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Full text of "Report of program activities: National Institute of Allergy and Infectious Diseases"

ANNUAL REPORT 
OF 
PROGRAM ACTIVITIES 
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 
Fiscal Year 1979 



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TABLE OF CONTENTS 
1979 ANNUAL REPORT 
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 

Director I-I 

Immunology, Allergic and Immunologic Diseases Program 2-1 

Microbiology and Infectious Diseases Program 6-1 

Extramural Activities Program I2-I 

Intramural Research Program I8-I 

SCIENTIFIC DIRECTOR I8-I 



Laboratory of Biology of Viruses I9-I 

Laboratory of Clinical Investigations 20-1 

Laboratory of Immunogenetics 2I-I 

Laboratory of Immunology 22-1 

Laboratory of Infectious Diseases 23-1 

Laboratory of Microbial Immunity 24-1 

Laboratory of Parasitic Diseases 25-1 

Laboratory of Streptococcal Diseases 26-1 

Laboratory of Viral Diseases 27-1 

Pocky Mountain Laboratory 

Laboratory of Microbial Structure and Function 28-1 

Laboratory of Persistent Viral Diseases 29-1 

Epidemiology Branch 30-1 

Rocky Mountain Operations Branch 3I-I 



ANNUAL REPORT OF PROGRAM ACTIVITIES 

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 

October 1, 1978 Through September 30, 1979 

Nineteen Seventy-Nine has been a busy year for the NIAID. This is re- 
flected in the reports of the program directors and branches and labora- 
tories which follow. We have cause for pride in the accomplishments of 
our scientific constituency and the momentum that is being built toward 
solution to many of the problems that have beset us and been insolvable. 

The Director has devoted much of his time to interaction with constituent 
groups. These efforts to inform them of the Institute's research programs, 
learn their views and enlist their interest and support have opened new 
pathways to information about the problems of the practicing physician and 
his views on priorities. In January 1979, the Institute held an open house 
which was attended by representatives of 24 professional societies and volun- 
tary health organizations. This all-day meeting was devoted to exchange of 
information and discussions. The Director also gave the following invited 
lectures during the year. 

Diabetes, Virology and DNA or It's a Long Way from Amphioxus. Octo- 
ber 1, 1978, Annual ICAAC Lecture, Atlanta, Georgia. 

Introductory Remarks. NIAID 30th Anniversary, October 27, 1978, 
Masur Auditorium, NIH, Bethesda, Maryland. 

The Influence of Infection of the Geography of Heart Disease. Novem- 
ber 13-15, 1978, T. Duckett Jones Memorial Lecture, 51st Annual Meeting 
of the American Heart Association, Dallas, Texas. 

The Second Century of Medical Bacteriology: Rediscovered Opportuni- 
ties. November 20, 1978, American Society for Microbiology Archives 
Collection, Catonsville, Maryland. 

From VD to STD (Sexually Transmitted Diseases). 'November 21, 1978, 
Medicine for the Layman, Masur Auditorium, NIH, Bethesda, Maryland. 

A Tribute to Miss Helen Hayes. November 28, 1978, Asthma and 
Allergy Foundation of America, New York, New York. 

Conference on Pharmaceuticals for Developing Countries. January 21- 
31, 1979, National Academy of Science, Washington, D.C. 

A Return to Nature: A Challenge for Outdoor Medicine. February 2, 
1979, Columbia Medical Society, Columbia, South Carolina. 

International Symposium on Potentiation of Immune Response to Vaccines. 
February 20-21, 1979, Wilson Hall, NIH, Bethesda, Maryland. 



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From Glendalough to the Great Famine: The Unexpected in Human His- 
tory. March 30, 1979, Annual Meeting of the American Epidemiology 
Society, Atlanta Hilton Hotel, Atlanta, Georgia. 

Can Curiosity Flourish in an Age of Diminishing Expectations? April 5, 
1979, Rosenstiel Basic Medical Sciences Research Center, Waltham, Massa- 
chusetts. 

The New York Academy of Medicine Medal for 1979 to Dr. Maclyn 
McCarty. April 19, 1979, The New York Academy of Medicine, New York, 
New York. 

INTRODUCTORY REMARKS: The Kinyoun Lecture. "The Role of Com- 
plement in Natural Resistance to Infections." April 24, 1979, Wilson Hall, 
NIH, Bethesda, Maryland. 

A Claim for Curiosity on a Vanishing Frontier. May 4, 1979, Center for 
Interdisciplinary Research in Immunologic Diseases (CIRID), University of 
California, Los Angeles, California. 

INTRODUCTORY REMARKS: The Kinyoun Lecture. "Cell Mediated Im- 
munity to Intracellular Parasites and Polymorphic Nature Transplantation 
Antigens." May 8, 1979, Wilson Hall, NIH, Bethesda, Maryland. 

Cystic Fibrosis and the Historical Precedent for Optimism. August 9, 
1979, The Cystic Fibrosis Foundation President's Conference, Dulles 
Airport, Virginia. 

"Role of the National Institute of Allergy and Infectious Diseases in the 
Conquest of Disease — A Tenacious Strategy to Match a Restless Tide." 
September 21, 1979, Wilson Awards Symposium: Prevention of Major 
Infectious Diseases: Current Concerns and Future Promise, Rochester, 
New York. 

Dr. Jack S. Whitescarver has attended a number of professional society and 
voluntary health organization meetings to meet with officers and others re- 
garding relationships between them and the Institute. He is assuming a 
major role in the development of fiscal and program information desired by 
these organizations, working closely with the Director and Program Directors 
in doing so. 

International Medical Research 

The absence of a full-time professional with responsibility for international 
medical research activities threw quite a burden on the Director and Deputy 
Director. A new initiative in the government to develop research and techno- 
logical exchanges and activities with the People's Republic of China (PRO 
required development of suggested program activities. With the signing of a 
formal agreement between the Ministry of Health, PRC , and the DHEW, the 
NIAID drew responsibilities for collaboration in infectious diseases, immunology 



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and recombinant DNA research. The activities now involving program staff 
have required representation at a number of meetings. As the year ends, 
definitive activities are evolving. 

The Director has also been heavily involved with the Office of International 
Health in reviewing possibilities for expanded relationships with medical re- 
search in India. This stems from requests by the Indian Medical Research 
Council and involved a trip to India for discussions. 

The Deputy Director was designated as Chairman of the Subcommittees on 
Biomedical Research and Infectious and Parasitic Diseases of the U.S. -Egypt 
Committee on Medical Research. In this role he attended the joint meetings 
of the subcommittees and committees in July where the entire collaboration in 
research was reviewed , pending applications reviewed and approved , and 
plans made for the allocation of the rapidly dwindling residual of PL 480 
funds. 

Also as the year ended, new initiatives in cooperation with the Japanese were 
developing. These stemmed from interests of Frank Press, Director of the 
Office of Science and Technology Policy, The White House, in increasing the 
investment being made by the Japanese in research and technology which 
potentially benefits the U.S. A July, 1979 trip in which the Director, NIH, 
participated led to agreement in several areas involving the NIAID. These 
were vaccine development, immunology, and recombinant DNA research. The 
Director is working with the Director, NIH, and staff toward specific propo- 
sals in these areas. Immunology will eventually wind up within the framework 
of the U.S. -Japan Cooperative Medical Sciences Program administered by the 
NIAID. Vaccine development may also go this route, but recombinant DNA 
will require new mechanisms for cooperation. 

Influenza 

The Deputy Director, along with the Director, MIDP, serves on an Inter- 
agency Working Group (IWG) for DHEW activities in the control of influenza. 

The IWG has coordinated responses to a number of questions regarding in- 
fluenza stemming from the Secretary's Conference in 1978, and the Congress 
and the OSTP. It has also maintained an exchange of information between 
the CDC, the NIAID, and the BoB/FDA on occurrence of the disease, virus 
strain characteristics and research activities. Other activities, including 
the Consensus Conference on Amantadine, are described in the MIDP report. 

Recombinant DNA Activities 

The Office of Specialized Research and Facilities continued to maintain high 
containment facilities in an operational state throughout FY 79. The labora- 
tory at the Frederick Cancer Research Center provided the main facility 
support for risk assessment experiments being conducted by Drs. Rowe and 
Martin of the Intramural Program. Those experiments have now provided 
the first direct evidence that some potential biohazards considered earlier 
are unlikely to occur. It is anticipated that those results, and others yet 
to come from continuing studies , will have a major impact on revisions of the 

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NIH Guidelines for Recombinant DNA Research. The 1978 Guidelines do not 
explicitly require P-4 containment for any permissable experiment. Conse- 
quently the laboratory in Frederick is being operated at lower levels but it 
can convert to P-4 operations on very short notice should a need arise. The 
Mobile Containment Laboratory has been downgraded to a P-3 facility and 
will not be operated again in the P-4 mode. 

During the year, the Architectural /Engineering studies for the National Bio- 
medical Containment Laboratory were completed in compliance with the original 
workscope. The A/E construction cost estimates were significantly higher 
than those developed by both staff and the prime contractor, Litton Bio- 
netics, Inc., at the beginning of the project. Review of the A/E estimates 
revealed them to be accurate and reasonable but significantly higher than 
estimating data references for general research laboratories; this reflects 
the extremely complex nature of the NBCL. Accordingly, the A/E has been 
asked to restructure the drawings to permit a basic bid package with "add- 
ons" to accomplish the construction of the entire building in stages. Receipt 
of the revised drawings is anticipated near the end of FY 79 and a decision 
as to whether to solicit construction bids is expected at that time. 

With the issuance in December 1978 of revised guidelines, the Secretary, 
DHEW, requested that the NIH prepare a Risk Assessment Plan. Since the 
hypothetical risks and technical basis of recombinant DNA research are pri- 
marily microbiological in nature, the responsibility for coordination and 
implementation of the plan was assigned to the Director, NIAID. In compli- 
ance with the Secretary's request, the NIAID published a proposed plan for 
public comment and had that document reviewed by the Recombinant DNA 
Advisory Committee. This first Annual Plan has now been published in the 
Federal Register. In implementing the Plan the NIAID will: (1) recruit and 
appoint an eminent scientist as a Special Assistant to the Director for Risk 
Assessment to provide leadership and coordination of all activities concerned 
with the evaluation of risks; (2) develop and issue such requests for appli- 
cations or proposals as are necessary to ensure the conduct of risk assess- 
ment research required to answer specific questions or to fill gaps in data 
being accumulated from other research; (3) prepare and send periodic re- 
ports to the RAC identifying questions, problems, and evaluations of scien- 
tific information pertinent to their various advisory functions ; and ( 4) 
respond to inquiries from scientists, the public, DHEW, or other government 
agencies regarding available data on risk assessment and evaluation of those 
data. In carrying out these responsibilities, the NIAID will enlist the 
services of existing NIH offices, committees, and people to provide informa- 
tion, to advise and evaluate, and to review, as appropriate, reports for 
completeness and accuracy. 

The consolidation by NIH of recombinant DNA administrative activities into 
one Institute was continued by the decision to transfer the Office of Re- 
combinant DNA Activities from NIGMS to NIAID. As FY 79 draws to a close, 
the operational aspects of the transfer have all been effected and it is 
anticipated that at the start of FY 80 final administrative approval will be 
received . 



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Office of Research Reporting and Public Response 

The loss of experienced professional and secretarial staff during this year 
weakened the efforts of ORRPR. Assistant Chief Margaret McElwain had been 
a member of our staff for more than 10 years; Ms. Bobbie Plocinik, our 
"resident immunologist," had been with us for more than five years, and 
Ms. Sylvia Glukenhaus had been ORRPR secretary for more than eight years. 
These personnel losses hitting ORRPR in one year resulted in a reduction in 
overall productivity, particularly in the area of research reports. 

We were successful in recruiting Mr. Thomas Coleman, former executive di- 
rector of the National Association of Hearing and Speech Agencies, to serve 
as an expert in medical communications. He will be developing the Institute's 
capability in the audiovisual and electronic media. He also will be available 
to work with the Director and other Institute personnel on communications 
projects, including "outreach" programs and minority efforts. Also, Ms. 
Lydia Woods Schindler has been employed under contract to work on a varie- 
ty of writing assignments with Dr. Krause. 

Publications — Much of ORRPR's staff effort this year went into producing 
publications which were a part of or an outgrowth of two NIAID task forces. 
As a result, NIAID now has the beginnings of a Scientific Monograph Series 
with a six- volume report of the Virology Task Force and a single volume 
reporting the Task Force on Asthma and the Other Allergic Diseases. Our 
staff devoted a great deal of time in editing, proffing and distributing these 
monographs to appropriate scientists and physicians. In addition, we helped 
to prepare and distribute summary reports of the Virology Task Force chair- 
man and the Asthma and Allergic Disease Task Force. The latter summary 
report contained NIAID Council Recommendations. 

One aspect of both Task Force charges was to produce a document aimed at 
the general public. The publication, Allergies and Asthma — An Optimistic 
Future , was written under contract by Patrick Young, freelance science 
writer. Approximately 70 illustrations were selected and developed by ORRPR 
staff to be included in this lay version, which is scheduled for distribution 
early next year. The manuscript for the lay publication in virology, written 
under contract by Elaine Blume Wilson, a former ORRPR staffer, has the 
working title of Intimate Enemies — An Introduction to Viruses . It also is in 
final stages of preparation and is being considered as an "occasional paper" 
from the Institute. 

Our staff assisted in the preparation of the Institute's chapter on "Tobacco 
— Its Role in Allergy and Immunity," for the DHEW publication, Smoking and 
Health: A Report of the Surgeon General . Copies of this chapter have 
been reprinted and are being distributed to the nation's allergists. 

