-passive of Kamon, 264 antibodies and, 110 bacterial vaccines and, 113, 114 clinical detection of, 119 determination by Schick test, 255, 259 early views on ciiemical concept, 143 ondocrines and, 103, 104 hereditary factors in, 107, 108 humoral theory of, 143 luxurj^ definition of, 108 passive, 121 placental transmission of, 109 prenatal vs. postnatal, 84 primary stimulus, 119 secondary stimulus, 119 theories of, in syphilis, 433, 434 Topley's views on inheritance and, 108 transmission of, through milk, 109 tuberculosis and, 538 variation in, following vaccination, 120, 121 Immunization, attenuated bacteria and, 112 inunction with toxoid, 263 measles and, 290 programs for, 117, 118, 264 value of, 119 recommendations, New York City Board of Health, 264, 265 scarlet fever and, 278 use of combined toxoids in, 263 Immunological specificity, 319-321 Immuno-transfusion, definition of, 281, 283 luactivation, procedure of, 417 Incompatibility of species and spe- cificity, 317 Incubation period, animal variation in, 473 definition of, 48 sensitizing, definition of, 473 theory of, 243 India ink, phagocytosis of, 90 Indicator tube, Eamon flocculation and, 248 Infection, acute, definition of, 31 beneficent, examples of, 31 chemotherapy in, 287, 288 chronic, definition of, 31 direct extension and, 77 624 SUBJECT INDEX Infection — Cont 'd effect of, on agglutinins for other bacteria, 389-391 establishment witliin tissues, 74 experimental, of chorioallantoic membrane, 133, 134 respiratory, 131-133 staphylococcus, 129 streptococcus in rabbits, 128 tubercular, 544-546 with pneumococcus, 131-133 factors affecting spore germination in, 295 in pulmonary, 77, 78 focal distribution and importance of, 31 genitourinary tract and, 81 hematogenous extension, 76 hypersensitiveuess due to, 504-537 intestinal factors in, 78-80 lines of defense, 70 localization of, 130, 545, 546 localized and generalized, 30 lymphogenous extension of, 75 mechanism of, 72 methods of penetration of surface barriers, 72 normal antibodies and, 84, 96, 146, 147 phenomenon of, 29 physiological factors and. 73 routes of dissemination, 74 secondary examples of, 32 subacute, definition of, 31 ulceration of intestine, 79 variation in antibody during, 415 Wherry's theory of, 74 with pathological change, 30 without pathological change, 30 Infectious agents, classification of, 29 local fixation of, 130 disease, definition of, 32 droplets, role of, 47 mononucleosis, heterophile antibod- ies in, 154 Inflammation, acute, 54, 55, 57 chronic, 57 Influenza, vaccination for, 112 Inheritance, blood groups and, 172 Inhibition phenomenon, explanation of, 358 reaction, use of, by Landsteinor, Inoculation, protective. 111 Insulin deficiency and resistance, 73 Intcrfacial tension technique, 217 description of, 217, 218 Intestinal infection, factors in, 78-80 pathogens, methods of spread, 43 Intestine, diseases and ulceration of, 79 Intracutaneous technique, 254, 571 Intradermal tests, 571 Intranasal vaccination, results from, 132 Invasive power, diphtheria bacteria and, 242 Iodized antigens, method of prepara- tion, 344, 345 specificity of, 345 protein, antigen modification in, 333 lodotryptophane, formation of, 333 Irradiation^ value in developing im- munity, 139 Isoagglutination, irregular, at low temperature, 183, 184 Isoantibodies, mechanism of action, 149 Isohemagglutinins, 149 time of appearance, 172 use of, in classification, 168-172 Isohemagglufinogens, inheritance of, 172 time of appearance of, 172 Isohemolysins, 149 paroxysmal hemoglobinuria and, 149 Isopliile antibodies, importance of, 187 J Jansky, blood group classification of, 169-171 Kahu precipitation tests, controls in, 455 description of, 452 and choice of, 455-457 inactivation of serum, 453 salt titration, 452 shaking, 453 spinal fluid procedure with, 456 standardization of antigen, 453 time of reading results, 455 test, presumptive, 456 primary stage and, 420 quantitative, 456 verification of, 435 reasons for, 457 Keratin, lack of species specificity of, 323 SUBJECT INDEX 625 KiKluffo's principles, serological di- agnosis, 4ol Kliue elimination test, 460 microscopic precipitation test, de- scription of, 457 test, agreement with other tests, 458 antigen preparation, 458 reading of, 458, 459 sensiti%'ity of, 458 sources of error in, 4(i0 technique of, 458 Kolmer antigen titration, protocol of, 430 antigens, preparation of, 429 titration of, 429, 430 unit of, 430 hemolytic unit of, 202 red cell unit of, 202 Kolmer 's technique, requirements met by, 415, 417 Kolmer-Wassermann test, 428-431 antigen used, 429 reporting results of, 430 Kupflfer cells, 90 Lo dose of toxin, definition of, 200 L+ dose of toxin, definition of, 200 variation in, 251 Lf dose of toxin, definition of, 248, 249 Lr dose of toxin, definition of, 254 Labile antigens, 38 Lacrimal secretions, importance of, 71 Landsteiner, blood group classifica- tion of, 169 early work on antigens, 332 Langhans ' giant cells, description of, 57 Lecithoproteins, antibodies and, 197 Lens protein, nonspecies specificity of, 322, 323 Leptospira icterohemorrhagiae, .".0, 36, 39 Leucocidiu, 33 streptococci and, 129 Leucocyte count, appendicitis and, 61 Ameth, 61 effect of iiistamine on, 66, 67 of infection on, 54-67 of splenectomy on, 66 of various substances on, <>(!, 67 in pneumonia, 61 juvenile neutrophiles, 62 Leucocyte count — Cont 'd meningitis and, 61 myelocytes and, 62 normal, 60 physiological conditions and, *il rhythmical variation in, 60 severe infections and, 61 stab forms, 63 Leucocytes, capillary permeability and, iJ6 degenerative forms, 63 differential counts, 60-66 Schilling c(mnt, 61 segmented forms, 64 Leucocytosis-promoting