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Those too large to be entirely included in one exposure are filmed beginning in the uppwr left hand corner, left to right and top to bottom, as many frames as required. The following diagrams illustrate the method: Lea cartes, planches, tableaux, etc., peuvent Atre fiimAe A dee taux de rAduction diff Arents. Lorsque le document eet trop grand pour Atre reproduit en un seul clichA, 11 est filmA A partir de I'angle supArieur gauche, de gauche A droite. et de haut en baa. en prenant le nombre d'images nAcessaire. Las diagrammes suivants illustrent la mAthode. 1 2 3 1 2 3 4 5 6 ,. .I~i-W. i £2^ ■PWB REGENT STUDIES UPON IMMUNITY. Bit J. 6. ADAMI, M.A., M.B., M.R.C.S., Fellow of Jesus Oollege, Oambridge. *•* [Reprinted from the " JMedicai Chronide," Noyfmber and December, 1891.] JOBR BETWOOn, FBIKTKB, MANCHESTER AMD LONDON. £ 'f fcAi'^ <'<> RECENT STUDIES UPON IMMUNITY.' I. The jvolution of our bacterial knowledge has been so rapid during the last few years, that already the ideas prevalent early in the eiglities are matters of ancient history, and the theories witli regard to bacterial action and the nature of immunity which then obtained are wholly out of date. Yet these theories were useful in their day, and, if possessing no other value, they are useful now as niai'king the progressive advances of a young science. Based upon very scanty observations, these older theories were disproved so soon as they were put to the experimental test. The "exhaustion" theory of Pasteur and Klebs, that bacteria use up certain materials in the economy, and that consequently further invasion and proliferation is pi'evented by the absence of tlie food material necessary for the growth of these bacteria, was sliown to be insufficient ulien it was determined that the bacilli of anthrax would grow easily in tlie body fluids of animals rendered immune to the disease, and not only in the removed fluids, but also, under certain conditions, within the bodies of the same animals. And Chauveau's "retention" theory, which owed its origin to the fact that microbes grown outside the organism tend to be destroyed by the products of their metabolism, and which supposed that products of bacterial growth present in the fluids of the body pre- vent a second development of the bacteria, was found to be equally weak. It is in the highest degree improbable that such soluble substances should not bo gradually excreted during the course of a very few days or weeks. But apart from this antecedent improbability, a further difficulty arises from the fact already mentioned, that microbes can grow easily in the removed body fluids of animals rendered refractory. Even if it be taken that the bacterial products are insoluble, then, like mercury, arsenic, and other substances wliich remain unexcreted, they must be stored up in certain organs, and the immunity conferred by them can be but local, confined to the organ or organs in which they are deposited; it cannot be generalised. * In reprinting this article I have t.ikon tho opportunity to make one or two rcrbal altera- tions where it seemed to me that the original might be onsidered ambiguous. 2 And here must be mentioned another of tlie earlier theories, which may be said to date back to Eichhorn in 1829, and which has received more recent support from Buchner (1877) and Wolffberg (1885). This may be termed the theory of local immunity, and supposes that during infection the regions attacked by any specific disorder are so affected that the weaker cells are destroyed, while those which have survived the bacterial invasion transmit their resistant properties to their descendants. But granting that this occurs locally, in diseases which have a purely local manifestation, the supposition does not explain the immunity acquired by unaffected parts of the same organ. Why, for example, in ordinary vaccination, is not only the vaccinated area but the whole skin and mucous membrane rendered resistant to variola 1 And assuredly the theory cannot be applied to explain what may occur in conuectiou with diseases like anthrax, in which subcutaneous inoculation of the attenuated virus, the bacilli remaining localised, suffices not only to give immunity against the cutaneous malady, but also prevents the onset of the disease either by way of the respiratory or of the intestinal tract. These earlier theories must, therefore, be dismissed ; some wider generalisation is demauded, a generalisation resting upon a more solid basis of facts, and, as the discussion at the recent Hygienic Congress in London abundantly proved, the question at present exciting the greatest interest among bacteriologists is whether Metchnikoff's cellular theory can adequately explain the known phenomena of the cure of infectious disease and the production of immunity,* or whether, in addition to, or apart wholly from the action of cells, the action of the bodily humours must