ORRPR has collaborated with NIH's Medical Arts Department in the design of 
a new format for our fact sheet series. The new format is half the former 
page size, easier to display and store, more attractive and economical. Six 
fact sheets have been printed in the new format. 



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Other publication efforts include a new fact sheet on hereditary angioedema, 
a backgrounder on staph infections, and a Q and A on genital herpes; major 
revisions in three pamphlets — "Asthma," "Sexually Transmitted Diseases," 
and "Drug Allergy" — and revisions of four fact sheets — "Malaria," "Toxo- 
plasmosis," "Viral Hepatitis," and "Rocky Mountain Spotted Fever." 

We continue to prepare columns for the "Search for Health" series distributed 
by the NIH to local newspapers around the country. Over the years, the 
response to these health columns has been excellent with thousands of 
follow-up requests for NIAID publications. This year's columns resulted in 
requests for our publications on pollen allergy, insect allergy, poison ivy 
allergy, and Rocky Mountain spotted fever. 

We also have made a special effort to supply allergy publications on a con- 
tinuing basis to the local chapter of the Asthma and Allergy Foundation of 
America for distribution at its public meetings, which are often attended by 
an ORRPR staff member. 

In our continuing cooperation with the Consumer Service (CIS) , ORRPR pro- 
vided copies of the backgrounders on "Acne" and "Q&A About Allergies," 
both of which were promoted in the spring and summer CIS catalogues. A 
CIS spokesperson has advised ORRPR that in response to requests approxi- 
mately 90,000 copies of "Acne" and 75,000 copies of "Q&A About Allergies" 
have been distributed this year. 

In total, more than a quarter of a million NIAID publications were distri- 
buted by ORRPR during the year. 

Public Inquiries — The number of public inquiry requests continues to in- 
crease. Approximately 700 individualized letters were prepared to respond 
to a variety of requests, including nearly 40 responses to the Congress. 

Scientific Meetings and Information — During the past year, ORRPR staff 
managed press briefings tied to the International Symposium on Pertussis, 
the International Symposium on Potentiation of Immune Response to Vaccines, 
and the results from the initial polyoma recombinant DNA risk assessment 
experiments conducted by NIAID scientists (Martin and Rowe) at Ft. Detrick. 

Working closely with Institute scientists, representatives of NIH's Office of 
Medical Applications for Research (OMAR) and a private contractor, ORRPR 
staff developed promotional materials and advance publicity for the NIH Con- 
sensus Development Conference on the Use of Amantadine. This conference, 
to be held on October 15, 1979, will be followed by a press briefing and the 
recommendations of the conference panel will be disseminated by ORRPR 
staff. 

Exhibits were produced and manned at the annual meetings of the following 
organizations: American Public Health Association (APHA): the Association 
of Military Surgeons of the U.S.; the American Academy of Allergy ; the 
American Society for Microbiology (AMS) ; and the American Lung Association/ 
American Thoracic Society. This represents an increased effort in reaching 



1-6 



members of NIAID "constituent" organizations. ORRPR staff also assisted 
NIAID's EEO Coordinator in developing an exhibit for the Minority Biomedical 
Science Symposium in Atlanta. We furnished pamphlets and information on 
infectious diseases for two panels of an NIH Year-of-the-Child exhibit, which 
is being sent to many meetings, including next year's APHA meeting. 

At the ASM meeting, ORRPR employed a videotape presentation for the first 
time. This included two short segments: (1) a description of NIAID's high- 
risk containment facility at Ft. Detrick; and (2) scientific footage, provided 
by Dr. Adel Mahmoud of Case-Western Reserve University, visualizing an 
antibody potentiated attack on trichina larvae by eosinophils. 

Last year, ORRPR staff developed our first patient-education slide /tape show 
on "What You Should Know About Asthma." Because of its success, we were 
developing a second show, "Coping with Your Allergy — At Home, At School 
and On the Job." This was previewed at our exhibit at the American Acade- 
my of Allergy meeting. It will be pretested in selected patient populations 
next year for suitability of material and its comprehension level prior to be- 
ing offered to the General Services Administration (GSA) for possible sale. 
GSA has reported that nearly 300 copies of "What You Should Know About 
Asthma" have already been sold to physicians, hospitals, and nonprofit or- 
ganizations. In addition, ORRPR has lent copies of the show to several 
groups, including the PTA, Bronco Junction (a camp for asthmatics) and 
local chapters of the Asthma and Allergy Foundations of America and the 
American Lung Association. 

Special Events — ORRPR handled publicity in connection with the Institute's 
30th anniversary celebration. Members of the Institute staff attending the 
formal ceremony in Masur Auditorium on October 27 received a special four- 
page feature of NIAID, prepared by ORRPR staff, which later appeared in 
the NIH Record's special anniversary issue. 

ORRPR worked closely with Dr. Krause and Dr. Gordon Wallace on the pro- 
gram, poster and plaque design for the NIAID Kinyoun Lecture Series. 
Established by Dr. Krause (with Dr. Fredrickson's verbal approval), this 
new series of invited lecturers will, for the most part, emphasize the inter- 
dependence of infection and immunity. The first two lecturers were Dr. 
Hans Muller-Eberhard and Dr. Rolf Zinkernagel, both from Scripps Clinic, 
La Jolla, California. 

EEO Activities and Functions 

Report from the Chairperson, EEO Advisory Committee — The EEO Advisory 
Committee consists of 16 voting members. Twelve were elected by the NIAID 
staff, two were appointed by Dr. Krause, and two are the NIAID EEO Coun- 
selors for the Bethesda campus. There is an additional EEO Counselor at the 
Rocky Mountain Laboratory and she participates in a newly organized EEO 
Advisory Subcommittee for RML. In addition to active participation by the 
RML Subcommittee, a Federal Women's Program Subcommittee has some mem- 
bers on the Advisory Committee and some other Institute members. The 
Federal Women's Program Coordinator (FWPC) for NIAID heads the FWP 



1-7 



Subcommittee and is an ex officio member of the Advisory Committee. Also 
participating as ex officio members are the Training Specialist and Coordina- 
tor for Employment of Handicapped and the Hispanic Employment Program 
Coordinator, along with the EEO Coordinator for NIAID. Members of the 
Office of the Director, Personnel Office, Information Office, Scientific Direc- 
tor's Office and others in various divisions of management participate in 
advisory capacities. The EEO Advisory Committee is directly advisory to the 
Director on all matters relating to EEO concerns, programs and objectives. 
Much of the committee's activities involve monitoring of the existing Affirma- 
tive Action Plan, encouraging completion of EEO program objectives and 
development of new EEO and affirmative action objectives. A summary of 
recent work is presented in this report. 

Reports from Subcommittees of the EEO Advisory Committee — Chairpersons 
for various subcommittees of the EEO Advisory Committee prepared final re- 
ports on activities conducted by their subcommittees during the past fiscal 
year. These detailed reports, which were submitted to the Chairperson of 
the EEO Advisory Committee, are summarized below: 

1. Subcommittee on Program Evaluation — The subcommittee recognized the 
need for a system to monitor the implementation of the Affirmative Action 
Plan (AAP) for the NIAID. It recommended that the Major Initiative 
Tracking System (MITS), or a similar system, be used by the EEO Advi- 
sory Committee to facilitate program evaluation in this regard. This 
tracking system entails (a) the selection of the most impacting objectives 
from the AAP for tracking, (b) the collection of monthly progress re- 
ports from responsible officials, (c) the charting of progress made with 
respect to the attainment of these objectives on a quarterly and annual 
basis, and (d) the dissemination of all charted information to NIAID em- 
ployees at the end of each fiscal year. By means of this tracking sys- 
tem, the EEO Advisory Committee should be made aware of problems 

and /or obstacles that may impede progress in implementing the AAP. 
The subcommittee acknowledged that the EEO Coordinator is involved in 
monitoring the implementation of the AAP. However, the EEO Advisory 
Committee, as an elected body, has a special obligation to carefully and 
systematically monitor affirmative action programs. This may be done in 
conjunction with the EEO Coordinator's office, but it should also be done 
by the EEO Advisory Committee separately. 

2. Subcommittee on Statistics — This subcommittee is responsible for col- 
lecting, interpreting, and reporting statistical information to the EEO 
Advisory Committee for further evaluation. In order to perform these 
functions , access to a vast source of statistical information must be ob- 
tained. Since this type of information is not presently available to the 
subcommittee , it is exploring the possibility of establishing a computer- 
ized personnel master file for NIAID. This file would be accessible to 
both the EEO Advisory Committee and the EEO Coordinator. The pur- 
pose of such a file would be to provide up-to-date information on current 
trends in hiring, promotions and transfers; it would serve as a means 
for detecting flagrant deficiencies in these areas. 



3. Subcommittee on Training — On the basis of the response to a question- 
naire submitted to employees of the NIAID, the subcommittee prepared an 
extensive report on training opportunities within the NIAID . Several 
barriers to training were identified and suggestions were made as to how 
these barriers might be overcome. The subcommittee's general recom- 
mendations included (a) the proper distribution of NIAID policy state- 
ments on training opportunities to all interested employees, (b) increased 
efforts to eliminate advancement barriers for research employees, (c) the 
requirement that all new employees be informed of NIAID-sponsored train- 
ing programs, (d) the encouragement of supervisors to discuss training 
possibilities with employees, (e) communication on possible continued 
employment to stay-in- school employees now on board, and (f) a request 
for training slots for employees in the animal care and secretarial job 
series . 

4. Subcommittee on Communication — The major function of this subcommit- 
tee is to disseminate information on EEO activities to employees within 
the NIAID and to seek ways by which this communications process and 
awareness of the objectives of EEO programs can be improved. The 
subcommittee is currently seeking input from employees within the NIAID 
on the format, agenda, and organization of the coming EEO Workshop at 
which a new five-year AAP will be formulated. 

5. Subcommittee on Awards — This ad hoc subcommittee reviewed several 
nominations for annual EEO awards. In accordance with NIH guidelines 
for service and dedication to the principles of EEO , three nominees were 
selected for awards and special recognition: Ms. Thelma Gaither, LCI; 
Dr. Richard Asofsky, LMI; and Ms. Edna Miller, OD . These individuals 
were presented with certificates and cash awards, in recognition of their 
outstanding contributions to the EEO program, at the NIAID All Hands 
Staff Meeting on June 21, 1979. 

6. Report from the Coordinator of the Federal Women's Program — Late in 
1977, nominees or volunteers for a Federal Women's Program Coordina- 
tor (FWPC) were requested of the NIAID staff. Selection was made by 
the EEO Coordinator (Vincent Thomas) with Advisory Committee concur- 
rence. Weltha Logan (Bio Lab Tech, LMI) was Dr. Krause's appointment 
for a two-year collateral duty, from September 1977 to 1979. 

A brief survey was conducted by Ms. Logan, in the course of a month's 
detail in January 1978, to assess the concerns of the women of the Insti- 
tute and to determine support for an NIAID Federal Women's Program. 
Major among the concerns listed by the responders were those of posi- 
tion management, career advancement, and training. 

A subcommittee of the Advisory Committee was the preferred organiza- 
tional choice of management , based on the desire to maintain all EEO 
concerns as an entity. Ten members, selected from among responders to 
the survey and representing various Institute programs and EEO pur- 
views, were appointed in September 1978. They received training in 
November 1978 to assist them in understanding their functions and to set 



1-9 



specific goals for the coming year. Working groups were established for 
(a) survey, (b) program, (c) communications, and (d) network. Also, 
an ad hoc group was established to formulate an amendment to the Advi- 
sory Committee charter describing the subcommittee. 

Prior to the establishment of the subcommittee, group discussion lunches 
had been scheduled periodically. Early in 1978, their purpose was to 
discuss possible goals for the program. Later, Ms. Rachelle Mandlebaum 
of the Employee Assistance Branch spoke on "Coping with Stress on the 
Job." Dr. Elizabeth Hearne of NIGMS discussed statistics on women in 
the sciences; and Alice Sargent, an organizational consultant, spoke on 
women in management. 

Presently the subcommittee is working mainly in two areas: program and 
survey. Programs approved and scheduled include: (a) two presenta- 
tions for Secretaries' Week on effective communications by Dr. Gloria 
Harris, offered to all of NIAID's office support staff; and (b) a two-hour 
seminar open to all of NIH, entitled "Women in the Workforce." Dr. Ann 
Briscoe, biochemist at Harlem Hospital Center in New York City, spoke 
on the problems faced by professional scientific women. To speak on 
issues which affect all women who work was Dr. Fierst, economist and 
member of the Interdepartmental Task Force on Women established by the 
White House. 

Survey questions have been formulated and sent around for review; 
these suggestions were revised for final approval by the EEO Advisory 
Committee and EEO Team. It is hoped that information received from the 
survey will initiate action items for the five-year AAP to be devised this 
summer. 

Meetings of the Federal Women's Subcommittee are scheduled for the third 
Wednesday of each month from 12:00 to 1:30, held alternately in the 
Westwood Building and Building 31. The meetings are open to visitors. 
Minutes of meetings are distributed to the Institute with the minutes of 
the NIAID EEO Advisory Committee meetings and posted for staff perusal. 

Special Projects Related to the Affirmative Action Plan. 