factor, de- scription of, 67 Leucopenia, different microorganisms and, 65 discussion of, 65, 66 diseases showing, 60 Leucopenic immunity, 65 index, 572 Leucotoxine, nature of, 67, 95 Leukins, 14(i, 147 Lipid, metabolism of, 93 Lipoids, antigens, 447 bacteria and, 355 importance of, compared with pro- teins and carbohydrates, 355 Lobar pneumonia, research on, 77, 78 stages in, 54, 55 Local fixation phenomenon, 130 545, 546 ' Loeb, adsorption experiments, 214 Long's synthetic medium, 504 Lower animals, serum sickness in, 310 Lungs, in anaphylaxis, 477, 478 in pneumonia, 54, 55 lymphatic drainage of, 76 Lymph channels, removal of infec- tion by, 75 drainage of lungs and pleura, 76 glands, filtration of bacteria by, 30 function as filters, 52 Lymphatics, diainage and, 75, 76 living skin and, 281 Lymphocytes, 58 Lymphogenous extension, infection and, 75 Lymphogranuloma inguinale, dura- tion of allergy in, 553 Frei test and, 553 Lymphoid tissue of: intestines. 78 79 respiratory tract, 71 Lynphile process, preservation of complement by, 158 626 SUBJECT INDEX Lysins, alpha, 14-1, 14(j beta, 146, 147 Bordet 's conclusions, 145 distribution of, in animals, 147 immune, 144 normal, 144, 14(5 Lysis, Bordet 's and Ehrlieh 's views of, 163 visible phenomena of cell, 16'.i Lysozyme, 146, 147 Lytic mechanism, Bordet 's explana- tion of, 145 M MA-100, Huntoon's, nature of, 508 Macrocytase, 87 Macrophages, 87 Malaria and diathermy, use of, in treatment of syphilis, 433 Mammary secretions, antibodies in, . 109 Man, anaphylaxis in, 485 Mantoux tests, grading of, 522 Manwaring theory of anaphylaxis, 490 Masked anaphylaxis, 477 Mass law, precipitin reaction in, 212 Mazzini microflocculation test, 466 Measles, degree of protection desired, 290 history of, in Faroe Islands, 49 immune globulin in, 118, 291 importance of epidemiological fac- tors in, 291 placental blood in, 291 preparation of convalescent serum for, 290 prophylaxis, Tunnicliff's serum in, 290 use of serum in, 289, 290 Meat industry, brucellosis and, 387 Mechanism, antigen-antibody, 199, 200 summary of, 219 Bordet 's explanation of lysis, 145 of agglutination, controversy over, 200 of infection, injury and, 72 leucocytes and, 72 lymphocytes and, 72 physiological, in defense, 82-85 serum sickness and, 310, 311 theory of toxin-antitoxin, 251, 252 Wassermann reaction and, 435 Medicolegal procedures, precipitin tests and, 228, 229 Meinicke's test, antigen for, 461 interpretation of, 462 principles of, 461 serum for, 462 specificity of, 461 technique of, 462 Membrane potential, 588 effect of sensitization on, 21J, 215 pyogenic, 55 Meningitis, meningococcus, 289 Meningococcus meningitis, serum therapy in, 289 polysaccharide in, 366 question of toxins from, 289 sulfonamide drugs and, 289 Mesenchyme, undifferentiated, 89, 91 Metabolism in anaphylaxis, 487 Metastasis, definition of, 32 examples of, 32 Mice, insusceptibility to diplitheria toxin, 243 Microbic association, 31 Microcytase, 87 Microphages, 87 Miliary tuberculosis, tuberculin test in, 518 Milk, antitoxin in, 258 precipitins for human and cow 's milk, 222 [iioteins, nonspecies specificity of casein, 323 species specificity of globulin in, 323 Mills-Reincke phenomenon, 49 Mirror reaction, 393 Mitachondria, staining of, 91 supra-vital stain of, 58 M, N, and P factors, human blood and, 181, 182 Modified and conjugated antigens, 332-349 Molecular orientation, 590 weights, pneumococcal antibodies and, 197 Monilia, carbohydrate haptens in, 325 Monkeys, agglutinogens in, 175, 176 anaphylaxis in, 484 Monocytes, Sabin's views on, 91 Monophyletic theory, 91 Moro 's percutaneous test, tuberculin and, 516 Mosaic structure of antigens, 121, 185, 193 Moss, blood group classification of, 169-171 Mouse method of pneumococcus typ- ing, 395 SUBJECT INDEX 627 Mouse — Cont \\ protection, antibody fractious and, 197 typhoid, experimental epidemics of, 50, 51 M.E.D., definition of, 254 Mucin, nidus of, in pneumonia, 78 Mucins, antigenic difference in, 322 lack of species specificity of, 323 role in pulmonary infection, 78 Mucous film, importance of, 71 membrane, protection of, 70 secretion, factors and, 78 Mudd, technique of, 217, 218 Multiple antigens, Durham 's concep- tion of, 319 Mumps, convalescent serum in, 292 Mutation, definition of, 41 Mycobacterium tuberculosis, varieties of, 59 Myelocytes, description of neutro- philic, 62 N N factor, incidence in Eskimos, 173 Natural antibodies, 109, 148 immunity, age and, 105 antibodies and, 109 endocrines and, 103 Neufeld 's theory of, 107 nutrition and, 106 passive acquisition of, 109, 258 racial differences in, 102 species differences in, 102 Topley's views on, 108 vitamins and, 105 Webster and Hodes' views on, 108 Nephritis, factors in acute, 82 Nervous system, importance of, 83 Neufeld 's quellung reaction, descrip- tion of, 395, 396 Neurosyphilis, serology of, 423 Neutral red, in supra-vital stain, 58 Neutrophilia, 65 Newborn calf, immunity in, 109 guinea pigs, immunity in, 258 New York City Board of Health, standards of, 418 New York, Park 's susceptibility tests in, 259 Neymann and Gager, Wassermann antigens of, 427 Nitrogen-fixing bacteria, 29 Nitroso compounds, 338 Noguchi, studies on venoms, 35 Noon pollen unit, 575 NonpaliMiiity, blood groufjs and, 177- 179 Nonprotein antigens, 359 Nonspecies specificity, 322, 323 Nonspecific reactions, 306 Normal antibodies, 147 bacteriolytic substances, 146, 147 Nutrition, resistance and, 106 O Obermayer and Pick, early work of, on antigens, 332 Old tuberculin, 504 Operative procedures, vasomotor rhi- nitis and, 566 Ophthalmic test, serum hypersensi- tiveness and, 308 Opportunists, definilion of, 47 Opsonification, nature of, 96 Opsonins, 