be invoked. Although the enthusiastic reception accorded to Metchnikoff at the Burlington House meeting may have been but a tribute to his ceaseless energy, a spontaneous and ungrudging acknow- ledgment of the respect due to him for his laborious studies upon the subject during the greutsr part of the last ten years, yet the heartiness of the welcome seemed almost to indicate that the bacteriological world is now nearly prepared to acoppt his theory with few reservations, and that, in fact, among thooe competent to weigh the evidence, phagocytosis has almost won the day. It is unnecessary nere to detail the observations upon intracellular digestion in the invertebrata and lower vertebrata which led Metchnikoff to study phagocytosis in its relation to the prevention of bacterial invasion; * The process of cura and resolution of infectious dise.ise and the processes conccrnod in tlie production of immunity are of tiie same order of affairs ; it is impossible to draw any sharp line of demarcation between the two. The conferment of immunity by means of attenuated virus ia attended by local inflammatory action, and often by a feljrilo state of short duration — such as Gamaleia has shown to be the inevitable attendant of successful inoculation of anthrax in sheep, and as, to come nearer home, constantly attends successful vaccination against variola: and similar disturbances attend the inoculation of powerful virus in animals already made refractory. Thus, to quote Metchnikoff, " immunitt/ U most often but ncovcri/ in operation from tin very onait of a disease." n nor need I give a resume of the separate researches of later yeure. It will he sufficient to state his theory as it at present stands, amplifying such portions of it as are seen to need fuller treatment. And the theory may be stated as follows : — (1) That two forms of leucocytes, present in the blood and lympii, the largo uninuclear " macrophages " and the sn^aller " microphages " which tend to the raultinuclear, are, under certain conditions, capable of incepting such bacterid as have gained entry into the system. (2) That the splenic corpuscles, endothelial colls, and sundry other cells of mesodermal origin, possess the same property, though it is called into action to a less extent. (3) That these phagocytes incept and destroy living and active microbes. (4) That the more acute the disease, the less the phagocytosis that can be determined ; the more chronic, the more extensive the phagocy- tosis ; or otherwise : the more virulent and rapidly proliferating the microbes, the less the potentiality of the phagocytes to incept and destroy them, and vice versa. (5) That this potentiality of the phagocytes depends upon the " chemiotaxis " exerted by the jjroducts of bacterial growth. In the case of the more vii'ulent microbes the leucocytes are repelled from the focus of infection ; there is a negative chemiotaxis, and thus, in the absence of phagocytosis, the proliferation of the microbes takes place without hindrance ; whereas, the less virulent microbes and their pro- ducts attract the leucocytes ; they exert a 2}ositive chemiotaxis, so that there is an emigration of leucocytes through the capillary walls to the focus of infection, and the leucocytes taking up the microbes arrest the infective process. (G) That the leucocytes and other mesodermal cells may become accustomed, and eventually attracted, to substances by which at first they are repelled, and thus a negative may be transformed into a positive chemiotaxis. Hence, as in acute zymotic diseases, the microbes may at first rapidly proliferate, and phagocytosis manifest itself only after a certain period, leading then to resolution of the disease. (7) That the cells having once acquired positive chemiotactic properties in relation to the products of any specific microbe retain and transmit these properties through a series of coll generations, the length of which varies according to the microbe, the extent of the primary reaction, and the idiosyncrasies of the individual. (8) That, consequently, immunity and the cure of disease are mainly brought about by the activity of special cells, the i^agocytes, — being primarily dependent upon the attraction existing between these cells and the products of bacterial metabolism. (9) Yet while this is the main factor it is quite possible, nay, probable, 4 that others, with the extent and nature of whose action we are nt present uimc(iuainted, aid in combating bacterial invasion. Among these may be mentioned the febrile reaction. The wise discernment that further obsorTatiuns and the consideration of a greater number of facts may lead to an expansion or modification of the theory has, it would seem, prevented MetchnikoiT from placing it in a succinct finite form, and so fossilising it. However, I believe that the above, while not Metchuikoffs "ipsissima verba," is a full and correct statement of his theory as it stands at present. In his