1. Recruitment Initiatives 

A. Outreach to Local Minority High Schools — A program was developed 
during the past fiscal year to stimulate minority and women's interest 
in biomedical careers. Students from four high schools in the Dis- 
trict of Columbia (Wilson, McKinley, Coolidge, and Spingarn) were 
invited to visit the NIAID; students from more high schools could 
have been invited, but the four- week D.C. teachers' strike created 
serious communication problems in this regard. Each visiting group 
was comprised of from 12-17 students plus at least one of their 
teachers. The program for the students consisted of a welcome and 
orientation session at which the Scientific Director presented back- 
ground information on research programs being conducted at NIH 



1-10 



and NIAID; also, there was a discussion of volunteer and salaried 
positions available for high school students within the NIAID. Then 
students were escorted to several laboratories where members of the 
scientific staff explained the type of research being conducted. 
Here, the students had an opportunity to observe experiments ac- 
tually in progress and the operation of instruments and /or techniques 
used in biomedical research. Although Dr. Lois A. Salzman, LBV, 
was largely responsible for planning and coordinating all aspects 
connected with this program, the individual efforts of many senior 
staff scientists contributed greatly to its success. 

B . Introduction to Biomedical Research — The NIAID recently hosted 
39 outstanding science students who participated in a seminar, 
Introduction to Biomedical Research . The students who came from 
20 states, the District of Columbia, and Puerto Rico were chosen by 
their colleges to participate in this special program held February 27 
to March 1. The seminar was part of the Minority Biomedical Sci- 
ences Program that assists financially and educationally disadvan- 
taged students to enter biomedical research and health-related pro- 
fessions . 

Dr. Zora J. Griffo, OD, and Dr. Richard M. Krause, Director of 
NIAID , welcomed the students and discussed career opportunities 
at NIH. Dr. Ciriaco Q. Gonzalez, Chief, MBS Program, DRR , empha- 
sized the need for minority participation in the biomedical research 
sciences. The students also heard from NIAID intramural scientists 
and Dr. Kenneth Sell, the Institute's Scientific Director. 

The students visited NIAID laboratories where they spoke with in- 
vestigators and observed various experiments in progress. They 
were also interviewed by researchers for their interest in NIH's sum- 
mer employment program ; 10 of these students were invited to return 
to NIAID in the summer to work in various laboratories. 

The program was organized by Dr. Kathleen Jaouni Cook, assistant 
to Dr. Sell, and Mr. Frank Fountain, NIAID 's EEO Coordinator. 

2. Community Affairs 

The Community Outreach Program — As a follow-up to the Personnel Of- 
fice's participation in a Career Fair held at St. Paul's Christian Communi- 
ty Church, 414 Tennessee Avenue, N.E., in June of 1978, the following 
activities were initiated and are now in progress: a tutorial reading 
service; an alcohol and drug abuse program; and safety and security 
workshops. 

3. Employee Development Programs — The Employee Development Specialist 
(Mrs. Edna Miller) instructed and coordinated a course in Laboratory 
Animal Care for 16 employees, as well as coordinated three seminars on 
the Factor Evaluation System. The latter were designed for administra- 
tive and supervisory personnel. Mrs. Miller has established a Mini 



1-11 



Information Center for all employees in her office (Room Bl-01, Building 
5) . Catalogs and a variety of information on educational opportunities 
for various career specialties are available. In addition to these activi- 
ties, tours of the Institute were conducted for summer employees, new 
employees, and administrative personnel; such tours are scheduled to be 
held at least four times each year. 

4. Employment of the Handicapped — The Coordinator for the Handicapped 
Persons Program (Mrs. Edna Miller) reported that three handicapped em- 
ployees successfully completed a course in Laboratory Animal Care in 
April 1979. The Coordinator has attended two courses so as to keep 
abreast of policies and laws concerning (a) the selective placement of 
the handicapped, and (b) cooperative training and employment programs 
for the handicapped. 

Report from the Office of the EEO Coordinator — The National Institute of 
Allergy and Infectious Diseases is undertaking a program of affirmative ac- 
tion to which good faith efforts will be directed for achievement of its 
Major Civil Rights and Affirmative Action Initiatives. Our goals and initia- 
tives are consistent with those of DHEW, PHS , and NIH. In addition, goals 
and initiatives have been established which are unique to NIAID. 

The EEO Coordinator has direct access to the Institute Director. Addition- 
ally, the EEO staff has direct access to all NIAID personnel, including the 
Executive Officer, Personnel Officer, and Associate Directors. There are 
Federal Women's Program and Hispanic Employment Program Coordinators as- 
signed on a part-time basis, each spending at least 20 percent of their time 
toward accomplishing program objectives. It has been difficult for both the 
FWPC and HEPC to actively pursue program objectives based on a 20 per- 
cent time allocation. All other EEO officials who have other principal assign- 
ments devote at least 20 percent of official time to EEO program matters and 
serve as technical advisors to the Personnel Officer. The Institute has an 
EEO Advisory Committee and three EEO Counselors. Adequate management 
and fiscal controls are established to track all resources devoted to the EEO 
program and expenses have been charged to, and tracked by, the EEO 
Coordinator and Budget Office. 

Our present recruitment sources do yield qualified minority and female ap- 
plicants who meet organization needs; however, there has not been an ade- 
quate number of minorities to select from in the M.D. and Ph.D. community. 
Our recruitment literature reflects the desire to reach all segments of the 
potential for employment, and this effort is also across internal NIH organi- 
zational lines to obtain maximum effectiveness and efficiency . All paid 
advertisement used for recruitment includes minority media coverage. These 
recruitment efforts are monitored to ensure equal treatment regardless of 
race, color, religion, sex, national origin, handicap, and age. 

Plans are being developed to increase the representation of Hispanics and 
Native Americans through our Outreach Programs. 



1-12 



NIAID's Extramural Activities Program, through training grants and con- 
tracts, participates in the Minority Access to Research Careers Program 
(MARC) which is designed to help minority institutions train greater num- 
bers of scientists and teachers in the biomedical discipline. Presently, two 
postdoctoral fellows are being supported through this program. No new 
hires resulted from this program during FY 1979. 

Minority Biomedical Support Program (MBS) is designed to increase minority 
participation in biomedical research, to develop a pool of minority scientists, 
to strengthen biomedical research capabilities of minority institutions , and to 
utilize the talents of minority biomedical investigators in the mission of the 
NIH. NIAID contributed $125,858 during FY 1979 as part of its support 
agreement. 

NIAID is supportive of the Cooperative Education Program and has two Black 
female biomedical student participants. 

Expert appointments are those appointments of individual scientists or pro- 
fessionals qualified in fields related to the program needs of NIAID. Two 
white females have received appointments. 

Intergovernment Personnel Agreement (IPA) — One Black female is presently 
in our Laboratory of Clinical Investigation. 

The Clinical Associates Program, class of 1979, provided two white females. 

NIAID is committed to increasing the participation of minorities and women in 
the scientific community and in those grades and occupational series where 
there is underrepresentation. No hiring goals were set for any of our re- 
cruitment programs during FY 1979. 

Sixty employees responded to a qualifications analysis questionnaire. The 
survey was forwarded to all nonprofessional employees. A consultant was 
hired for this project. During the follow-up, approximately 45 NIAID em- 
ployees were interested in a qualifications analysis. Workshops were con- 
ducted which focused on specifically identified career fields, i.e., personnel, 
administration, program analysis, etc. Employees were counseled as to their 
specific qualifications, what was needed for entry into primary career fields 
of their interest, and exposed to the prospective opportunities at NIH in 
those fields. Two such surveys were conducted during FY 1979, one by 
NIH. The skills file is referred to when considering applicants for all vacan- 
cies. 

Three workshops were conducted on the Factor Evaluation System. The con- 
tent of these workshops was designed and geared to the needs /interests of 
the audience. A film was shown and a discussion was held on how this sys- 
tem would affect those individuals in the auditing and classification of their 
positions . 

Training opportunities are available for all employees to facilitate their 
career goals. All denials of training must be supported in writing. An 



1-13 



analysis of training sponsored by the Institute is ongoing to determine if 
there is any maldistribution by sex, race, grade, etc. 

The majority of this Institute's supervisors and managers have received 
supervisory and EEO training. An assessment is being conducted to examine 
our supervisory and management practices and to identify our needs for im- 
plementing training in specific areas of concern. The Institute is committed 
to the assurance that all managers and supervisors will receive training in 
accordance with established principles. 

This Institute supports the NIH Upward Mobility Programs including the Merit 
Promotion plan. At present we have no internal training programs or posi- 
tions. However, NIAID provides training for other Institutes' Management 
Interns. Career counseling is available to all employees through our Em- 
ployment Development Specialist. 

The EEO Plan is updated annually. The various program evaluations are in- 
corporated into the plan as changes or additions at that time. The evaluation 
is conducted by the EEO Coordinator, the Director, the EEO Advisory Com- 
mittee, and the EEO Team. Other management officials participate when the 
topics to be discussed require their input. An AAP Update Workshop is 
planned for the transition year. One of the objectives is long- and short- 
range AAP objectives. The EEO Advisory Committee will receive EEO Train- 
ing for Advisory Committee Members. This will also facilitate monitoring and 
evaluation of our EEO Program. 

Currently, there is no program in effect to cross-train the EEO Specialists 
or the Personnel Management Specialists. The Personnel Management Spe- 
cialists are scheduled to attend the EEO courses in accordance with one of 
our AAP objectives. One Equal Opportunity Specialist progressed to the 
Management Analysis field. 



1-14 



Annual Report 

Immunology, Allergic and Immunologic Diseases Program 

Table of Contents 



Director 1 s Report 2 

A. Administrative Summary 2-1 

B. Scientific Summary 2-5 

Allergy and Clinical Immunology Branch 3 

A. Scope 3-1 

B. Awards and Support Levels 3-2 

C . Program Areas and Highlights 3-2 

Genetics and Transplantation Biology Branch 4 

A. Scope 4-1 

B . Awards and Support Levels 4-1 

C. Programs Areas and Highlights 4-2 

Immunology and Immunochemistry Branch 5 

A. Scope 5-1 

B. Awards and Support Levels 5-1 

C . Program Areas and Highlights 5-2 



REPORT OF THE DIRECTOR 

IMMUNOLOGY, ALLERGIC AND IMMUNOLGIC DISEASES PROGRAM 

Fiscal Year 1979 

A. Administrative Summary 

1. Organization and Function 

The Immunology, Allergic and Immunologic Diseases Program (IAIDP) 
was initiated on October 1, 1976, coincident with the reorganization of 
NIAID into major programs characterized by professional function. With 
this change the IAIDP assumed the responsibility for research, training, 
and conference activities that formerly were assigned to the Allergy and 
Immunology Branch of the Extramural Programs and contract activities in 
transplantation immunology previously administered within the Collaborative 
Research Program. 

During FY 1979, the following organization served the IAID Program: 

Office of the Director 

Director-Sheldon G. Cohen, M.D. 

Special Assistant to the Director-Dorothy D. Sogn, M.D. 

Assistant for Program Management-George R. Jenkins, B.S. 

Collaborative Projects Section 

Head-Daniel I. Mullally, M.D., M.P.H. 
Program Officer- John G. Ray, Jr., Ph.D. 

Allergy and Clinical Immunology Branch 

Chief-Robert A. Goldstein, M.D., Ph.D. 
Immunology and Immunochemistry Branch 

Chief-Bernard W. Janicki, Ph.D. 

Genetics and Transplantation Biology Branch 

Chief-Henry Krakauer, M.D., Ph.D. 

Serum Bank Manager-Katherine A. Hopkins, R.N. 

Each of the three IAIDP Branches assumes responsibility for adminis- 
tering investigator initiated research grants and collaborative research 
contract awards, developing programmatic initiatives and new projects, 
serving on trans-NIH coordinating committees and taking active roles in the 
development and conduct of workshops and conferences in areas of the bio- 
logical disciplines and medical specialties with which they are concerned. 



2-1 



All three Branches are also involved with programs related to Research 
Career Development Awards and training, i.e., individual and institutional 
fellowships. In addition, the Allergy and Clinical Immunology Branch 
administers Allergic Disease Academic Awards which are designed to further 
the careers of mid-level investigators preparing for careers in Academic 
Allergy Research and the Young Investigator Research Grant Awards which 
are designed to assist and encourage investigators in early stages of 
their careers to develop research interests and capabilities in clinical 
aspects of immunology. 

The LAID Program also assists investigators in their research by 
providing certain reagents that are not otherwise available from commer 
cial sources. Such reagents are intended either for reference standard 
materials such as human and mouse histocompatibility typing sera and 
various purified allergens. Program staff has the responsibility for 
maintaining adequate inventories of reagents as well as for developing new 
sources and identifying new materials for acquisition. 

A new program activity, initiated in FT 1979 is designed to evaluate 
the efficacy of skin testing with major and minor determinants of penicil- 
lin prior to prophylactic administration of this drug or its analogues. 
Using multi-institution contracts for clinical testing data will be pro- 
vided on the predictive value of newly developed diagnostic reagent 
material through the collaborative efforts of many physicians working in 
diverse clinical situations with patients manifesting a variety of 
infectious illnesses, some rare. It is anticipated that the use of common 
protocol with standardized material prepared and furnished to participa- 
ting investigators will help to establish the criteria necessary for 
developing penicillin pre- tests as an important practical diagnostic tool 
and accelerate its clinical application. Of especial concern are the 
problems faced by patients with positive histories of penicillin reactions 
presenting indications for therapy with benzylpenicillin, ampicillin, 
carbenicillin, phenoxyethylpenicillin, phenoxymethylpenicillin and amoxi- 
cillin. Contracts for participation in this field trial were awarded to 
six geographically dispersed university sections having interdisciplinary 
programs in allergy and collaborative efforts with infectious diseases 
sections (Mayo Foundation; University of Rochester; Northwestern Univer- 
sity; University of Colorado,- University of Washington, and University 
of California at Los Angeles) and one interagency agreement was 
established with the Allergy Section of Walter Reed Army Medical Center. 
The Clinical Centers Program consisting of Centers for Interdisciplinary 
Research on Immunologic Diseases (CIRID) and Asthma and Allergic Disease 
Centers (AADC) continues to focus upon multidisciplinary collaboration, 
interactions between basic and clinical investigators and the application 
of research leads in the biomedical sciences to clinical problems in diag- 
nosis, treatment and prevention. Project Directors have been exploring 
possibilities in their regional areas to increase the involvement of their 
staff in outreach areas of education, referral services and the socioeco- 
nomic aspects of disease and disability. The AADCs now total 16 in number 
with three new Centers added to this network, State University of New York 
at Stony Brook; for the study of urticaris, angioedema and vaculitis; 



2-2 



State University of New York at Buffalo; for the study of asthma, and 
University of Kansas; for the study of respiratory disease and systemic 
lupus erythematosus. 