152 Opsonocytophagic test, use of, in un- dulant fever, 387 Optimal proportion technique, 229- 232 coarse test, 231 fine test, 232 proportions, venoms and antiven- oms and, 230 Organ specificity, 322 Orifices, importance of blood supply, 80 Osier, on sj-philis, 421 P, and P„ fractions of Landsteiner, '325 Pain, importance of, 83 Pancreatitis, anatomical factors and, 79 stone in ampulla of Vater and, 79 Pandemic disease, definition of, 47 Parasites, classification of, 29 Goodpasture's classification, 29 Kendall 's classification, 29 Paratyphoid fever, Widal in, 382, 386 organisms (see Salmonella) Paresis, treatment of, 83 Paroxysmal Iiemoglobinuria, isohemo- lysins and, 149 Wassermann test in, 150 Passive imnmnity, acquired from mother", 258 examples of, 257 experimental infection and, 129 mammary secretions and, 258 nature of, 121, 122 628 SUBJECT INDEX Passive immunity — Cont 'd placental transmission of, 1U9 virus disease and, 289-292 sensitization, animal variation in, 475 definition of, 475 difference in immune sera in, 484 duration of, 475 incubation period fur, 475, 470 Pasteur, rabies virus passage and, o7 Pasteurella tularensis, agglutinins for Br. abortus and, 391 Patch tests, 571 Pathogenicity, definition of, o6 virulence and, 36 Pathological change, types of, 30 P-ehlorobenzovl chloride, anaphylaxis and, 472 Penetration, mechanism of, 72 Perennial vasomotor rhinitis, 56(5 Perforation, result of, 79, 80 Peritoneal exudate, cells in, 58 Peritoneum, absorption from, 138 Peritonitis, cellular response in, 13S defensive mechanisms in, 138 experimental, 138 Permeability changes, anaphylaxis and, 491 Pertussis, vaccination against, 116 Peyer's patches, ulceration of, 79 Pfeitfer 's immunity unit, description of, 144,' 145 phenomena, description of, 144 pH, cellular agglutination and, 214 etfect of, on flocculation of toxins, 250 on precipitin test of, 233 Phagocytosis, dyes in, 87, 88 factors in, 95 fate of infectious agents, 97 Menkin's work and, 95 quantitative aspects of reference to, 101 reference and theories of, 97 (Smith's concept of, 542 Theobald Smith's views on, 52 Phenomenon, Arthus, 471 Dale, 478 Dienes, 529 Mills-Eeincke, 49 Pfeiffer's, 144 Theobald Smith, 471 Phosphatids, composition of tuber- culo-, 356 tubercle formation and, 59 Physical allergy, 570 Plivsiological mechanisms, role of, 82- 85 Phytotoxins, Ford 's work on, 34 Picryl chloride, anaphylaxis and, 473 von Pirquet and Schick, theory of serum sickness, 310 test, description of, 515 P-K reaction, 560 Placenta, transmission of antibodies through, 109 types of, 110 Plasma cells, function of young, 79 typhoid bacteria in young, 44 proteins of, 193 Plasmodium cathernerium, immunity mechanisms for, 135 Pneumococcal antibody, concentration of, 285, 286 flora of nasopharynx and, 194 molecular weights of Types I and II, 197 polysaccharide, anaphylactic shock with, 474 conjugated to horse globulin, 361 Type I polysaccharide, variation in animal response to, 197 Pnouiuococcidal antibodies, age and, 105 Pneumococcus, C substance of, 359 discovery of soluble specific sub- stance of, 285 of types of, 284, 285 distribution of types in carriers, 55 immuno-chemical studies of, 356- 358 infection, immunity mechanisms in, 131-133 pneumonia, history of serum treat- ment in 285 polysaccharide. Type I, difference in antibody response to, 115 vaccination with, 115 typing, 394-396 reasons for, 394 technique of, 395 Pneumonia, acute inflammation in lobar, 54 allergy and, 551 enzyme treatment of, 286, 287 experimental, 78 history of serum treatment in, 285 immunization against, 115 leucocyte count in, 61 lobar, conclusions of Blake and Cecil, 77 pre-existing humoral immunity and, 110 resolution in lobar, 55 SUBJECT INDEX 629 Pneumonia — Cont ' d results of serum treatment of, 28C route of infection in lobar, 77, 78 stage of engorgement in, 54 types in lobar and bronchopneu- monia, 284, 285 Poliomyelitis, active immunization for, 292 antibodies and immunity to, in monkeys, 110 use of convalescent serum in, 291, 292 Pollen count, Blackley's picmeer work, 565 exciting agents, 575, 57(5 Pollen extract, preparation of, 576 standardizing of, 576 unit of, 575 extracting fluid for, 576 Polypeptide, hapten nature of, 367 Polypliyletic theory, 91 Polysaccharides, bacterial, 354 chemistry of pneumococcal, 356-358 Forssman's antigen and, 153 O and Vi content of, 363 pneumococcal, 195 Shigella dysenteriae fractious of, 364 synthetic as haptens, 360, 361 tubercle bacilli and, 354 Populations, variation in susceptibles in, 255 Portal of entry into body, 47 Portals of infection, 75 Position isomers, specificity and, 340 PPD, chemical nature of, 509 surveys with, 521 Precipitates, solubility of, 593 Precipitation, Bordet's theory of, 200 Heidelberger and Kabat's theory of, 200 theories of, 199, 200 Precipitin reaction, tuberculin and, 354 serum, titration of, after Nuttall, 227, 228 after Uhlenhuth, 227 test, biological relationships and, 223 Cannon's technique, 229 early application of, 223, 225 investigations, 223 effect of pH on, 233 of salts on, 233 of thiocyanates on, 233 estimation of haptens and pro- teins with. 232 Precipitin test — Cont 'd iniiibition of, by tuberculin, 530 introduction of cholesterol, 444 Nuttall 's optimum proportions, 228, 230 range of specificity, 229 reasons for diluting antisera, 235 syphilis and, 444-4(i8 unit of Dean and Webb, 232 use of, in determining antigenic specificity, 225 Precipitinogen, definition of, 222 Precipitins, 222-241 bacteria vs. animal proteins, 224 Cannon's method of titrating, 201, 229 coexistence of antigens and anti- bodies in blood, 311, 313 definition of, 151, 208, 222 discovery of, 222 effect of