last "Etude 8ur I'immunite, ' which has come into my hands after going to press, M. MetchnikofF introduces a further expansion of the theory ren- dering it yet more complete. To this modification I shall refer in the second portion of my paper. That the theory has shown itself capable of expansion is evident by the stress now laid upon chemiotaxis as com- pared with the standpoint of a little more tlian a year ago. It will be well to state more fully the observations which have led to this expansion. Already, in 1883, in his paper upon iutracellular digestion as per- formed by mesoderm cells of the invertebrata, MetchnikofF noted that these did not take up indiscriminately every particle that came across their path, but exercised a choice, incepting some and leaving others untouched. And in the succeeding year, when discussing the relation- ship of phagocytes to the anthrax bacilli, he attributed acquired immunity to the " progressive habit exhibited by phagocytes of assimi- lating substances which at first they avoided."* But until some law based upon numerous determinations could be laid down as to this selective action, these observations and the explanation of them could not be regarded as adequate, and there remained a distinctly weak place in the theory. It is to the botanists that we owe the first advance. Engelmann, in 1881, determined that certain chemical substances excite the lower motile organisms and showed that oxygen energetically attracts certain bacteria. Later, Stahl made a series of important investigations upon the Plasmo- dium of iEthalium septicum, an organism living in tan pits — in infusion of oak bark. Placed on the surface of a glass moistened with water, this Plasmodium remains motionless until a drop of infusion of oak bark is allowed to fall near to it ; now the plasmodium moves rapidly towards the infusion. Other liquids bring about a different result — thus, a drop of one-half per cent solution of glucose, placed so as to be just in contact with the organism, causes it to move rapidly away from the drop; and so it is with solutions of different salts. What is of especial interest is that Stahl found that if this experiment with, for example, the glucose solu- tion, be repeated frequently, the plasmodium no longer flies away; but, on * Slctchnlkoff, Virehoto't Archiv, Bd. XCVII, p. 518, 1884. the contrary, move?, at lust, towards tho li(iuiil, just as it does i»rimarily in tho cnso of tho drop of iufusion of oak bark. Passing over certain observations of do liary we como to numerous researches made since 1885, by W. Pfoffor, to whom espoci'illy we owo our knowledge of chemiotnxis among the lower plant furms ; wiiile equally important though not so extensive aro tho researches of Rosen, who pointed out that tho spores of C'lytridium zygnomatis are attracted by the prockicts of decomposition of tl o cells of Zygnema ; of Zopf, upon the attraction of zoospores of certain Chytridiacea) towards poUon grainn, and of Stange upon tho cheralotactlc movemouts of Myxomycetes and the zoospores of Saprolegnia. Pfeffer showed that certain substances exert an almost specific action upon some vegetable organisms; that, for instance, malic acid has a remarkable attraction for the seminal filaments of ferns and solaginoUais ; that nutritive media exert a similar action upon motile bacteria ; and, again, that this chemiotaxis is determined by the specific chemical nature of the substance, and not by movements of diffusion of tho liquid. Continuing his observations upon chemiotaxis in connection with bac- teria, ciliate infudoria and volvocinete, Pfeffer made out that a positive chemiotaxis is exerted by peptone (energetic), asparagin, creatin, taurin, sarcin, carnin, urea (feeble), dextrin, and for certain organisms (and here to a high degree), glucose; also in another class of substances by sali- cylate of sodium and morphine; while a negative chemiotaxis occurs with free acids, alkalies and salts (lactate of iron and zinc sulphate). If then the lowest organisms exhibit such chemiotactic properties, it became a matter of considerable interest to observe whether, by using similar means, like peculiarities could be determined for the leucocytes — cells whose every property finds its counterpart among those possessed by the amoeboid animal and vegetable organisms. The first definite observations made in this direction were those of Pekelharing in 1889.