The rapid developments and progress in research on physiologic 
function, chemistry and genetics of immunocompetent cells nave not only 
been important for the fundamental knowledge revealed but additionally 
have pointed to areas where clinical relevance may be. Accordingly, this 
year we have instituted efforts to facilitate interactions and exchanges 
between the Directors of NIAID supported clinical centers (CIRID and AADC) 
and Directors of Program Projects in Lymphocyte Biology. The effective- 
ness of this program has been sufficiently productive to move in the 
direction of expansion through the addition of one new "Lymphocyte Biology 
Center" at Jewish Hospital of St. Louis/Washington University, bringing 
the total of participating institutions to five. 

Our mission as the designated lead Institute in the study of immune 
mechanisms has alerted us to the work of other NIH B/I/D concerned with 
organ and disease oriented interests involving dysfunctions of the immune 
system. Accordingly, we have developed joint program efforts especially 
related to conference activities and definition of research initiatives 
with NIAMDD (dermatology, diabetes), NEI (occular immunopathology) , NCI 
(tumor immunology) and NHLBI (hypersensitivity lung diseases). 

During FY 1979, the work of the Task Force on Asthma and Other 
Allergic Diseases was completed. One hundred and fifty contributors and 
consultants participated in the work of twelve panels. The final report 
of their Task Force dealt with the state-of-the-art and put forth specific 
recommendations for extended study in twelve specific areas concerned with 
social economic aspects, basic mechanisms, asthma, rhinitis, special 
problems of allergic children, farmer's lung and related disorders, the 
expanding problem of occupational/environmental asthma and related respira- 
tory diseases; proposed development of integrated consultative and 
research facilities, dermatologic allergy, allergic emergencies, drug 
allergy, allergic and immunologic aspects of other diseases, and educa- 
tional needs. 

The Scientific Report has been published and distributed to the 
community of health professionals concerned withclinical and investigative 
endeavors in asthma and allergic diseases. An interpretative summary 
version is in the process of completion for distribution to the concerned 
lay community of health action groups. 

Staff members of the IAIDP cooperated in the preparation of the 
updated Report of the Surgeon General on Smoking and Health. For this 
DHEW publication we assumed the responsibility of developing a state-of- 
the-art review concerned with the effects of tobacco and its products on 
the immune system in a chapter entitled "Tobacco - Allergy and Immunity" . 

The phase of data collection and correction of Kidney Transplant 
Histocompatibility Study (KTHS) has been completed and the first stage of 
evaluation and analysis is currently being carried out by the Naval Medical 



2-3 



Research Institute for the IAIDP. This work consists of characterization 
of donor and recipient populations, of selection, operative and post- 
operative procedures, of outcomes, and of certain elementary correlations 
among them. 

The analysis of the data will be extended under contract with the 
Peter Bent Brigham Hospital, Doctor C.B. Carpenter, Principal Investigator, 
through a series of in-depth studies of specific issue in renal transplan- 
tation. These studies will be directed by groups of scientists representing 
the KTHS contractors and program staff. 

2. Budget 

The following shows the distribution of support by award 
mechanism for the activities of the Program during FY 1979. 

Immunology, Allergic and Immunologic Diseases Program 
FY 1979 Awards 



Award Mechanism 



Number 



Amount 



Research Grants 450 

Career and Career Development Awards 58 

Subtotal 508 



$ 46,195,862 

1,744,640 

$ 47,940,502 



Fellowship Awards 
Training Programs 



Subtotal 



42 

34 _ 

76 $ 



539,221 
2,311,604 
2,850,825 



Contracts 



Total 



54 
638 



2,373,061 
$ 55,118,775 



Total costs, estimated using a 40% overhead for indirect costs. 

Conference Support 

During FY 1979, the following meetings, conferences, and workshops 
relevant to the activities and functions of the Immunology, Allergic 
and Immunologic Diseases Programs were supported. 

October 1979 Mediation of Effector Function by Antibodies. 
Bethesda, Maryland 

Immune Mechanisms in Renal Diseases, 
Bethesda, Maryland 

Conference on B Lymphocytes in Immune Responses, 
Scottsdale, Arizona 



2-4 



November 1978 Asthma, Allergic and Immunologic Centers Workshop, 
Bethesda, Maryland 

January 1979 The Role of Non-HLA Tissue Antigens in Human 
Transplantation , 
Bethesda, Maryland 

February 1979 Review of Kidney Transplant Histocompatibility Study, 
Bethesda, Maryland 

1979 Conference on Biological Recognition and Function, 
Keystone, Colorado 

Conference on Cell Surface and Malignancy, 
Keystone, Colorado 

The Regulatory Role of Macrophage Products in Immunity, 
Brook Lodge, Michigan 



March 



May 



1979 Second International Lymphokine Workshop, 
Ermatingen, Switzerland 



September 1979 Third International Workshop on Nude Mice, 
Bozeman, Montana 



Workshop on Immune Mechanisms in Cutaneous Disorders, 
Brook Lodge , Michigan 

B. Scientific Summary 

Central to the activities and interests of the Immunology Allergic, 
and Immunologic Diseases Program (IAIDP) is the immune system, its func- 
tions in the maintenance of health, its involvement in immunologic and 
allergic diseases and its genetic relationships in transplantation 
phenomena and organ tranplantation surgery. Within the programmatic 
activities of allergy, diseases of the immune system and tissue transplan- 
tation is original work focusing on the structure and function and the 
basic biology of the immune system and its application to pathophysiologic 
changes. Responsibility for involvement in these biomedical areas is 
accomplished through investigator initiated research projects, collabora- 
tive research and development contracts, clinical centers concerned with 
asthma and allergic disease (AADC) and Interdisciplinary Research on 
Immunologic Diseases (CIRID), Lymphocytes Biology Program Projects 
("Lymphocytes Biology Centers"), procurement and distribution of research 
resource and standard research reagent materials, and the maintenance of a 
serum bank for histocompatibility testing. 

Designed to accomplish these objectives, the IAIDP is organized into 
three biomedical and scientific Branches: Immunobiology and Immuno- 
chemistry, Allergy and Clinical Immunology and Genetics and Transplantation 



2-5 



Biology. It is from the work of these three programmatic functional 
entities that the IAIDP has gained an insight into important developments 
and aspects of the state-of-the-art of both basic and clinical endeavors in 
immunology and allergy. A review of studies in immunobiology , immuno- 
chemistry and immunogenetics reveals not only progress in the generation of 
fundamental knowledge but also important potential applications of basic 
research data to problems concerned with health and disease through the 
function of the immune system. Investigations in asthma, allergic 
mechanisms, immunologic diseases, biology and inflammation, immunopathology 
and transplantation biology present promising leads for the prompt appli- 
cation of technologic advances to developing improved methods of diagnosis, 
treatment and prevention of relevant disorders. 

An understanding of immunocompetency will depend upon the ultimate 
definition of the origin, differentiation pathways and functional roles of 
the various subsets of lymphocyte cell populations. It is therefore 
important to focus studies upon both the biology of progenitor cells and 
the identification of mechanisms of functional maturation. The most 
promising leads in this area are emerging from cell surface molecular 
markers on lymphocytes of both bone marrow and thymus derivation. Critical 
to the functional adequacy of differentiation and function of immunocompe- 
tent cells may be their specific enzyme contents. Keeping alert to 
expressions of defects in critical points of both T and B lymphocyte 
development may offer leads to deal with immunodeficiency diseases by 
appropriate reconstitution. The study and identification of cell surface 
antigenic markers, especially in embryos undergoing differentiation, can 
offer clues to immunologic association and involvement in the regression of 
cell maturation processes leading to neoplasia. Accordingly, it will be 
important to direct such studies to the investigation of both cell surface 
markers and cytochemical processes. 

One of the most important benefits of the ascendency of research in 
cellular immunology is the increasing insight gained on cell to cell inter- 
actions. This is especially important in the potential application of 
incoming knowledge on both suppressor and helper influences of T lymphocyte 
cell subsets on antibody producing B lymphocytes in immune defense to 
microbial agents, hypersensitivity phenomena and transplantation reactions. 
Of especial pertinence is the regulatory role of products of the major 
histocompatibility complex on the functional role of other antigens 
expressed on the cell surfaces in the immune response. In searching for 
key factors for reconstitution in immune aberrations, a pertinent research 
approach which must be extended concerns new knowledge pointing to the 
necessity to match for functional interactions based on cells having same 
or self markers . 

It becomes increasingly clear that immunocompetence depends upon a 
programed series of events among cells of different types to complete the 
process of recognition, uptake and processing of antigen and regulation of 
the critical signal to those cells involved in either humoral immunity 
through specific antibody production or cell mediated immune processes. 
However, it is not sufficient to recognize only physical contact between 



2-6 



macrophages and lymphocytes, and lymphocyte to lymphocyte of different 
subsets. It will be important to search out and identify cell-derived 
soluble factors that can be utilized in manipulation of the immune system. 
Central to this is the need to understand regulatory mechanisms whether 
through feedback or suppressor dysfunction in hopeful anticipation of the 
ability to design mechanisms and specific approaches to the control of both 
allergic and autoimmune diseases. Thus, such technologic developments that 
can lead to the separation and isolation of reactive cells with their 
subsequent cloning in culture are encouraging developments. 

One of the early developments in the study of cell-mediated immunity 
was the identification of soluble products of lymphocytes termed lympho- 
kines. Demonstrated only by their biologic activity in in vitro systems, 
this finding continues to give an implication of T cell reactivity apart 
from systems giving evidence of immunoregulatory functions. Though lympho- 
kines have been purported to be responsible in a variety of immunologic 
reactions including inflammation, homograft rejection, delayed hypersen- 
sitivity, tumor and other cell killing, progress in this research area will 
require chemical identification. Another critical point is that lymphokine 
activity has thus far only been appreciated in in vitro systems. We will 
therefore be carefully looking at work that suggests the possibility of 
suppression of in vivo manifestations of cell-mediated immunity. 

Though the direction of research endeavor in recent years has progres- 
sively moved to studies at the cellular level, important work continues on 
immunochemistry attempting to define the molecular components of the immune 
system utilizing chemical approaches. Supplementing earlier studies on the 
chemical composition and structure of humoral and secretory immunoglobulins, 
structure of antigens and the kinetics of immunocomplex formation are 
sophisticated investigations at the molecular level of cell surface 
components. Additionally, we see the emergence of a newly developing 
field, immunopharmacology, centering on the identification, characteriza- 
tion, isolation and attempted synthesis of chemical regulators of immune 
function. Elucidation of these factors can contribute to our understanding 
of immune dysfunction and measures to correct altered mechanisms in immuno- 
logic and allergic diseases. To understand the role of antibody synthesis 
in physiologic and pathologic situations, it will be important to have a 
clear definition of basis of antibody diversity. Accordingly, immuno- 
chemical studies offer the possibility for ascertaining whether antibody 
diversification depends upon a germ-line or a somatic process. An 
important indication is the likelihood that antibody diversity can arise by 
somatic cell mutation. 

The past year has seen an escalation in technologic advances in an 
area bridging immunobiology and immunochemistry, especially concerned with 
the development of hybridomas. Cell fusion techniques and selection are 
being employed for the insertion of antibody producing characteristics into 
cultured malignant plasma cells so that by in vitro methods large 
quantities of pure immunoglobulins may be recovered. Another intriguing 
approach has been the development of methods for inserting functional 
messenger RNA into cultured cells. By these techniques, investigators now 



2-7 



may be able to obtain an unlimited source of homogenous antibodies and 
other immunologically- important molecules for study. The application of 
recombinant DNA technology to the problem of suppling large amounts of 
immunologically active reagents also is feasible and is being watched with 
considerable interest. 

Another technologic advance has been the development of a fluores- 
cence-activated cell sorter which can be utilized to prepare pure 
populations of human mononuclear cells from peripheral blood for functional 
studies. In addition to the contribution that this methodology has made to 
immunobiologic studies it may be utilized in the detection of neoplastic 
lymphocytes types and, in turn, be utilized to monitor therapy in patients 
with lymphomas . 

The major objective of research in immunogenetics is the acguisition 
of sufficient understanding of the immune processes that lead to acceptance 
or rejection of a graft to render human organ transplantation a successful 
therapeutic modality. Indeed much of the knowledge of immunogenetics has 
resulted from attempts to define the genetic and antigenic relations that 
determine histocompatibility, i.e. from "tissue typing." 

In all the mammalian species studied, histocompatibility is governed 
principally by an array of genes in a well-delimited region of a single 
chromosome, the so-called major histocompatibility complex (MHC). It is 
designated H-2 in mouse, the most exhaustively studied animal, and HLA in 
man. The genes of the MHC code for a series of cell surface antigens, most 
of which have been identified by serologic techniques as a result of their 
ability to stimulate antibody production and some by their ability to 
induce the proliferation of nonsyngeneic lymphocytes. 