heat on, 224 Kraus' work on, 222 measurement of (ring test), 201 nature of, 224, 225 Nuttall 's conclusions in, 224, 225 optimal proportions of Dean and Webb, 229-232 prerequisites and preparation of sera for, 225-227 quantitative technique of Nuttall, 228 source of precipitate, 224, 233 specificity of, 320 turtles and, 484 Preservation of complement, 158, 159 Preserved human blood, reference to studies on, 284 Primary host, effect of removal of, 49 stage, results of serological tests in, 420 Programs, immunization, 117, 118 Protein, A and B fractions of im- mune, 196 ucetylation of, 334 aliphatic groups in, 328 antigens in vegetable, 324 factors in specificity of, 325, 326 of serum, 321 fractionation of, 193 fractions from bacteria, 41 of normal and immune, 195, 196 ketone form. 327 nature of animal, 321, 322 nonspecies specific, 322, 323 number of compounds possible from amino acids, 327 plasma, 193 630 SUBJECT INDEX Protein — Cont 'd precipitin tests and differentiation of, 223 red cell, 322 structure of, 337 and properties of, 325-329 tissue, 322 Proteoses, antigenic property of, 525 Proteus 0X19, cross reaction for, 154 Proteus X19, use of, in diagnosis, 389 Protoxoid, definition of, 251 Protozoa, metabolism of, 34 Provocative Wassermann, 432 Pseudoagglutination, phenomenon of, 184 Pseudoreactions, Schick test and, 255 Ptomaines, nature of, 35 Puerperal sepsis, causes of, 81, 282, 283 Pulmonary infection, factors in, 77, 78 Puncture tests, 571 Pus, definition of, 55 Pyelonephritis, factors in, 82 Pyemia, definition of, 31 Q Quarantine laws, object of, 49 Quellung reaction, description 395, 39G of. E R and 8 colony types, 37 Rabbit serum, effect of formalin on, 344 hypersensitiveness to, 308 intracutaneous test for hyper- sensitiveness to, 308 test for hypersensitiveness to, 308 Rabbits, anaphvlactic shock in, 479, 480 " experimental tuberculosis, immun- ity in, 544 streptococcus infection in, 128, 129 variation in antibody production in, 385 Rabies, effect of animal passage on, 37 Racemization, effect of, on antigens, 326 Eacemized proteins, properties of, 327 Racial freedom from disease, causes of, 50 immunity, 102 Ramon tlocculation, effect of pH on, 250 of temperature on, 250 soui'ce of precipitate, 251 test, absence of Danysz phenome- non in, 250 reliability of, 250 Rats, anapliylaxis and, 482 insusceptibility to diphtheria toxin, 243 zone phenomenon in, 252 Reaction, age and toxoid and, 260- 263 Brodie, in cats, 312 chemical vs. colloidal, 585 crop, 484 delayed serum, 309 factors in cell sensitization, 162 serum, 306-316 yhvvartzman, 552 Reagins, atopic discovery of, 150 biological or normal, 435 comparison with antibodies, 563 definition of, 560 distribution of, 149 effect of atopen injection on, 563 of heat on, 564 facts about, 562 naming of, 150 natural lipoidophilic, 150, 151, 434, 435 passive transfer of, 560 precipitin tests for syphilitic, 444- 468 tests for lipoidophilic, 444-468 verification test for normal, 457 Recapitulation of specificity chapters, 374-378 Red cell haptens, nature and distribu- tion of, 185 nature of, 195 suspension, unit of, 416 cells, proteins of, 322 unit of, 202 Refractory state, reasons for, 476, 477 Relationships, host-parasite, 47, 74, 133, 134, 542 Resistance, age and, 105 antibodies and natural, 109 factors in, 50, 51 Neufeld 's theory of, 107 nutrition and, 106 vitamins and, 105, 106 Resolution in pneumonia, mechanism of, 55 SUBJECT INDEX 631 Respiratory defense, Blooiiificld 's tlio ory of, 71 infection, method of spread, 47 tract, agents affecting defense of, 133 defensive mechanisms of, 71 Reticulo-endothelial system, 87 blockade of, 94 classification, 88 function of, 93 Reticulum cells, 89 Revaccination, indications for, 120 Reversed anaphylaxis, 477, 480, 484 Rheumatic fever, allergy and, 551 Richet and Portier, anaphylaxis and, 470, 471 Ricinus communis toxin, 35 Rickettsiae, immunization against, 110, 111 King technique, precipitin test and, 201 test, limitation of, 229 range of specificity, 229 technique of, 228 Zinsser's reasons for diluting antigen, 235 Robinis pseudoacacia toxin, 35 Romer, intracutaneous technique of, 254, 571 Rooster sera, use in precipitin test, 226 Rosenau and Anderson, anaphylaxis, studies of, 471 Rosenthal test, 40(5 Rough and smooth colonies, Bordet 's studies of, 43 Rouleaux formation, 184 effect of dilution on, 184 inhibition of, 184 Routes of dissemination, infection and, 74 S •Sabin's views on monocytes and clas- matocytes, 57 Sachs-Georgi reaction, 465 Salmonella aertryeke, antigenic carbo- hydrate-phosphatide com- pounds from, 115 enteritidis, antigenic carbohydrate- phosphatide compounds from, 115 relationship to E. typhosa, 391 group, antigenic pattern, 384 Salt concentration, effect on agglu- tination, 214, 215 formation, ways of, 342 Salt-forming groups, antigens and, 341 Salts, effect on precipitin test of, 233 Sanitary measures and disease con- trol, 49 Sauer's vaccine, 116 Scar tissue, formation of, 57 Scarlatinal antitoxin, government le- quirements for, 280 therapeutic dose of, 280 Scarlet fever antitoxin, measurement of, 201 effect of antitoxin on, 277 liemolytic streptococcus and, 271!, 277 inununization against, 118 natural immunity in, 102 prophylactic ' immunization, 278 results of immunization against, 278 Schultz-Charlton test in, 277 serum treatment in, 280 streptococci, confusion over clas- sification, 277 fractions of, 277 toxin, 35 types of, 280 whole blood in treatment of, 28] Schick test, history and definition of. 255 interpretation of, 265 recommendations of Taylor and Maloney, 255 results of, after immunization, 260-264 Schick toxin, j)seudoreactions and, 255 susceptibles in New