* Introducing under the skins of frogs small masses of cotton wool, some moistened with indifferent fluids, others with anthrax cultures (the frog being refractory to anthrax), and removing these after some hours, Pekelharing found that tho wool impregnated with the bacilli contained very many more leucocytes than did that steeped originally in indifferent fluids. A few months later, in February, 1 890, Massart and Bordetf pub- lished what is really the first, as it is tho most important, systematic re- search upon the excitability of the leucocytes. These observers employed what was essentially Pfeffer's method, and what was, it may be noted, but a modification of that introduced some years ago by Councilman to deter- mine an allied question, namely the formation of pus by irritants, such * Pekelhnrlng. La Smaine Mhlieale, No. 22, 1880, p. 184. + Massart et Bordet. Journal de la Societc KoyaU, des Science! MedicaUt et Katuralea de Bruxdles, 18!)0. as turpentine and oil of mustard, in the absenco of bacteria,* Into tho abdominal lymph apnces of frogs thoy introduced capillary tubefi, closed at ono end, and tilled with the substance whose action was to be examined. They employed f )r their observations cultures of staphylococciis pyogenes albus, and of tho bacilli of fowl cholera, enteric fever, anthrax, etc., and at tho end of twenty-four hours they found tiiat musses of leucocytes had entered tho tubes. Cultures of the staphylococcus were pre-eminent in attructinfi; the leucocytes ; tho j)ure nutrient media, on the other hand, had comparatively little attractive power ; hence it is the bacteria or their products which call into play a positive chemintaxis. That it is tho soluble products was shown by employing the sterilised fluids from which tho microbes had been removed by filtration. Thus Massnrt and Bordet concluded that the products of bacterial metabolism attract leucocytes towards the focus of infection. They further discovered that chloroform and chloral hydrate temporarily suspend the movement of the wandering cells towards tubes containing positively chemiotactic me chloroform (watery solution), jequirity (2 per cent), glycerine, bile, bacillus of fowl cholera. (2) Indifferent, exercising a faint attraction : dlHtilled water, weak solution of sodium and potas- sium salts, carbolic acid, ontlpyrine (1 per cent), phloridzine (1 per cent), glycogen (1 per cent) peptone (1 percent), beef broth, blood, aqueous humour, carmine in siispension. (:!) rossesslng a positive chemiotaxis : sterilised and non-sterilised cultures of pathogenic and non-pathogenic bacteria— staphylococcus, B. pyooyancus, B. anthracis, B. typhoid abd., B. prodigiosus, etc. 8 permits me to refer. He finds that the serum of a young rat has just as much power of attracting the leucocytes of the mouse as serum coming from an adult rat. Nevertheless, the latter has the power of preventing the development of anthrax in mice. Serum from a very young rat can exhibit no such action. While acknowledging that the evidence is now strongly in favour of Metchuikoff's contention that a negative may eventually be changed to a positive chemiotactio state, it is hard to see in this a complete elucidation of sundrv difficulties. Take, for instance, a case that Metchnikoff has made his own— the case of recurrent fever. Here, during the access of the fever, spirilla swarm in the blood, while none are to be discovered within the leucocytes or other phagocytes ; the crisis occurs, and in a very few hours, not a single free spirillum is discernible, but a fair number are to be seen within the cells of the spleen, there undergoing degeneration. It is difficult to comprehend that a negative has been so abruptly changed into a positive chemiotaxis. It is difficult not to opine that there is some factor added to and beyond chemiotaxis and phagocytosis, determining the crisis in acute zymotic disease. I have already laid stress on the fact that Metchnikoff freely admits the possible action of some other factor or factors not as yet included in his philosophy, and here it seems to me that other factors must be invoked. Otherwise, the more one studies the phagocyte theory and compares it with those which have been brought forward within the last year or two to replace or augment it, the more, as I shall proceed to show, one sees that it fulfils the conditions of a good theory and embraces the widest circle of phenomena. II. The literature which has owed its origin to Metchnikoff's studies upon, phagocytosis and immunity has already attained to such proportions that it is impossible within the limits of a comparatively short article to do more than touch upon some of the main points that have been raised, and to attempt to show wherein is the strength and wherein the weakness of the counter theories. Undoubtedly phagocy^-es are not always readily determinable. Leucocytes in general, as all know who have studied films of pure blood under the microscope, break up with extreme ease. Thus, unless special precautions are taken, and special methods employed, it is quite possible to see only free micro-organisms in the field, when, previous to trans- mission to the slide, all had been confined within phagocytes. Thus, whereas a few years ago there were many prepared to deny that phagocytosis occurred, now, when the methods to be employed are more 9 V'' fully understood, there are few or none who deny its frequent occurrence, and the question is not so much whether