The genes of the MHC are very highly polymorphic. In man, the two 
most extensively studied loci are designated A. and B. An ever growing list 
of serological specificities, presumably reflecting antibodies against 
specific antigens, currently about 70, have been identified. Until 
recently, matching to attempt to obtain histocompatibility in organ trans- 
plantation was restricted to these loci. Such matching has very 
substantial predictive power value for renal graft acceptance when donor 
and recipient are related. Whether matching at the A and B loci improves 
graft survival when donor and recipient of the kidney are not related is 
not at all clear, especially in the highly heterogeneous U.S. population. 
In the case of transplantation between related persons, matching at the A 
and B loci implies very strongly matching over the entire MHC. In man, at 
least three other loci have been identified. The C locus codes for sero- 
logically determined antigens, but has relatively little effect on graft 
survival. The D locus codes for antigens determined by cellular methods: 
lymphocytes disparate at the D locus stimulate each other to proliferate. 
Matching at the D locus is particularly critical in bone marrow transplan- 
tation. Indeed, the mixed lymphocyte reaction involving cells of donor and 
recipient may be characterized as a transplant in the test tube. It is 
usable in renal allografting from a living donor, but, because of the time 
it currently requires, it can not yet be used when the donor is a cadaver. 



2-8 



Under intense investigation at the present time through an International 
Workshop sponsored by NIAID (IAIDP) is the role of the DR locus which is 
very closely linked to the D locus in renal transplantation. The initial 
reports from Europe are very encouraging, but the data beginning to become 
available on the experience in the U.S. are much more ambiguous. 

Overall, it is clear from work in human organ transplantation as well 
as in animal models that matching at the MHC does not guarantee graft 
survival even when donor and recipient are related, unless, of course, they 
are identical twins. There are many other loci on various chromosomes 
which code for cell-surface antigens which, should they be polymorphic, 
could function as histocompatibility antigens. In addition, evidence for 
tissue-specific alloantigens is accumulating. Current histocompatibility 
testing focuses on lymphocytes as the antigen-bearing cells. The expres- 
sion of antigens on various tissues is not uniform. The existence, 
therefore, of tissue-specific histocompatibility antigens is to be expected. 
It does, however, increase the difficulties of matching donors and 
recipient very substantially. 

Alternative maneuvers are being attempted to improve graft survival. 
Among selection procedures there are tests of general reactivity of the 
recipient to antigenic stimulation. For example, the vigor of the response 
to challenge with dinitrochlorbenzene prior to transplantation correlates 
significantly with the vigor of graft rejection. Pretreatment of the 
cadaveric kidney to reduce its immunogenic ity is under active investigation. 
Preculture of thymic cells and of pancreatic islet cells has been found to 
sufficiently enhance their survival after implatation. There are now 
numerous reports of the beneficial effects of blood transfusions on the 
survival of renal allografts, whether from cadaveric or living related 
donors. Our own Kidney Transplant Histocompatibility Study has shown that 
the much- feared consequence of blood transfusions, the sensitization of the 
recipient to a variety of donors, with the consequent increased difficulty 
of finding an appropriate donor, does not in fact occur with significant 
frequency. 

Given the practical near impossibility of obtaining true histocom- 
patibility, manipulation of the immune system of the recipient, and of the 
engrafted donor cells in bone marrow transplantation, is the sole remaining 
approach to maintenance of the graft survival. The mainstays of immuno- 
suppressive therapy are adrenal cortical steroids and cytotoxic agents, 
principally azathioprine. Because of their toxicity, the transplanter 
treads a narrow path between graft rejection and patient death. A clear 
goal is the minimization of immunosuppression. There is thus a constant 
search for alternative strategies and more specific suppressants. One 
approach under investigation, with a clinical trial just now begun, 
involves massive irradiation of the recipient's lymphoid tissue with either 
sparing of the bone marrow or followed by transfusion with previously 
collected autologous marrow. The post-irradiated state in which the 
lymphoid organs are reseeded with lymphoid precursors is apparently one of 
great susceptibility to induction of tolerance, for example of a graft. A 
more selective immunosuppressant, under study for a number of years, is 



2-9 



anti- lymphocyte or anti- thymocyte globulin. The weight of the evidence is 
for its efficacy. Yet its effect is to improve graft survival only some- 
what, and it is not certain that, like steroids and cytotoxic agents, it is 
not so generally immunosuppressive as to endanger the life of the trans- 
plant recipient without adequately enhancing the probability of graft 
survival. Thus, in one study, its abandonment led to improved patient 
survival without appreciable increases in graft losses. 

A still more specific immunosuppressant, the cyclosporin family of 
drugs, whose target is a lymphocyte subpopulation, is now coming under 
careful study. There is very clearly great need for such highly specific 
agents , given the great complexity and delicate balance in the regulatory 
mechanisms that govern the immune response. It is of no use to so disturb 
these mechanisms with immunosuppressants as to produce an uncontrolled 
active response. Yet this does occur on occasion. It is, further, clearly 
necessary when intervening massively in the function of the immune system 
to carefully monitor the activity of its various regulatory components. 
The availability of reagents which identify various lymphocyte subpopu- 
lations that function as helpers, suppressors and effectors made this 
possible in the mouse recently. Similar reagents are now becoming 
available for man. Before long, the careful tailoring of immunosuppressive 
treatment of the transplant recipient should become possible. Together 
with careful selection to avoid, for example transmission of potentially 
lethal viral infections as in the case of cytomegalovirus with the 
transplanted organ and pretreatment of the recipient, solid organ trans- 
plantation should significantly improve its status as an effective form of 
therapy. Bone-marrow transplantation is considerably more difficult 
because, in addition to the possible rejection of the marrow by the 
recipient, there frequently occurs an immune reaction of the marrow against 
the recipient. Here also success is predicated on careful and selective 
immunosuppression. Significant advances are being made. 

In recent years, numerous statistical associations between disease 
syndromes and HLA types have been established. These associations have 
practical diagnostic utility. However, much more interesting are the 
theoretical implications. In the mouse, genes that control response to 
various defined antigens have been mapped to the MHC. In addition, the 
serologically identified antigens have, in certain cases, been involved in 
the presentation of foreign antigen in the recognition phase of the immune 
response. Thus, there is promise that the association of the disease with 
a cell surface structure will lead to an understanding of factors that 
determine susceptibility to the disease. 

While studies in basic immunology and immunogenetics have been respon- 
sible for rapidly expanding knowledge of fundamental biologic processes, at 
the same time they have been increasingly generating data to provide 
insights into mechanisms of allergic and immunologic diseases. During 
recent years the escalation of rewarding productive investigation into 
allergy, clinical immunology and immunopathology has been a direct product 
of a maintained awareness of the potential of basic experimental leads and 
our ability to seek their application to important clinical problems. An 



2-10 



understanding of etiologic and pathophysiologic factors of disorders of the 
immune system in turn has changed the direction of our approaches to work 
directed to the development of improved methods of diagnosis, prevention 
and treatment of allergic and immunologic diseases. Research focused on 
pharmacologic, biochemical, and molecular aspects of immunology by eluci- 
dating of functions and abberations of the immune system is providing the 
necessary base of information for promising approaches to deal with hyper- 
sensitivity, inflammatory and immune deficiency disorders by immune 
manipulation and immunopharmacologic techniques . 

A number of the multifactorial influences contributing to the 
expression of asthma as a clinical disorder are being more clearly defined. 
Along with the established role of inhalant and food allergens there is 
increasing evidence of the sensitizing role being played by chemical and 
viral agents. The multiplicity of allergenic, inflammatory and infectious 
etiologic entities appear to have a common denominator of pathophysiologic 
action effected through the release of chemical mediators such as 
histamine, slow reactive substance of anaphylaxis, eosinophil chemotactic 
factor and platelet activating factor. It is expected that ongoing immuno- 
genetic studies will help to explain the mechanism of genetic predisposi- 
tion to allergic involvement of the respiratory tract and other target 
organs of allergic processes. 

Because asthma continues to be an incompletely understood disease com- 
plex, it is necessary to direct attention to investigative approaches from 
several different standpoints. Among pathologic factors to be delineated 
are the physiochemical character and control of mucus secretion, the pro- 
tective role and possible alterations in bronchial mucosal barriers and 
bronchial epithelial permeability, and structure and function studies 
involving mast cell distribution and smooth muscle of the bronchi. Since 
neurogenic influences play an important role in the control of bronchial 
smooth muscle tone, it will be important to more clearly establish contri- 
butions by improperly functioning autonomic nervous control and excessive 
cholinergic activity, nonadrenergic inhibitory nervous system influences in 
the tracheobronchial tree and whatever specific afferent fiber receptors 
may be involved in the physiology of reflex mechanisms. Though smooth 
muscle spasm has been indicated as an important cause of airway obstruction 
in asthma, the relative roles of pathophysiologic involvement within the 
larger central airways or in the smaller periphreal airways are still to be 
determined. Developing improved methods for the treatment of asthma will 
depend upon information that can be obtained from extended approaches on 
the study of the pathophysiology of airway obstruction, the neurogenic 
control of bronchial smooth muscle and the pharmacodynamics of asthma. In 
the area of diagnosis we are looking to the answers that will be provided 
by standardization of provocation testing based upon the challenges of 
exercise, tartrazine and aspirin inhalation, occupational exposures and 
pharmacologic agents. Studies under way which hopefully may contribute to 
preventive aspects include pathogenic factors of bronchial hyper-reactivity, 
food and environmental restrictions in infancy, the natural history of 
asthma, and the effect of immunotherapy on established asthmatic patterns. 



2-11 



Along with asthma there is evidence for immune mechanisms in a variety 
of other bronchopulmonary conditions associated with occupational and 
environmental factors, e.g., chronic bronchitis, fibrosis, granulomatous 
disease and hypersensitivity pneumonitis. Allergic reactions appear to be 
central to the pathogenesis of many types of occupational asthma and hyper- 
sensitivity pneumonitides. Among the isocyanates, (e.g., TDI), cotton 
linters and wood dust, experimental studies are focusing on a delineation 
of the respective irritant, allergic and pharmacologic pathogenic proper- 
ties. In addition to occupational and environmental asthma, immune 
mechanisms are being shown to play important roles in the production of 
certain fibrotic and granulomatous lung processes associated with inorganic 
dusts, e.g., beryllium, silica and asbestos fibers. Important relevant 
investigations include those concerned with the study of basic immune 
mechanisms, clinical expressions, epidemiologic studies especially in the 
plastics, pharmaceutical and wood industries. It is anticipated that 
specially designed materials and methods for bronchial provocation, 
respiratory function tests, immunologic skin tests and serologic studies 
correlated with environmental monitoring will help us assess important 
causative factors and measures for their control. While the largest number 
of instances of hypersensitivity pneumonitis occur following occupational 
exposure, (e.g., moldy fodder in farmer's lung, moldy sugar in bagassosis, 
bird droppings in pigeon breeders disease, mold spores in maple bark 
stripper's disease) there is increasing evidence that sensitizing organisms 
contaminating forced air heating, humidification and air conditioning 
systems produce comparable pulmonary disease risks in homes and offices. 

Important areas of ongoing research on hypersensitivity pneumonitis 
require the isolation of well defined antigens, controlled inhalation 
exposure, pathogenic mechanisms involving disposition of antigen, various 
parameters of the immune response, and immunopathologic and toxicity 
studies. Pertinent points for extended investigation will be focused on the 
immunochemical characteristics of suspected offending organic dusts and the 
definition of environmental factors involved in the generation and 
dispersion of such causative agents. 

An understanding of immediate hypersensitivity will require a precise 
definition of structural molecular characteristics of basophil and mast 
cell receptors for IgE and the regulation of IgE synthesis and secretion. 
In turn, an important goal of research is the development of methodology to 
block IgE fixation and release mechanisms. In this connection studies of 
eosinophil biology and eosinophil-mast cell relationships can be expected 
to provide insights into the potential residing within such cellular 
mechanisms for modulation of hypersensitivity reactions and/or possible 
secondary cytotoxic tissue damage. Since mast cell hyperproliferation and 
eosinophil migration are prominent accompaniments of helminth tissue 
invasion, studies dealing with the immunologic and immunopathologic aspects 
of parasitic infection assume increasing importance. 

Important areas of definition of basic immediate hypersensitivity 
mechanisms relate both to chemical mediators of inflammation and inter- 
actions between immunocompetent cells. Approaches to developing 



2-12 



pharmacologic agents capable of preventing or modulating allergic inflam- 
mation have been aided by emerging information on the two types of 
histamine cell receptors (HI and H2) and their specific antagonists capable 
of blocking this amine 's bronchial vascular effects. Additionally, infor- 
mation on the surface characteristics of subclasses of lymphocytes may make 
it possible to selectively impair the function of suppressor, helper or 
cytotoxic lymphocytes and possibly to approach the treatment of both 
allergic and immunodeficiency disorders by immune modulation or reconsti- 
tution. 