York, 259 time of disap}>earance of, 255 use of, 119 young adults and, 256 urban and rural populations and, 255, 259 Schilling counts, 61-65 hemogram, 64 phase I, 65 phase II, 65 phase III, 65 School cliildren, tuber<'ulin surveys in, 520, 521 Schultz-Charlton blanching test, defi- nition, 277, 279 Scratch tests, 571 Sensitization, factors in cell sensitiza- tion, 162, 4S(; in utero, 562 nature of antibody in, 486 Sensitized cells, definition of, 146 632 SUBJECT INDEX Sensitizers, definition of anaphylac- tic, 151 hemolytic unit, 202 Sepsis, puerperal, definition of, 81 Septic infection, leucocyte count in, 61, 64 Septicemia, definition of, 31 factors in, 283 immuno-transfusion in, 283 streptococci and, 283 Sera, convalescent and immune, 276- 305 fractions of, 195, 196 inactivation of, for Kahn test, 453 some diseases used in, 276 use of, in virus disease, 289 Serodiagnostic tests, corandttee of U.S.P.H.S. on evaluation of, 430 Serological classification, divisions in, 351, 352 errors due to presence of extrane- ous material, 353 lack of standard procedure for, 352 diagnosis, Kilduffe's basic prin- ciples in, 431 methods, limitation of, 351 Serum, inactivation of, 487 reactions, 306-316 acquired hypersensitiveness and, 307, 308 antibacterial sera and, 308 chill-producing factor in, 306, 307 classification of, 306 delayed, 309 immediate, 306, 307 incidence of alarming symptoms and death in, 307 ophthalmic test for, 308 specific, 307 status lymphaticus in, 306 test for hypersensitiveness, 308 toxin-antitoxin and, 308 sickness, 309 atopic reagins and, 311 j3 isoagglutinins and, 311 discussion of antigen-antibody theory of, 311-314 heterophile antibodies and, 311 incidence of, 309 incubation period, 313 lower animals and, 310 precipitins and, 310, 311 relapses in, 309 theories as to, 310, 311 various sera and 309 Serum — Cont 'd therapy, mechanism of, in pneu- monia, 287 treatment, results in pneumonia, 286 Shift to the left, degenerative, 64, 65 regenerative, 64 significance of, 64, 65 Shiga dysentery bacilli, endotoxins of, 34 Shigella dysenteriae, studies on an- tigen complex of, 364 Shock organs, allergy and, 559 specific serum reactions and, 307 Shwartzman, reacting factor, 552 reaction, general, 552 nature of, 552 preparatory factor, 552, 553 Side chain theory, Ehrlich's, 197, 19S Simple chemical compounds, anaphy- laxis and, 472 Sinuses, defensive mechanism of, 71, 72 type of epithelium of, 71 Skin, bacteria on, 47 barrier to invasion, 70 reactions, anaphylactic vs. tubercu- lin, 528 recording allergic, 577 reference to lymphatics of, 281 tests, Brucellosis and, 387 classification of food allergens and, 567, 568 description of Francis test, 397 for antibodies, 397, 398 intracutaneous, 254, 571 introduction by Blackley, 565 M.E.D., and L^ dose of toxin in, 254 scarlatinal toxin and, 279 Smallpox, Kansas City epidemic data of, 111 vaccination against. 111, 112 Smooth muscle, crop reaction of, 484 Soil, pathogenic agents in, 47 preparation for infection, 81 Solubility of precipitates, 593 Soluble specific substances, bacteria and, 354 chemistry of, for Types I to XXXII, 357, 358 of pneumococcal, 356-358 reference to Brown's studies of, 285 relationship of Type XIV to group A substance, 358 Solutions, size of molecules in, 584 SUBJECT INDEX 63^ Somatic antif^eus, common to E. typhosa and other or- fi^anisras, 391 Soiidoiii 's interpretation of leuro- eoevte count, fil SOT and SOTT or PPD, nature of, 307, 509 .Spatial lelationships, antigens and. Species, bacterial tvpes witliin, 320, 321 differences within, 375 homogeneity and, 351 immunity, 102 rehitionsliip, precipitins and, 320 serological classification and, 351, 352 types and, 351 types within, 320, 321 Specific carbohydrates, V. cholerae and, 364 microbic association, examples of, 31 I'eactions, natural allergy and, 307 soluble substances, bacteria and, 41, 285, 356-358 substances, bacteria and, 354 surface, 586 Specificity, biological and antigenic, 317-331 chemical constitution and, 348 examples of, in nature, 374 factors in antigenic, 321 fertilization and, 317 genetics and, 318 hormones and, 318 immunological, 317, 319-321 incompatability of species and, 317 Landsteiner 's views, 195 nonspecies, 322, 323 organ, 322 position isomers and, 340 precipitin test and, 225 precipitins and, 320 role of haptens in, 234 spatial relationships and, 339, 377 species and type, 375 vs. immunological, 350 sterioisomers and, 377 Speed of mixing, effect on colloids, 593 Spinal fluid, effect of serum on coll count, 289 Spontaneous bovine tuberculosis, studies o f Theobald Smith, 541, 542 S])or('s. factors affecting germination in infection, 295 Stab forms, description of, 63 Standard reagents, Kolmer's sugges- tion for, 415-417, 428-431 Standardization, toxin and antitoxin and, 247 Staphylococcus, experimental infec- tion with, 129 polysaccharides in, 3(16 Stem cells, function of, 91 Stereoisomers, antigens and, 339 Stone in ureters, result of, 82 Streptococcus, Dochez N Y 5 strain of, 278 erysipelas and, 281 erysipelatis, studies of, 282 experimental infections with, 128 group and type specific substances of, 365, 366 hemolytic scarlet fever and, 276 infection, antibodies and, 129 passive imnmnization and, 129 Lancefield 's grouping of, 365, 366, 396 partly labile antigens of, 38 rheumatic fever and, 551 scarlatinal toxin, unit of, 279 Strychnine, antibodies to, 348 Subgroups, blood groups and, 179- 181 importance of quantitative differ- ences in, 179 Submaxillary glands, ducts from, 70 Sulfonamide compounds, use of, 288 effect on Lancefield group D, 288 references to, 303-305 use in endocarditis, 288 Suppression phenomenon, diiodotyro- sine and, 344 