phagocytosis plays a part in the prevention of disease, as whether it plays the most important part— whether there are not other factors equally or more impoiiant. It is but natural that when the ph:igocyto8is observed was insufficient to account for the number of microbes that had undergone destruction, attention should have been turned to the possible bactericidal action of the blood plasma, and of the humours of the body in which this destruction had taken place. And many workers were led to turn their enquiries into this direction, first among whom may be mentioned von Fodor, Emmerich and di Mattel, Nuttall, Buchner, Nisseu, and Lubarsch. In 1887 von Fodor* found a gradual diminution in the number of bacilli to be obtained by making successive plate cultures at short intervals after inoculating sterile rabbits' blood with growths of anthrax. The blood evidently exercised a bactericidal property. In the following year Nuttallt published some much more extensive and satisfactory obser- vations. Taking the blood serum of the frog in the first place, and afterwards employing that of a long series of animals, including man, he showed that by carefully observing the micro-organisms immersed in the fluids upon the warm stage of the microscope it was possible to follow the gradual degeneration and death of the micro-organisms in the absence of any cellular action. He found that a temperature of 55' C. destroyed the bacteria-killing power, and was of the opinion that serum and other body fluids owed their bactericidal eff'ect to the presence of some ferment. This paper was followed by another from the same laboratory at Breslau, in which Nissen; showed that numerous other pathogenic and non-pathogenic bacteria were destroyed by defibrinated rabbits' blood, as for example, the spirillum of cholera, the typhoid bacillus, Friedlander's pneumonia bacillus, and the coccus aquatilis, while others were little affected and some grew abundantly. Such were, among others, the pyogenic staphylococci and the microbes of fowl cholera and of swine erysipelas. Some of the former were killed so rapidly that Nissen con- cluded that the swiftness of their destruction outside the body was in itself an argument against phagocytosis, and instituted experiments to show that a similar swift destruction occurs within the blood vessels of the organism— but inasmuch as he made no series of cultures from the organs in whose capillaries the injected microbes might have been arrested, his results are, in this respect, of but little value. Emmerich ■" von FoAov.— Deutsche med. ]f'ochenschrift, 1SS7, p. 745. t Nuttall. " Experimente fiber dio biikterienfelndlichen Elnfliisae des thierisclicu Korpcrs." —Zdtsch: f. Hygiene. Vol. IV., 1SS8, p. 35;i. {Nissen. "Die baoterienvorniohtendo Eigenschaft des B\\ites."—ZeUscKr.f. H'jgieM, \ol. VI., ISSO, p. 487. 10 and di Mattei,* had, some months previously, argued that the rapidity with which the injected micrococci of swine erysipehis disappear from the blood is evidence that there can be no question of phagocytic action^ and that therefore there must be some anti-bacterial substance developed by the cells of the body and free in the humours. There is not space here to give in detail Metchnikoff's very able refutation of the argument of Emmerich aud di Mattei ; suffice to say that using still smaller quantities of the virus than did these observers, and employing refractory and not susceptible animals, thus again making his case stronger, Metchnikoif showed that in place of the microbes being all destroyed in from fifteen to twenty minutes some at least were still alive aud capable of giving cultures after several hours, indeed for as long a period as four days, and he found that in two and a half hours there was already definite phagocytosis obsei-vable, some phagocytes containing as many as twenty-eight bacilli, f These observations and others of a similar nature showed conclusively that outside the body the humours have an unmistakable power of destroying many forms of micro-organisms. The next question to be determined in order to establish the humoral theory was whether any relationship is to be made out between the insusceptibility of an animal to a given disease, and the degree of bactericidal power of its body fluids. It was discovered, in the first place, that the different fluids of the body possess bactericidal powers differing in degree. Thus, f jr example, the blood serum is generally speaking the most active of all, the aqueous humour the least active. But the fact is not opposed to the humoral theory, for in the susceptible animal it requires a larger injection of pathogenic microbes into the circulation to induce disease than is required in the case of inoculation into the anterior chamber of the eye. At the same time, the fact that the aqueous humour is deficient in leucocytes, while the blood contains them in quantity, makes this argu- ment support the phagocyte theory