In the area of drug reactions we are looking to the possibility of 
providing tests of predictive value in allergic reactivity. Additionally, 
the morbidity due to allergic drug reactions could be considerably reduced 
by attention to such factors as isolation, purification and possible 
elimination of the defined antigenic moiety. Identification of the actual 
chemical determinants for haptens responsible for drug allergy will assume 
increasing importance in our search for the development of specific 
diagnostic reagents in drug allergy situations. In addition to concen- 
trating on immunological detection systems potentially capable of detecting 
cellular or humoral immunity applicable to drug sensitization an equally 
important approach is being directed to developing sensitive in vitro 
systems. Another approach which may eventually be helpful in reducing the 
incidence of serious drug reactions to specific drugs awaits ongoing 
studies on the identification of HLA or other genetic markers that may be 
associated with allergic reactivity based on altered drug metabolism, 
alterations in reactive metabolite information or innate immune responsive- 
ness. Additionally important in the area of drug allergy are approaches to 
understanding the mechanisms of drug modulation of the immune response 
associated with exacerbation of autoimmune disease during the drug therapy. 
We also are looking to those studies on immunotherapy in allergic disease 
based upon the induction of tolerance as models for the design of 
tolerogenic analogues of therapeutic agents having allergic potential. 

The importance of allergic mechanisms and immune system function has 
been demonstrated in several dermatologic disorders, especially atopic 
dermatitis, allergic contact dermatitis, urticaria and angioedema, the 
vesiculobullous diseases (pemphigus, dermatitis herpitiformis, bullous 
pemphagoid) necrotizing vasculitis and lupus erythematosus. Conversely, 
the study of these skin diseases has added considerably to our under- 
standing of basic immune mechanisms. An appreciation of the ready access 
of skin for study of disease expressions in systemic immunopathologic 
processes merits our continued attention. We have been especially 
concerned with studies directed to identifying disorders and their 
mechanisms where skin may be primarily involved as target tissue by immuno- 
humoral and/or cellular reactants, or where cells of the integument may 
serve as natural sources of sepcific antigens in immune processes. 
Important pathogenic mechanisms have been shown to include IgE involvement, 
activation of the complement cascade and formation and deposition of immune 
complexes. Atopic dermatitis once believed to be solely an immediate 
hypersensitivity disorder has now been shown to involve complex mechanisms 
including T cell abnormalities and possible immunodeficiency factors. As a 



2-13 



multifactorial disorder, work on this disorder is being concentrated on 
genetic predisposition and cyclic AMP cell biologic functions interacting 
with IgE mechanisms. In urticaria and angioedema where the role of 
chemical mediators of inflammation and vascular permeability can be 
demonstrated, the importance of complement activation and associated 
vasculitis are providing helpful approaches to the design of specific 
pharmacologic agents. 

The importance of immune mechanisms has been demonstrated in a variety 
of connective tissue, renal, gastrointestinal, endocrine, hemotologic and 
neoplastic conditions. Among those of established definition meriting 
immunopathologic experimental approaches are rheumatoid arthritis, 
hepatitis, inflammatory bowel diseases (ulcerative colitis and Crohn's 
disease), intestinal malabsorption syndromes, neurologic disorders 
(Guillain-Barre syndrome, myasthenia gravis, multiple sclerosis), erythro- 
blastosis fetalis, glomerulonephritis and interstitial nephritis, systemic 
lupus erythematosus, agranulocytosis, juvenile diabetes and T and B cell 
lymphomas. Over the past several years significant advances in basic 
immunology have exerted a major impact on our study and understanding of 
this broad range of diseases. Accordingly, many can now be classified into 
those related to specific immune mechanisms, e.g., immunodeficiency, immune 
complex deposition and autoimmunity. Accordingly, we are utilizing highly 
sensitive methodologies for the identification of etiologically important 
antigens, antibodies and immune complexes involved directly or indirectly 
in their suspected pathogenesis and techniques for identifying and 
fractionating various subpopulations of lymphocyte cells involved in 
immunologic reactivity and immunoregulation. Fruitful approaches include 
the identificaion of antigens and immune complexes in vasculitis related 
disorders and the identification of antibodies against cell surface 
receptors. The identification and functional delineation of subpopulations 
of lymphoid cells is expected to allow for a greater understanding of the 
primary and secondary immunodeficiency disorders and in turn an explanation 
of some immunoregulatory abnormalities in autoimmune diseases. We, there- 
fore, continue to direct our programmatic concerns to technological 
advances emerging from basic research to provide useful tools for probing 
disease states and applying basic immunologic investigative data to 
clinically relevant disorders. 



2-14 



ALLERGY AND CLINICAL IMMUNOLOGY BRANCH 

A. Scope 

This Branch is concerned with the etiology, pathogenesis, diagnosis, 
prevention and treatment of both naturally occurring and acguired allergic 
and immunologic diseases . 

Relevant studies of allergic diseases supported by the Branch include: 
(1) factors, both primary and predisposing, which contribute to the produc- 
tion of asthma, such as extrinsic allergy, infection, abnormalities of the 
sympathetic nervous system, cellular and chemical mediators of inflammation, 
and pharmacologic agents,- (2) immediate type hypersensitivity and its 
disorders (allergic rhinitis, atopic dermatitis, urticaria and angioedema) ,- 
(3) allergic phenomena affecting respiratory, gastrointestinal and 
cutaneous tissues; (4) allergic reactions and disorders caused by insect 
bites and stings, foods, airborne allergens, and infectious agents,- (5) 
humoral antibody, particularly IgE, and the chemical mediators released by 
the interaction of antigen and antibody on target cells,- (6) therapy and 
prevention of allergic disorders and hypersensitivity reactions by immuno- 
therapy with specific antigens or drugs,- (7) mechanisms of delayed hypersen- 
sitivity and contact dermatitis,- (8) mechanisms of drug reactions and 
chemical sensitization,- and (9) isolation and chemical characterization of 
the active fractions of known allergenic agents; and (10) epidemiologic and 
environmental studies designed to ascertain those agents or substances 
which may be of clinical relevance to allergic individuals — either as 
causal or contributory to their disease process. 

In the area of immunologic diseases, the Branch activities are focused 
on studies of the underlying mechanisms of disease and the application of 
basic knowledge to the etiology, prevention and management of immunologic 
disorders. Either or both of two disciplinary approaches. Clinical Immun- 
ology and Immunopathology , are involved in this effort. Studies in Clinical 
Immunology are directed toward acquired and inherited diseases associated 
with dysfunctions of the immune system. Immunopathology studies include 
genetics, cytology, biochemistry, physiology and pharmacology of the immune 
system. Relevant areas supported by the Branch include studies of (1) 
immune deficiency diseases arising from primary defects in development or 
maturation of the immune response or secondarily resulting from disorders 
affecting immune responses, (2) clinical manifestations mediated by 
products of lymphocytes, (3) diseases associated with immune complexes and 
autoimmune phenomena, (4) immunodermatology , i.e., immune disorders 
involving the cutaneous system, and (5) immunotherapy of disease processes. 

The ultimate goal of the Allergy and Clinical Immunology Program is to 
promote the acquisition, translation, and application of research findings 
to the diagnosis, prevention, and treatment of allergic and immunologic 
diseases. To achieve this goal, the Branch is responsible for and manages 
Programs of Institute Emphasis (PIE) in Asthma and Allergic Diseases, 
Immunologic Diseases, and Immunology Centers and Program Projects. Colla- 
borative clinical as well as basic research studies are supported among 
Centers and Program Projects in an effort to take advantage of the 

3-1 



nationwide character of these programs and to facilitate achieving our 
goals. To support these efforts, the Branch funds targeted contract efforts 
and also obtains and distributes selected research reagents and materials 
to investigative allergists and clinical immunologists. 

B. Awards and Support Levels 

Allergy and Clinical Immunology 

FY '79 Awards 

Award Mechanisms Number Amount 



Research Grants 
Career Awards and 
Career Development Awards 
Subtotal 

Fellowship Awards 
Training Awards 

Subtotal 



Contracts 



Total 



151 

21 
172 

16 
24 
40 

10 
222 



$ 15,930,164 

783,711 
$ 16,713,875 

216,015 

1,611,585 

$ 1,827,600 

452,156 
$ 18,993,631 



Total Costs, estimated using a 40% overhead for indirect costs. 

The distribution of these awards by area of study is approximately 30% 
for asthma and allergic diseases and 70% for immunologic diseases. Approxi- 
mately 34% of these awards were for competing new or renewal applications; 
the remainder represents commitments to support awards made in prior years. 

Support for the PIE activities is included in the research grant 
category. During FY '79, the Branch supported these activities with awards 
for 15 Asthma and Allergic Disease Centers at a total cost of $1,787,467, 6 
Clinical Immunology and Immunopathology Program Projects at a total cost of 
$3,093,499, and 4 Centers for Interdisciplinary Research in Immunologic 
Diseases at a total cost of $1,184,446. 

C. Program Areas and Highlights 

1. Asthma and Allergic Disease Centers Program (AADC) 

A network of 15 Asthma and Allergic Disease Centers, located through- 
out the United States, is actively engaged in a collaborative fashion to 
gain new knowledge and insights into the field of allergic disorders 
including those manifestated in the upper and lower respiratory tract, 
skin, and other organs such as kidney, gastrointestinal tract, etc. 
Research, both fundamental and applied, is conducted; and through this 
multi- faceted approach, methods have already been developed to improve the 
diagnosis and treatment of asthma and other allergic diseases. Although 



3-2 



the emphasis of the Centers Program is directed toward a better under- 
standing of the basic mechanisms involved in the various allergic disorders, 
they also have provided the field of allergy with unique academic resources 
in the discipline of allergy. 

By serving as referral centers for patients in their areas, by 
engaging in collaborative clinical trials to assess diagnostic and thera- 
peutic discoveries, and finally by acting as an important resource for 
training academic allergists for the future , they have had a profound 
impact on the delivery of health care to allergic individuals. Separately 
each of these Centers have also been successful in competing for additional 
support through either the regular grant program, training grants, or 
research career awards. 

At the Eighth Annual AADC Workshop in Bethesda on November 30 to 
December 1, 1978, directors and professional staffs from the 15 Asthma and 
Allergic Disease Centers (over 125 participants) met at NIH to review 
progress by grantees in this Centers Program. While the presentation of 
scientific papers (more than 50) provided the focal point of interest, the 
ultimate success of this workshop can be measured better by the broad and 
open discussion and the significant opportunities for investigators to 
exchange ideas for future study. A measure of the importance of this 
meeting came from the overwhelming requests by people outside the Centers 
Program to participate. 

Brief highlights of some of the activities conducted by our Asthma and 
Allergic Disease Centers follows. 

John Salvaggio, AI 13401, Tulane University and his colleagues have focused 
on Environmental/Occupational Hypersensitivity lung diseases. Purification 
of coffee and castor bean allergens enabled them to perform studies showing 
that workers in the coffee industry were sensitized by exposure to sacks 
that had previously been used to transport castor beans. Persons with 
asbestosis and silicosis have been found to have a high frequency of anti- 
nuclear antibody, a finding which may lead to better understanding of the 
pathogenesis of fibrotic lung disease. Finally, studies of mold sensitive 
asthma have progressed with clarification of antigenic relationship among 3 
species of aspergillus — an important effort in attempting to clarify and 
control environmental sources of these allergens. 

R oy Patterson, AI 11403, Northwestern University has continued efforts to 
reduce the allergenicity of ragweed antigen E while retaining antigenicity 
by use of polymerized fractions,- preliminary clinical trials with this 
material has demonstrated efficacy when compared with monomer preparations 
but with greater safety. Expanded trials in collaboration with other 
centers are underway. 

Ric hard Hong, AI 10404, University of Wisconsin and colleagues have 
continued studies investigating the role of viral infections in predis- 
posing or causing asthma. 



3-3 



Osca r L. Frick, AI 11010, University of California at San Francisco 
similarly has been in following children prospectively in order to clarify 
the meaning of a coincidental association between certain virusinduced 
colds and flu- like symptoms and the onset of allergy during the first year 
of life. They found in a small group that antibodies to influenza- like 
viruses and certain environmental or food materials (dog, cat, house dust, 
grass or cow and soy milk) increased at time of onset of allergic symptoms. 
Studies were conducted to learn whether certain viruses can act to alter 
the immune system so as to trigger the onset of allergy. 

Kenneth P. Mathews, AI 10391, University of Michigan has continued studies 
of "vasomotor rhinitis" in an effort to define the varied mechanisms by 
which people may be affected. Clinical trials are also being conducted 
assessing efficacy of intranasal treatment with gluteraldehydeaggregated 
ragweed extract in treatment of ragweed hay fever--which if successful 
could greatly reduce morbidity and cost to patients. 

P hilip S. Norman, AI 10304, Johns Hopkins University and colleagues have 
continued studies of modified allergens or allergoid (formaldehyde modified 
whole extract and allergen). Also they provided fundamental data which 
permitted commercial release of venom extracts in prevention of anaphylaxis 
from bee stings. A double blind controlled trial is in progress to study 
the effects of Rinkel immunotherapy — no difference has been found to date 
between this and standard immunotherapy. 

G erald J. Gleich, AI 11483, Mayo Foundation and colleagues, taking advan- 
tage of ongoing studies to refine allergens and develop sensitize 
diagnostic tests, have shown that efficacy of ragweed pollen immunotherapy 
is associated with reduction of activity of IgE antibodies in the radio- 
allergo-sorbent test, thus giving a means of precise monitoring of efficacy. 

2. Centers for Interdisciplinary Research in Immunologic Diseases (CIRID) 

In September 1978, 4 Centers for Interdisciplinary Research in Immuno- 
logic Diseases were funded by NIAID. This program is designed to foster 
integration and coordination of research projects in clinical immunology 
being pursued in other clinical specialties (e.g. dermatology, pulmonary 
medicine, hematology, nephrology, rheumatology, infectious diseases and 
otorhinolaryngology) with those in basic research (e.g., immunochemistry, 
microbiology, virology genetics, biochemistry, pharmacology, general physi- 
ology, and pathology). An important additional component of these Centers 
is the funding of specific projects' in demonstration/outreach activities, 
which are designed to impact on the health care of the community in a 
tangible and immediate way by bringing research advances directly to the 
patient. While it is obvious that during the start up year efforts are of 
necessity preliminary in nature, the following is intended to provide an 
idea of how progress is anticipated in this area. 