explanation of, 358 Heidelberger and Kendall 's ex- planation of, 234 Landsteiner 's, 376 description of, 234 Suprarenal cortex, function of, 103 involution of, 103 Suprarenalectomized rats, effects of toxins on, 104 Surface antigens, masking effect of, 195 coating, requirement for agglutina- tion, 217 potentials, 587 tension, 5S() wetting, 589 Sui'gery, vasomotor ihinitis, iji}(} Surveys, tuberculin, using OT and MA-100, 517-520 using PPD, 521 634 SUBJECT INDEX Susceptibility, factors in, 50, 51 mechanisms of, K'i4 Suspension, red cells, 202, 416 Suspensoids and emulsoids, 585 Swallowing reflex, impoitance of, 71 Swine influenza, etiology of, 31 Symbiosis, 29 Synergistic substances, flocculating antigens and, 447 Synthetic medium. Bureau of Animal Industiy's, 505 Long's, 504, 505 toxin formation and, 24G Syntoxoid, definition of, 251 SjT)hilis, cerebrospinal lues, 42.'! complement fixation in, 419-443 eifect of malaria on, 31 of malarial or diathermy treat- ment on, 433 etiology of, 419 history of, 419 serology of, 423 immunity to, 433, 434 incubation and primary lesion of, 419 Kilduffe's basic principles and, 431 laboratory diagnosis in primary stage, 419, 420 methods of spread, 419 naming of, 419 Osier's saying as to, 421 paresis, 423 periods of latency in, 422 precipitin tests in, 444-468 serological results in treated, 421 serology in untreated, 421 of tertiary, 422 summary of complement fixation in, 436-438 tabes, paresis, and cerebrospinal, 423 third generation and, 419 transmission by transfusion, refer- ence to, 439 umbilical bloods in, 422 value of blood tests in primary stage of, 420 various names for, 419 Wassermann 's original technique, 424-426 Syphilitic reagin, eft'ect of treatment on content of, 432 history of precipitin tests foi-, '444 importance of more than one test for, 466 incidence of, in piimarv stage, 420 Syphilitic reagin — Cont M principles of floeculation tests for, 445, 446 specificity of, for Treponema pallidum, 435 status of floeculation tests for, 445 Systems, homogeneous and lieterogen- ous, 584 T Tabes, serology of, 423 T.A.T., determination of suscepti- bility with, 256, 259 dosage of, 259 history of early use of, 258, 259 preparation of, 259, 260 Theobald Smith 's studies with, 258 use of goats in preparation of anti- toxin for, 260 Teclinique, agglutinin titer and, 201 basic requirement, 205 empirical requirement, 205 interfacial tension, 217, 218 of Mudd, 217, 218 measurement of cohesive force, 21() optimal proportion, 230-232 original Bordet-Gengou, 401, 402 precipitin titer and, 201 lecommendations of Surgeon Gen- eral's Committee, 205 Widal, 380, 381 'I'eeth, apical infection of, 72 Temperature, importance of, in hem- agglutination, 181 Tendon sheaths, importance of, 77 Tertiary syphilis, serological findings "in, 422 Testiculai- protein, nonspecies speci- ficity of, 322, 323 Tests, diagnosis of allergy by, 570- 573 Dick, 278, 279 floeculation, antigens employed, 447-449 floeculation tests in syphilis, 444- 468 grading of Mantoux, 522 liypersensitiveness and rabbit se- rum and, 308 iiitra<'utaneous, for serum hyper- sensitiveness, 308 Schultz-(;harlt(m, 277, 279 Tctaiuis, Abel's tlieoiy of toxin patli- way, 33 active-passive immunity, 264 SUBJECT INDEX 635 'I'etaiius — Coiit M antitoxin, reasous i'ur iut'tfectivo- ucss of, 29;> unit of, 200, 201 cause of, o3 discovery of etioloyic agent of, 242 etiology of, 2!l.'I propiiylaxis, IK!, US, 2!):; theories of nie<'lianisnis in, 293 therapy ami antitoxin, 2()4, 29.''> toxin, discovery of, 242 pathwav to cord, 293 to CNS, 33 toxoid, immunization with, 11(5, 118 Theobald Smith phenomenon, 471 Theories of adsorption, 162 Theory, Arrheuius and Madsen 's, of toxin-antitoxin, 252 as to filterable forms of bacteria, 43 Bernstein's triple allelomorph, 178 Bordet's, of toxin-autitoxin, 252 two phase, 209 Breinl and Haurowitz, of antibody formation, 198 Duclaux's, on mechanism of coagu- lation, 209 Eaton's bacterial toxin formation, 245 Ehrlich 's, of toxin-antitoxin, 252 Gruber's, of agglutination, 200 Heidelberger and Kendall 's, of precipitin reaction, 212, 213 humoral, 143 Manwaring's of anaphylaxis, 312 mechanisms of anaphylaxis and, 492, 497 monophyletic, 91 Nageli "s, as to lack of constancy of characters, 41 Neufeld's regarding resistance, 107 of constancy of characters, 41 polyphyletic, 91 Sabin's, of antibodv formation, 198 serum sickness and, 310, 311 unitarian, of antibodies, 198, 199 Therapeutic agents, antitoxins, 257 Therapy, underlying principles of, 84 Thermal reactions, 3>0(i Tliiocvanates, etfect on precipitin test of, 233, 2.".4 Thyroglobulin, antigenicity of, 323 Tillett and Francis, C substance of. 359 Timothv bacillus, chemistry of, 3i55 Tissiic extiacls, tiocculalion tests and, 447 iuununily, 12S-I40 pi-oteins, specificity of, 322 summary of Cannon's work, 130 Tissues, establishment of bacteria in, 74 Titer, diagnostic tularemia, 383 tyi)hoid fever, 380 undulant fever, 383 Widal test, 380-382 variation in, 414 agglutinin, 414 hemolysin, 414 infection and, 415 Titration, hemolytic, anticomplement- ary, antigenic, of bacteri- al, 406-408 precipitin after Nuttall, 227 after Uhlenhuth, 227 Tolerance, in toxemic diseases, 139 Tonsillar ring, nature and importance of, 71 Tonsils, drainage of, 75 Toxemic diseases, examples of, 32, 33 tetanus, 293 Toxin, and antitoxin, intracutaneous titration of, 254, 255 discovery of bacterial, 242 doubt regarding meningococcus ex- otoxin, 289 effect of diphtheria on animals, 242, 243 of physical and chemical agents on, 24() Ehrlich 's spectrum, 252 high potency production of, 246 measurement of, by Ehrlich, 247, 248 M.R.D. and L, doses of, 254 Ramon 's method of titrating, 248 source and nature of diphtheria toxin, 245 standardization of, bv flocculation, 249 tetanus, pathway to CNS, 3;; unit of streptococcus scarlatinal, 279 Toxin-antitoxin, flocculation, source of precipitate, 251 specificity of, for toxin, 251 reaction, Ehrlich 's theory of, 251, 252 serum reactions and, 30S Toxins, action of, in gaseous gan- g?