equally well. It is when we attempt to solve the problem more directly that the humoral theory is found to be insufficient, and we owe to those who first propounded the theory the most striking demonstrations of its insufficiency. Thus Nuttall showed that the blood serum of the susceptible rabbit is more bactericidal towards the anthrax bacilli than is that of the sheep which has been rendered immune, and Behring and NissenJ pointed out that the same micro-organism develops without delay in the serum of such refractory animals as fowls, frogs, cats, and sheep that have been rendered immune, * Emmcricb and di Mattei. — Fortschritte der Medkin, VI. p. 720. + " In the torpedo, injecting anthrax bacilli, Lubarsch found phagocytoo after three quarters of an Iiour in pretty nearly every preparation he made, many phagocyteB containing from 20 to 30 bacilli."— «i(. /. Bakt., Vol. VI., 1SS9, p. 630. X Behring and Nissen.— Zdtsc/if./. Hygiene, Vol. VIII., 1890, p. 421. I 11 is variable in its development in the susceptible rabbit and the refractory ■ dog, and is only surely stopped in its development and destroyed by the serum of the rat — a very insusceptible animal. To these observations may be added those of Charrin and Roger,* who found that the rabbit is highly refractory to quarter evil, the guinea-pig most susceptible, yet the microbe of the disease grows more easily in the rabbit's than in the guinea-pig's blood serum. In his studies upon the action of the rat's serum Metchnikofff added a still more adverse case. He discovered that the blood scrum of a rat which had succumbed to anthrax had bictericidal powers equal to that of a rat immune to the disease. Evidently, therefore, there is no constant relationship to be made out between the extent to which an animal is refractory and the degree of the bactericidal power possessed by its blood-serum. Indeed, few satis- fiictory cases have been brought forward in support of such relationship. It has been found that the vibrio Metchnikovi is not killed by the serum of susceptible guinea-pigs, is killed by that of guinea-pigs which have been made refractory^ ; again, that while the growth of the quarter evil microbe is, as above stated, not abundant in guinea-pigs' serum, it is still more scanty in that of the vaccinated guinea-pig,§ and that in the serum of rabbits made refractory towards erysipelas there is as abundant a growth of the streptococcus as in that of normal rabbits, but now the coccus becomes attenuated,ir and similar phenomena are observed with the B. pyocyaneus in rabbits' serum. One or two more examples might be given, but still not enough to prevent there being more exceptions than illustrations in case we attempt to-propound a law as to the direct relationship between immunity and the bactericidal power of the body fluids. A further difficulty, to which attention has already been called, is the fact that the destruction of microbes in the extracted body fluids is much more rapid than in the humours within the organism. Not only is it more rapid, but it is more extensive. Thus Lubarsch || obsei'ved that in order to kill a rabbit at least 16,000 virulent anthrax bacilli are required if the injection bo made intravenously — directly into the circulating blood ; a lesser quantity, 10,000, for example, only causes a transient disturbance ; or otherwise, the whole circulating blood can cnly destroy 10,000 bacilli or so at a time. Now, one cubic centimetre of rabbits' blood serum can destroy in a few minutes an equal or even much greater amount. * Cliarrin and Roger. — Comptes rcndus dc la Sociile di Biologic, 18S9. t ilctchnikoff.—Annalcs de I'lnstitut Pasteur, IS'.tO, p. 193. t Behring and Nlseeii.— Zoc. cit.. Coiiarmod by Motschnikoff, Anncdes dc I'lmtitut Pasteur, 1891, September number. § Cbarrln and Roger. — Comptes reandus dc la SociHi de Bioloyie, 1S90. % Roger. See Bouchard. " Essai d'une Thuorio do I'lnfection."— X. Internat. Med. Congress, 1890. II Ln\3a,rach.—CaUralblatt.f. Bacle)-iologie,, VI., 1889, p. 841. 12 Here, then, there is a remarkable want of relationship between the- action of the humours outside the body and within. We are forced to acknowledge that if a factor at all in the production of immunity, the bactericidal property of the humours is not of primary importance, and the next question to be dealt with is how to reconcile the undoubted action "in vitro" with what occurs "in corpore." A most, important accompaniment of the clotting of blood and the production of serum is the breaking down of leucocytes, and, as in all the lymph spaces of the body, leucocytes can, and do, enter, so all the- body fluids (as distinguished from the secretions) contain a larger or smaller number of these cells— the blood and lympL the most, the aqueous humour the fewest. Thus all of these body fluids when removed become modified in that now the leucocytes break down and their soluble contents become set free. Can this account