At UCLA ( John Fahey, AI 15332 ) an evaluation of a supplementary program of 
nonmedical intervention on the course of childhood asthma is being con- 
ducted by (E. Lewis, M.A. Lewis & G. Rachelfsky) . A curriculum has been 
designed to teach children and their parents what asthma is, to recognize 

3-4 



initial symptoms, and then to utilize self -management techniques such as 
relaxation and breathing exercise to terminate or minimize each episode; to 
recognize indications for use of certain pharmacologic agents; and finally 
to conduct field trials. Early evidence from an initial group of 10 
children and their families suggest that the desired level of information 
can be acquired and more importantly that families are enthusiastic in 
their participation. 

John Leddy, AI 15312, University of Rochester has designed and implemented 
a 12session concentrated course in Clinical Immunology for physicians and 
other medical personnel (e.g., nurse practitioners) affiliated with out- 
lying community hospitals in the large semi- rural region served by the 
University of Rochester. It is hoped that this will lead to better compre- 
hension and therefore diagnosis and treatment of immunologic diseases in 
rural upstate New York. To prove this point a means to assess the benefit 
to the patient will be undertaken not only by sequentially testing partici- 
pants, but also by tracking referrals and as well open lines of 
communication fully between the CIRID and the regional community. 

Joseph Bellanti, AI 15321, Georgetown University and his collaborators have 
sponsored a symposium on public concerns of immunization (October 1978) as 
part of a broader effort to facilitate the transfer of basic science know- 
ledge to the general public. They have also initiated an Asthma Education 
Intervention Project emphasizing self -management techniques for pediatric 
asthmatics and their parents. They have further initiated study of 
prevalence of asthma and other allergic/ immunologic disorder and cost and 
utilization of health services in the metropolitan Washington, D. C. area. 

Char les W. Parker, AI 15322, Washington University St. Louis has 
initiated a program whose intent is similar to that of the Rochester CIRID, 
in that it is designed to enhance knowledge of immunologic disorders in the 
urban community served by the CIRID. 

It should be mentioned in closing that while this report has emphasized the 
outreach/demonstration activities of the 4 CIRIDs , the largest proportion — 
more than 90 percent--of their efforts continue to relate to basic and 
clinical research. 

3. Program Projects in Mechanisms of Immunologic Diseases 

Program Projects were established to encourage the development of 
collaborative basic science and clinical investigation in immunologic 
diseases. As such this program is designed to further investigate under- 
lying mechanisms of disease and to enhance basic knowledge relevant to 
eiology, prevention and management of immunologic disorders. Studies are 
effected from either one of two disciplinary approaches: clinical immun- 
ology or immunopathology. Clinical immunology studies are directed toward 
acquired and inherited diseases associated with dysfunctions of the immune 
system. Immunopathology studies include specific areas of genetics, cyt- 
ology, biochemistry, physiology, and pharmacology of the immune system and 
its disorders . 



3-5 



In FY 1979 there were 7 active grants in this program. The following 
will be an attempt to present sufficient detail from each to show the depth 
and breadth of this program. 

The project headed by Frank J. Dixon (AI 07007 - Scripps Clinic and 
Research Foundation) is designed to gain a better understanding of the 
molecular basis of diseases caused by aberrant immune responses. Its focus 
is upon 1) normal controls of immune responses, 2) the means by which 
unusual presentation of an antigen, particularly associated with chronic 
viral infections, may initiate pathologic responses, 3) the chemistry and 
function of the several mediator systems which are activated by immunologic 
reactions and cause injury, and 4) the multiple complex immunopatho logic 
events found in spontaneous or experimental immunologic disease. The 
diseases most immediately related to this research include glommerlar 
nephritis, systemic Lupus erythematosus, rheumatoid arthritis, other auto- 
immune diseases, acute and chronic viral infections, allergic and anaphy- 
lactic reactions, allograft rejection, and disorders of complement and 
Hagemann factor occurring in mediator systems. 

Measurements of immune complexes and biologic fluids appear to be 
providing useful information helpful in monitoring the clinical courses of 
patients and guiding therapy. It is hoped that isolation of immune com- 
plexes may contribute to the identification of pathogens and diseases of 
unknown etiology, and to the development of practical diagnostic and thera- 
peutic measures. An example of the value of such studies relates to the 
belief that the immune complexinduced degenerative vascular lesions seen in 
murine Lupus may have relevance to degenerative arterial disease in man; 
supported by the knowledge of increasing occurrence of coronary artery 
disease and myocardial infarction in humans with Lupus, confirmed by pre- 
liminary studies in autopsies, as well as examination of the deposition of 
immune complexes in arterial walls. It is thus thought that repeated 
vascular deposition of immune complexes associated with incidental immune 
reactions might provide a significant component in the genesis of athero- 
sclerotic disease. 

Studies by Joseph Feldman as part of this program project have deter- 
mined that survival times of skin grafts, repeatedly applied to BN and 
Lewis rats, were unaffected by the levels of circulating immune complexes, 
and in rats grafted with allogeneic kidneys (vascular graft) and in dams 
repeatedly impregnated, no circulating immune complexes were detected. The 
signifance of these studies is related to the desire to examine the hypo- 
thesis of the aging process and neoplasic (which occur with increased 
freguency in aged hosts) may well be associated with some membrane changes 
(microviscosity, cholesterol, phospholipid) and with cytoskeletal membrane 
interactions that revealed deficiencies in immune surveillance and response. 

In vivo studies by Charles G. Cochrane have examined the participation 
of the Hagemann factor system and complement component in the development 
of immunologic injury, which were made possible by biochemical analyses of 
the individual components and their mechanisms of action. With the avail- 
ability of components of the Hagemann factor system in both rabbit and 
human, together with methods for their detection, definitive experiments 

3-6 



were made possible to detect the active participation of such components, 
thus indicating that any deficiency in this pathway will abrogate humoral 
mediated destruction of virus-infected cells. 

Studies by Curtis B. Wilson have concentrated on examining the role of 
monocytes and macrophages in immune-complex induced glommerular nephritis 
in rabbits. An extension of previous studies regarding the contribution of 
monocytes and macrophages have shown that monocytes and macrophages can be 
recovered in large numbers by tissue culture of isolated glommeruli taken 
from rabbits with the most proliferative forms of experimental serum sick- 
ness glommerular injury. It is hoped that results of these studies will 
enable characterization of differing patterns of immune complex localiza- 
tion in kidneys and efforts have been made to relate these to host genetic 
factors which could influence the susceptibility of individuals to immune 
complexinduced glommerular injury. 

William 0. Weigle has continued studies examining the activation of T 
and B cells and the interaction of those cells with macrophages in the 
induction of immune tolerance and autoimmunity. These studies led to the 
description of a model of immunologic tolerance to human gammaglobulin 
which is maintained in the absence of demonstrable suppressor cells. Other 
studies showed that the tolerance state induced in B cells in adult mice to 
human gammaglobulin is the result of central unresponsiveness rather than 
of antigen blockade. A thymus replacing factor (TRF) has been produced by 
the activation of spleen cells with concanavalin A, the cell type in the 
spleen responsible for the generation of thymus replacing factor is a T 
cell, that is, LYT 1+ and LYT 2-. Preliminary studies indicate that TRF 
obtained from mice sensitized with sheep red blood cells support a primary 
response in nude mice. Further studies by H ans Muller-Eberhard into the 
mechanism and functional role of complement have shown that the isolated 
component mixture containing the proteins of the alternative and of the 
membrane attack pathway is capable of generating bactericidal activity, 
thus showing that the system is complete and antibody is not required for 
alternative pathway initiation. These observations relate the cytolytic 
alternative pathway to natural resistance to infection. Thus, in vivo, 
although bacteria are usually phagocytized and killed intercellular ly, 
complement is required for opsonization (C3B) and to some extent for intra- 
cellular killing (C5 through C8) . Individuals with homozygous deficiency 
of one of the precursor proteins of the membrane attack complex have , as 
expected, an increased susceptibility to infections, particularly of the 
Neisseria type. Thus, these reported structural and functional analyses of 
the membrane attack complex have provided a basic understanding of the 
manner in which complement lysis envelope the viruses and kills bacteria in 
animal cells. 

Hugh 0. McDevitt, AI 11313, Stanford University has been engaged in an 
overall effort to examine Humoral, Cellular and Genetic Mechanisms in Auto- 
immunity. Studies concerned with the relationship between the major histo- 
compatibility complex (MHC) have demonstrated the presence of immune 
complexes in patients with two related forms of arthritis, ankylosing 
spondylitis and Reiter's syndrome. The suggestion is made that these 
patients may have an abnormal immune response to certain bacterias, which 

3-7 



leads to formation of immune complexes which may be the cause of the 
arthritis. Other diseases being studied for possible genetic suscepti- 
bility include rheumatoid arthritis, multiple sclerosis, diabetis mellitus, 
and systemic Lupus erythematosus. Further studies aimed at improving MHC 
typing are being conducted by F. Carl Grumet in this program. 

A. Cacin has been following seroepidemiologically persons with enteric 
infection and Reiter's syndrome, finding that Shigella flexneri 2a and 
flexneri 1 cause Reiter's disease. 

Samuel Strober has been conducting studies assessing the use of total 
lymphoid irradiation in treatment of auto-immune diseases and in the pre- 
vention of organ graft rejection. Four of 6 patients whose rheumatoid 
arthritis failed to respond to all conventional therapies had significant 
improvement in their disease activity which lasted from 6 — 18 months. 

Roy Patterson, AI 11759, Northwestern University and his colleagues 
have concentrated their efforts on immune aspects of lung disease. Main- 
tenance of a colony of rhesus monkeys giving consistent asthmatic responses 
to ascaris as well as control animals has permitted investigation of 
response to prostaglandins and histamines as well as other allergic 
mediator substances. Studies of airways physiology as well as peripheral 
immune responses have been made possible. Dog models of allergy have also 
been utilized. Studies of human asthma have investigated physiologic 
responses to inhaled antigens and methacholine. Hay fever patients were 
found to respond normally in contrast to asthmatic subjects when challenged 
with methacholine; similarly however, they responded to asthmatics when 
challenged with antigen-this response was not mediated by cholinergic 
reflexes (atropine pretreatment made no difference). This group also has 
shed further light on the role of trimellitic anhydride (TMA) as an occupa- 
tional hazard which may produce not only asthma but pulmonary infiltrative 
disease. Their development of accurate diagnostic tests will permit exten- 
sion of these findings to a large number of potentially exposed workers. 

Other investigations of environmental hazards have included the obser- 
vation that di-2-ethyl-hexyl-phthalate was found to migrate from polyvinyl 
chloride tubing into human blood in dialysis patients. Studies are under- 
way to learn whether this finding can be related to the clinical obser- 
vation that such foreign substances could produce the accelerated form of 
atherosclerosis in dialysis patients or be responsible for other vascular 
damage . 

F red S. Rosen, AI 05877, Children's Hospital Medical Center, Boston 
and his coworkers have been examining the reactions and metabolism of 
immune proteins and cells. Continuing studies of genetic deficiency of 
complement components led to the description of the first homozygous (C3 
deficient individuals and that C3 nephritic factor was an IgG molecule (an 
autoantibody to C3bBb) . Further they documented a polypeptide produced in 
plasma of patients with hereditary angiodema, affects the postcapillary 
venule of man and is generated from the second component of complement by 
action of plasmin. Patients with acquired or common varied agammaglobu- 
linemia were found to have no B cells or to have B cells that were 



3-8 



unreactive in the presence of lymphocyte mitogenic factor. Restoration of 
function in some of these individuals has been accomplished with use of 
steroids. Bone marrow transplants were found to restore function in 
Wiskott-Aldrich Syndrome and adenosine deaminase deficiency. Finally, the 
dermatomyositis-like syndrome of agammaglobulinemia was found to result 
from infection of the CNS with ECHO virus. It should be mentioned that all 
of these studies are being accomplished in the setting of a large referral 
center for study of immunologic disorders, especially in children. 

David G. Marsh, AI 13370, Johns Hopkins University has continued 
studies of the Genetics of Immune Response in Man. Studying a group of 400 
allergic patients and their families since 1971 has resulted in the obser- 
vation of significant associations between specific HLA types and specific 
IgE mediated allergic responses to 4 different highly purified pollen 
allergens. In addition an IgE regulator gene, not linked to HLA, has been 
found to exert an additional control over specific IgE responses. Continued 
studies are planned to test the hypothesis that under immunogenically 
limiting dosage exposure, response to a specific antigen is regulated by 
relatively few immune responses (IR) and immune suppressor (IS) genes and 
that all IgE antibody responses may be controlled by a gene which regulates 
the overall synthesis of IgE to any specificity. 

4. Mechanisms of Allergic Diseases 

Studies within this program have been concerned with investigations of 
the etiologic factors, pathogenetic mechanisms, diagnostic measures, and 
therapeutic approaches related to the effective management of allergic 
disorders. 

The release of inflammatory molecules from tissue mast cells is a 
necessary step in the evolution of allergic reaction of the immediate type. 
It is interesting to note that such reactions may also be relevant in other 
immune responses, such as those implicated in graft rejection, poison ivy 
and immune kidney diseases. 