-ene, 295 636 SUBJECT INDEX Toxins — Cont 'd and antitoxins, 241-275, 276-305 bacterial, discovery of, 32 definition of, 33, 241 effect of CI. welehii and others on tissues, 297 flocculation of purified, 250 gaseous gangrene and, 295 history of active immunitv studies of, 258, 259 incubation period of, 29 ti insusceptibility of mice and rats to, 243 kinds of, 241 optimal proportions of ricin and antiricin, 230 proportions of venoms and anti- venoms, 230 pathology in lower animals due to, 243, 244 phytotoxins, 34, 35 similarity of, from streptococci of erysipelas and scarlet fe- ver, 278 symptoms in susceptible animals caused by, 243 theory of incubation period, 243 true, 241 zootoxins, 34, 35 Toxoid, age and reactions to, 260, 263 alum, 263 dosage of, 265 diphtheria, 117 elimination of, 264 history and method of preparation, 261 history of, 247 immunization with, by inunction, 263 immunized with combined, 263 number of injections necessary, 264 plain, dosage. New York Depart- ment of Health, 265 preparation of, 247 Eamon, 261 specifications of U.S.P.H.S., 262 tetanus, 116, 118 Toxone, definition of, 251 TPT, Seibert's, 507 Trachea, function of cilia, 133 lymphatics of, 76 Transfusion, immuno-, 281, 283 reference to transmission of syph- ilis by, 439 Transpleural mobilization, protection and, 128 Trauma, definition of, 72 Treatment, use of active-passive im- munity in tetanus or diph- theria, 264 Treponema pallidum, demonstration of, 420 effect of malaria on infection with, 31 Triple allelomorph theory, 178, 179, 188 vaccine (T.A.B.), use of, 115 Trypan blue, effect on anaphylaxis, 490 Trypanocidal effect, zone phenomenon and, 252 Trypanosome lewesi, 30 antibodies and, 152 effect of immune serum on, 253 Trypsin, anaphylaxis and, 476 Tryptophane, iodizing of, 333 T : S ratio, in normal and immune an- imals, 132 Tubercle bacillus, increase of viru- lence of B.C.G., 549 tissue reactions to fractions of, 524 varieties of, 59 formation of, 57 phosphatid stimulation and, 59 role of, in tuberculosis, 542 Tuberculin, active substance of, 505, 509 allergy, and anaphylaxis, possible explanation of dissimilari- ties, 526, 527 antigen-antibody mechanism the- ory, 529, 530, 532 Birkhaug's views on, 541 comparison of, with immune re- sponse, 528, 532 comparison witli anaphylaxis, 526-532 Dienes' work on, 526, 527, 528 discussion of antigen-antibodv concepts of, 530-532 effect of histaminase on, 532 epithelium and antibodies, 531 general conclusions concerning, 532, 533 intravenous vaccination and, 541 necessary factors for, 524-526 chemical nature of PPD, 509 diagnostic tests with, 515, 516 dosage of MA-100 employed, 517 of Mariette and Fenger, 519 effect of age upon, 522 fractions of, 354 importance of molecular weights of, 508, 509 SUBJECT INDEX 637 Tuberculin — Cout 'd inhibition of precipitin roaetiuu by, 530 isolations of fractions from, 531, 532 kinds of, 504, 509 Long's synthetic medium and, 504 Mantoux test with, SKi methods of production, 515 moleeuhir weight of, 354 Moro's percutaneous test with, 510 objections to OT, 504 OT, BE, and TR, 504 patch test with, 510 von Pirquet test with, 515, 510 precipitin reaction and, 354 properties of, 353 active substance, 505 reaction. Babes and Proca "s ex- planation of, 529, 530 Bordet's explanation of, 529 comparative statistics, 519 conclusions of Mariette and Fen- ger, 519 evidence of infection attack rate from, 520 grading of Mantoux tests, 522 incidence in normals, 518 of human or suspected cases, 518 Koch's explanation of, 529 Mariette and Fenger's results, 518, 519 miliary tuberculosis and, 518 negative reactors witli tubercu- losis, 518 non-correlated i-eactions, 518 von Pirquet and Schick's theorv of, 530 reading of, by Funk and Hunt- son, 517 results wdtli PPD, 521 site. of, 530 Slater and Jordan 's survev le- sults, 520 Seibert's SOT, SOTT or PPD, 507 views on structure of, 525 SOTT, molecular weight of, 530 source of, 40 spermatocyte unit of, 514 standardization, advantages and intracutaneous method, 513 classiticatiou of methods of, 510 complement fixation in 512 criticism of complement fixation method, 513 Tuberculin standardization — Cont'd Dreyer and Vollum 's method, 511 Frankfurt method of, 510 Koch's method of, 510 Long's spermatocyte method, 514 objections to lethal tests, 512, 513 skin test method of, 511 U. S. Bureau of Animal In- dustry method, 511 studies on molecular weight of, 50!' surveys with MA-100, 508, 517-519 systemic and skin reacting frac- tions of, 509 test, effect of labor and puerperium on, 518 exanthematous disease and, 518 TPT of Seibert, 506 variation in antigenicity of, 525 Wolff-Eisner's test with, 516 Tuberculo-anaphylaxis, Corper's com- parison with tuberculo-al- lergy, 532, 533 Tuberculo-carbohydrate, tissue re- sponse to, 524 Tuberculo-phosphatid, tissue reactions to, 524 Tuberculo-phosphatids, composition of, 356 Tuberculo-protein, tissue response to, 524 Tuberculosis, allergic theory of im- munity in, 539 allergy vs. immunity in, 539-547 BCG vaccine and, 548-550 complement fixation in, 413 development of hypersensitiveness, 59 discussion of allergy and immunitv in, 546, 547 experimental studies of, 541-546 factors causing localization, 545 hypersensitiveness in, 504 immunity in, 538 immunity mechanisms in, 544-546 immunization against, 112, 113 incidence determined