for the bactericidal properties of the serum and other fluids ? Curiously enough, those who first called attention to those properties were the first to adduce facts in favour of this supposition. Buchner* who, from the beginning, acknowledged that his demouatratiou of the bacteria-killing power of blood serum did not refute, but only modified, the phagocyte theory, showed that if the cells and proteid matter of defibrinated blood were, by keeping, allowed to fall, the serum lost its power, Avhile the layer of corpuscles at the bottom of the vessel possessed it. And Nissenf unwittingly brought forward what is as strong a proof as could be desired in favour of the same contention, a proof to which I have seen no reference, and which I would, therefore, dwell upon in some detail. Blood may be prevented from clotting either by the action of peptone or by adding to it a solution of magnesium sul- phate. In the former case, peptone plasma, as Nissen shows, destroys leucocytes, and this peptone plasma has bactericidal properties as strong, or almost as strong, as ordinary serum ; in the latter case the leucocytes remain intact, but the plasma has absolutely no destructive influence. And so Nissen argued, that in one case the destruction is undergone in the absence of leucocytes, in the other, although leucocytes are present in abundance, no bacteria are killed, hence there is here a proof positive of the correctness of the humoral theory as opposed to that of phagocytosis. But what the experiment proves, and proves most prettily, is that where the leucocytes are prevented from breaking down and liberating their contents, there the plasma has little or no bactericidal action, and that thus the bacteria- killing property of plasma or serum is due to the dissolution of leucocytes. * Buchner.—Centmlblatt f. BaKteriologie, 1888, Vol. V. p. 817, and Vol. VI. p. 1 and p. 561, and ArcMv fib- Hygiene, Vol. X., 1890, p. 130. f Nlsaen.— Zoc. c't., p. 600. 13 Many observers have shown that the intracellular digestion of the lower organisms is brought about by the formation of vacuoles around the food particles, these vacuoles being idled with a digestive fluid whereby the food is broken up and rendered soluble and assimilable. In the phagocytes, as Metchnikoff pointed out years ago, a similar pi-ocess obtains in relation to the incepted bacteria when these are destroyed. So that clearly the phagocytes, under favourable conditions, elaborate substances capable of killing and breaking up the microbes. This view that the leucocytes contain a bactericidal substance or sub- stances is strengthened, if it be not fully established, by Hankin's* dis- covery of a bacteria-killing globulin present in the lymphatic glands of the dog and cat, and also obtainable from the spleen. The former organs and, to a large extent, the latter are composed of what may be termed potential phagocytes. We thus arrive at the conclusion that the leucocytes and probably other mesodermal cells function as phagocytes under appropriate condi- tions within the organisms, and kill, or it may be, only modify the mi- crobe by means of their digestive fluids ; that outside the organism, where there has been destruction of the leucocytes, the soluble constituents of these cells are liberated into the blood serum, lymph, etc., which now gain the power of killing or modifying the microbes present. It is to the fact that Metchnikoff is now prepared to receive this doc- trine and to extend his theory in this direction that I referred in the earlier part of this paper, for, in his last communication,! he shows that the same attenuation and alteration in the mode of growth of the vibrio Metchnikovi is observable when the vibrio has been acted upon by phago- cytes within the body of " vaccinated " guinea-pigs, and when it has been acted upon outside the body by the blood serum of like animals. He points out that in the latter case the change may be due to substances derived from the broken down leucocytes. Having arrived thus far, the next problem to be dealt with is whether the dissolution of leucocytes within the organism is capable of aiding the action of the intact leucocytes and other phagocytes in preventing the growth and extension of pathogenic microbes. Such dissolution is, we know, of constant occurrence under normal conditions. During the pro- cess of digestion, for example, not only is there a very definite increase in the number of the white corpuscles of the blood, but as Heidenhaiu has shown, there is a most active breaking down of the same, and in the course of infectious disease it would seem that the production and destruc- tion is largely increased wherever there is a well-marked reaction on the part of the system. That there is increased formation is evidenced by the proverbial "buSy coat" of febrile blood in certain states ; and having • U&nUa.— Proceedings of the lio'ial Societ;/, Vol. XLVIII, 1S90, p. 93. t Metchnikoff.— .