by autopsv, 523 infection attack rate, 520 Krause 's theory of, 539 Lurie 's study on allergy and im- munity in, 544-54() nonprogressive primarv lesions and. 543, 544, 547" Park's studies on BCG, 549, 550 possible site of antibody produc- tion. 531 638 SUBJECT INDEX Tuberculosis — Cent M primary lesion in cliildien and, 547 type in, 538 leduction of, 522 Rich 's theory in, 539 spontaneous bovine, 542 studies of allergy and inununity in, 541 summary of Lurie 's studies on, 544 surveys, 517-521 Mariette and Fenger's, 508 Theobald Smith's studies on, 541- 543 tuberculin desensitization and, 540 value of skin test in, 518 vole vaccine and, 113, 550 Tularemia, 298 agglutinin response in inuuunized rabbits, 388 cross reactions in, ;'>90 infection in normal animals^ 389 nature of, 388 Tunnicliff's immune serum, value in measles, 290 Turgensen, statistics of, on smallpox vaccination, 111 Turtles, anaphylaxis in, 483, 484 2-4 dinitrochlorobenzene, anaphylaxis and, 473 Two-phase theory, Bordet 's, 209, 210 Tympanitis, value of, 138 Tyndall effect, 585 Type I SSS, 357 II SSS, properties of, 357 III SSS, 357 XIV pneumocoecus, relationship to group A substance, 358 specificity, haptens and, 234 Types, species and, 320, 321 Typhoid, bacteria, young plasma cells as host cells for, 44 fever, effect of vaccination on, 115 incidence in armies, 115 urinary excretion of Shwartzman substance in, 553 vaccination against, 114, 115 young plasma cells in, 134 inoculation, agglutinin response to, 384, 385 protein, Pj and Pj fractions, 41 Typhosus, bacillus, antigenic proper- ties, 115, 121, 3()3, 391 P, and P, fractions, 41 Typhus fever, Felix- Weil reaction and, 389 Tyrosinediazoarsanilic acid, modified antigens and, 346 U Ulcer, common location of, 79 Ulceration of intestine, diseases caus- ing, 79 Ultracentrifuge, use of, in virus stud- ies, 40 Umbilical bloods, serology of, 422 Undulant fever, laboratory proced- ures in, 387 Unit, Dean and Webb 's optimal pro- portion, 232 Pfeififer's immunity, 144, 145 Unitarian theorv of antibodies, 198, 199 United States Army, typiioid in, 115 Urban and rural populations, varia- tion in susceptibies in, 255 Uremia, factors in, 82 Ureteral impairment, result of, 82 Ureters, impairment of, 82 Uterus and cervix, factors lowering resistance of, 81 infection of, 81 Vaccination, antibody response and immunity, 385 army and, 115 data on, 120 programs f oi-, 117, 1 18, 264, 265 results of intranasal, 132 Kickettsiae immunity and, 110, 111 type of agglutinins in, 385 variation in agglutinin titer follow- ing, 384, 385 virus immunity and, 110, 111 Vaccine, attenuated bacteria as, 112 autogenous, 113 bacterial polvsaccharides as, 115 BCG, 548, 549 influenza virus and, 112 killed bacteria in, 113 mixed, 113 original meaning of, 113 pertussis, 116 preparation of, from virus cultures, 111 the "vole tubercle bacillus," 550 value of Br. abortus, 551 value of "cold vaccines," 114 Vaccines, antigenic variation in, 120, 121 bacterial, use of, 114 specific immunization witli, 114-118 Vaccinia, inmiune serum in, 292, 293 Variability, fluctuating, 42 SUBJECT INDEX 639 X'aiiula, use of convali'scont scniiii in, 292 N'Msnmotor rhiuitis, t'at'ts about ]iei- eniiial, 566 Vegetable proteins, antigenic content, 324 Wells and Osbnine 's work on, 324 Verification test, Kahn's, 435, 457 Vernes ' test, 465 Vi antigen, 121 inagglut inability and, 384 presence in Halnionella paratyphi G, 38 role of, 351 N'ibrio cholerae, antigenic composition of, 364 Virulence, capsule formation and, 37 correlation of colony type and, 37 definition of, 36 factors altering, 37 host factors and, 39 measurement of, 36 Virus disease, use of serum in, 289 fixation of, by lymphocytes, 72 infiuenzae, immunization with, 13 2 Viruses, age and resistance to, 105 chemical analyses of, 39 defense against, 139 immunization against, 110, 111 nature and characteristics of, 39 result of x-ray studies of, 40 size of, 39, 40 Stanley's work and, 39 theory of origin of, 40 tissue response in, 59, 60 Vital staining, 88 mechanism of, 89 Vitamins, resistance and, 105, 106 \'ole vaccine, ilata on, 113 tuberculosis immunity and, 550 W Wassermann antigen, introduction of cholesterol, 427 normal reagin and, 150, 151 stages in improvement of, 427 dispute over specificity of, 435 provocative, 432 reaction, mechanism of, 435 snake complement and, 161 test, early history of, 423 early technique, 424, 425 introduction of alcoholic tisstie extracts, 426 Wassermann test — Cont 'd Kolmer's antigen for, 429 modification of, 428 nonspecific reactions, 434, 435 paroxysmal hemoglobinuria and, 'l50 reporting results of, 430 stages in development of, 426- 428 titration of Kolmcr antigen for, 430 value of, in primary stage, 420 various modifications of, 427 Wassermann-fast cases, 432 Welch, hypothesis of, 37 Well's criteria of anaphylaxis, 485 Wherrv's tissue hydration theorv. 74 White cells, different kinds, 56 Whooping cough, vaccination against, 114, 116, 117 Widal, antigens in, 379, 380 controls in, 382 cross reactions with Salmonella organisms and, 384 errors in, 382 interpretation of, 381, 382 in vaccinated individuals, 386 microscopic method, 381, 382 _ normal and diagnostic titers, 383 organisms and, 384 percentage of positives in tvphoid, 415 techniques employed, 380, 381 time and temperature of incuba- tion, 386 use of H and O antigens in, 384 use of S. gallinarum or S. pullor- um in, 391 Vi antigens and, 384 Wounds, factors in gas gangren<», 295 tetanus bacteria in, 296 X X-ray, use in virus studies, 40 Y Yeast, gum, substances in, 325 soluble specific substance from, 325 Z Zone phenomenon, illustration of, 252, 253 Zootoxins, 34, 35 n [■■'yyj-M VmWltKKK