Ti mothy J. Sullivan, AI 10090, Washington University , in studies 
investigating the mechanism by which mediators are released from mast 
cells, has detected reactions which appear to control the release of his- 
tamine. The formation of 1,2 diacylglycerol (DAG) appears to be a key 
step, and drugs of the me thy Ixan thine class have been shown to inhibit the 
formation of DAG and mediators in parallel. From these studies it is hoped 
that drugs can be developed to control mast cell mediated inflammation. 

K imishige Ishizaka, AI 10060, Johns Hopkins University , also looking 
at the mechanism of mast cell secretion, found that bridging of receptor 
molecules rather than polymerization of cell-bound IgE molecules was 
responsible for mast cell triggering. 

L awrence Lichtenstein, AI 07290, Johns Hopkins University , has shown 
that human basophils have an adenosine receptor which is linked to 
adenylate cyclase and increases cyclic AMP. When this receptor is occupied 
by adenosine, histamine release is stopped. Interestingly, this receptor 



3-9 



is antagonized by theophyllin, a drug commonly used for the treatment of 
allergic disorders. 

M arshall Plaut, AI 12810, Johns Hopkins University , has studied the 
role of histamine type 2 receptors in vivo in man by use of the drug, 
cimetidine (an H2 blocker), and found no significant changes in either 
immediate type or delayed type skin reactions. Continued studies such as 
these should help clarify the role of mediators in acute allergic responses 
and further permit development of pharmacologic manipulation of these 
immune responses. 

St ephen Wasserman, AI 00254, Harvard University , has shown that 
heparin is present in human mast cells and that in a rat model of allergic 
disease, mast cell enzymes have been found and released after immunologic 
challenge. These enzymes, beta glucuronidase and hexosaminidase, could 
destroy connective tissue and explain prolongation of problems in human 
allergic diseases. Finally, he showed that disodium cromoglycate, which 
prevents mast cell mediator release, proved effective in the treatment of 
systemic mastocytosis. Andrew Grant, AI 12621, University of Texas Medical 
Branch, Galveston, examining histamine release from basophils, has 
continued studies assessing the role of C5A, a small fragment from the 
fifth component of complement, which has been found to bind directly to the 
blood basophil, inducing release of histamine. 

The role of reaginic antibody, also known as IgE, which circulates in 
the blood, is well established in the allergic response. Many investi- 
gators have been examining ways to determine how this immune response is 
regulated, in order to learn whether or not this response could be 
abrogated in some general way in order to benefit allergic individuals. 
For example, Kimishige Ishizaka, AI 11202, Johns Hopkins , showed that the 
IgE antibody response in the mouse is dependent upon the dose and nature of 
antigen--that is, induction of antigen-specific suppressor T cells is 
involved in the transient IgE antibody response found in the genetically 
high IgE responder animal. 

A ndrew Saxon, AI 15251, UCLA , found that control of B lymphycyte 
production was dependent upon the presence of T lymphocytes; that radiated 
T lymphocytes provided helper function without suppression of IgE at high 
T-to-B cell ratios. 

A lbert Kalisker, AI 13592 , has shown among mouse strains normally 
insensitive to ragweed antigen, who have been treated with moderate doses 
of cyclophosphamide, results in an enhancement of their ability to produce 
high anti-ragweed IgE titers. By way of explanation, it is thought that the 
drug eliminated from those mice cells which were involved in the actual 
suppression of IgE production. Other investigators such as Rebecca Buckley, 
AI 12026, Duke University, has also been examining the role of suppressor 
cells in the IgE response. Samuel B. Lehrer, AI 14891 , additionally has 
been assessing and detecting IgE antibody producing cells in different 
lymphoid organs, and selectively suppressing IgE production in the mouse 
with anti-IgE. Alec Sehon, AI 14526 , has carried the studies of IgE to 
penicillin allergy, showing that suppression of IgE antibodies to a 

3-10 



constituent of penicillin, that is, the benzyl penicilloyl (BPO) group was 
induced by the administration of non- immunogenic conjugates of BPO with 
normal gammaglobulins of the corresponding species . David Katz at Scripps 
Clinic has shown that IgE levels can be altered by a humoral factor which 
may either suppress or enhance the igE response in genetically different 
animals. It is hoped that this may lead to an ability to alter man's 
response to external allergens and enable investigators to modulate the 
immune response in such a way as to abrogate any high Ige responders. If 
this material can be deemed safe for clinical trials, it could be an alter- 
nate and universal "therapy" for allergy. 

Other studies related to IgE antibody include those of Frederick J. 
G rundbacher, AI 12360, Peoria School of Medicine , who has shown that low 
levels of IgA may constitute a predisposing factor to the development of 
allergies in children. F loyd J. Malveaux, AI 05385, Johns Hopkins has 
shown that the total number of IgE receptors per basophil is closely 
correlated with the level of circulating IgE. This relationship implies a 
genetic association between serum IgE and the number of basophil IgE 
receptors with a modulation of the number of receptors by circulating IgE. 

Charles W. Parker, AI 10405, Washington University , has been studying 
the IgE receptor on the rat basophilic leukemia cell line, which has main- 
tained in tissue culture. This receptor has been solubilized and purified 
and may permit possible design of analogs to this receptor which would 
compete with the receptor for IgE, thus inhibiting its ability to respond 
when confronted with an external allergen. 

The interaction of IgE with mast cells and basophils has been amply 
demonstrated. Hans L. Speigelberg, AI 10734, Scripps Clinic and Research 
Foundation, has shown that IgE binds to a subpopulation of bone marrow 
derived lymphocytes. Peripheral blood contains approximately 4 percent IgE 
binding cells in contrast to tonsils and spleens which contain four to five 
times as many. While it is unclear as to the biologic role of the lympho- 
cytes binding IgE, studies are underway to determine the number of IgE 
binding sites binding B cells in patients with allergies and other 
disorders. 

5. Asthma and the Other Allergic Diseases 

Studies within this program have been concerned with investigations of 
the etiologic factors, pathogenetic mechanisms, diagnostic measures, and 
therapeutic approaches to the effective management of various allergic 
disorders. Among some of the diseases covered are asthma, allergic 
rhinitis, hay fever, hymenoptera sensitivity, drug allergy, as well as 
basic studies in allergic dermatitis. 

Approximately 9 million Americans have asthma, and although they may 
present with a variety of clinical presentations, in essence, it consti- 
tutes a disorder of reversible airways obstruction. This obstruction has 
three major elements: 1) contraction of bronchial smooth muscles, 2) 
increased or retained secretions of mucus, and 3) inflammation of the 
respiratory tract mucosa. In individual asthmatics attacks can be provoked 



3-11 



by exposure to specific allergens, and some of these individuals have other 
allergies such as allergic rhinitis. On the other hand, many asthmatics do 
not have a demonstrable allergy, and in these individuals a variety of 
infectious, nervous (adrenergic and cholinergic), genetic or inflammatory 
factors may play a primary predisposing or contributory role. The program 
on asthma is structured to investigate all of the major parameters contri- 
buting to describing the mechanisms of causation in this disease. Areas 
under investigation include: the natural history, allergens, broncho- 
provocation challenge, animal models, pulmonary function testing, chemical 
meditors, cell mediators, agernergic agonists and antagonists, parasym- 
pathomemedic agents, drug allergy, diagnostic methodology, and therapeutic 
agents . 

Efforts to elucidate what causes asthmatic reactions in individuals 
have included studies like those of Stanley P. Gallant, AI 00304, University 
of Utah , who has examined the neutrophils of asthmatic individuals, showing 
that the number and quality of beta agernergic receptors to a variety of 
pharmacologic agents are essentially normal. Further dissection of the 
pathways of how while blood cells from asthmatic individuals respond should 
be helpful in elucidating means of pharmacologic action. The role of 
respiratory viral infections which induce asthma has continued under the 
leadership of Richard Hong, AI 10404, University of Wisconsin , whose colla- 
borators have shown that respiratory virus infection, particularly with 
rhino virus, may produce transient airway obstruction similar to asthma, 
and that the white blood cells from these individuals with rhino virus 
illness do have an impaired inhibition of lysosomal enzyme release from 
neutrophils. These observations have been extended to influenza virus, 
which has been shown to induce changes in the intracellular levels of 
cyclic AMP. Hopefully, these findings may provide leads for using either 
measures of peripheral blood leukocyte responses as indicators of the 
asthmatic condition, or to monitor therapeutic modalities. Other investi- 
gators are similarly engaged in these kinds of studies. 

During the past several years, broncho-provocation and inhalation 
challenge techniques have been refined to study how to induce asthmatic 
attacks, as well as being employed to corroborate the diagnosis of asthma 
and to ascertain the constituents of the chemical, cellular, and physio- 
logic events in the ontogeny of the asthmatic response, as well as to 
evaluate pharmacologic agents . 

Againdra Bewtra, AI 14233, University of Iowa has established that the 
sensitivity of airways to methacholine and histamine are comparable in 
asthmatics. Further studies have demonstrated that the chemical compound 
SCH-1000 protects against methacholine challenge on both the large and small 
airways; however, it protects against histamine only on the larger airways. 
Studies of responses to allergens are underway. Similar broncho-provocation 
studies have been conducted in asthmatic patients by Roy Patterson, AI 
117 59, Northwestern University with inhaled antigen, such as ragweed 
antigen E, as well as with methacholine. He has found evidence that 
asthmatic subjects appear to be more sensitive to inhaled antigen when 
tested after a period of natural environmental exposure than before. It has 
been impossible to date to correlate pulmonary function findings with 

3-12 



clinical results of immunotherapy. Since the inhalation of allergen per se 
can be a highly sensitizing event, as well as a provoking event, studies of 
broncho-provocation challenge with use of allergens have been impeded some- 
what because of these undesirable side effects. 

As an alternative, Dr. Patterson has been following a colony of asth- 
matic monkeys which appear to be to date the best animal model of asthma 
which may simulate the human disease condition, especially insofar as 
airways are concerned. For example, this antigen- induced asthma occurs only 
in selected monkeys, which like human subjects, have hyper-reactive airways. 
Dr. Patterson has shown that living antigen reactive mast cells reside free 
in the bronchial lumens of these monkeys, as well as in dogs and man, and 
that these cells can transfer the asthmatic type airway responsiveness to 
non-reactive animals. 

A variety of chemical mediators, such as prostaglandins, histamines, 
and other agents, which have been postulated as being important in 
describing human airways responses, have been tested in this animal model 
and shown to simulate the human asthmatic condition. Such a model will 
provide for evaluating therapeutic agents prior to tests in humans and 
offers the unique opportunity to perform repetitive studies under 
controlled conditions that otherwise could not be undertaken in humans. 

Additional basic studies which may prove relevant to the asthmatic 
condition include those of H. Benfer Kaltreider, AI 12296, University of 
California, San Francisco , who has studied immune functions of the lung, 
showing that sensitized lymphocytes capable of killing foreign cells could 
be induced to appear in lung tissue by either local or intraperitoneal 
immunization in animals. These killer lymphocytes have been found to be 
critical for the control of virally- infected or neoplastically induced 
cells. Additionally, alveolar macrophages may exert a suppressive effect 
on the responses of lymphocytes to several immune stimuli, and this appears 
to occur through substances such as prostaglandins, which may be released 
by the macrophages themselves. His efforts are at understanding the 
mechanisms of non-cellular and cellular suppression of local lung lymphocyte 
function, and thereby develop strategies for combating hypersentitivity 
reactions in the lung. Direct work studying alveolar macrophage function 
in asthmatic individuals is inhibited by the difficulties of performing 
bronchial lavage in individuals with hyperactive airways. However, studies 
are underway in several laboratories at this time, which should shed 
further light on these areas. 

Similar studies of basic immune functions of the lung have been per- 
formed by Barbara A. Nichols, AI 00294 , who has used the parasite model of 
Toxoplasma gondii to study the interactions between this parasite and lung 
macrophages . 

Ragweed allergy is a disorder affecting approximately 15 million 
Americans. For most individuals it is a mild seasonal disorder commonly 
called hay fever, which is characterized by eye irritation and nasal dis- 
charge; symptoms are usually relieved by antihistamines; however, in some 
individuals, although not life threatening, are a source of considerable 



3-13 



morbidity. In others ragweed has been indicated as being causal in 
producing an asthmatic-like syndrome. 

Most of the advances in understanding allergic rhinitis have occurred 
in this model, particularly in the areas of etiology, diagnosis, patho- 
genesis, treatment and prevention. Further progress can be expected to 
lead to greater understanding of factors involved in all allergic diseases. 

Pertinent in this regard is the difficulty in obtaining purified 
allergen preparations. Despite the fact that there are at least 100 
different proteins in ragweed, only five have been demonstrated to produce 
allergy in man. J. Donald Capra, AI 12796, University of Texas at Dallas 
has determined the chemical structure of the smallest of these five 
proteins, termed RA 5, and is completing studies on defining the structure 
of further components. More recent studies are making an effort to 
determine the amino acid sequence of cedar and sage allergens. T. P. King , 
AI 14422 , has prepared conjugates of purified Antigen E with non-immuno- 
genetic polymers. These conjugates of Antigen E show reduced allergenic 
activity, yet retain the immunosuppressive property of the native antigen, 
and would not have been possible without the clarification of antigen 
structure. He is continuing further studies in the immunochemical charac- 
terization of vespid venoms with similar goals. 

The clarification of the structure of individual allergens is impor- 
tant both chemically and biologically. Commercial suppliers of pollen for 
use in human desensitization work may vary in their source, so if an 
individual is receiving treatments in various parts of the country, there 
may be increasing or decreasing response to treatment. In addition and 
more importantly, if an allergist clinician should change the supplier of 
his pollen extracts or if the supplier should obtain pollen from a 
different region of the country, the potency of the extract might be 
markedly different. Efforts are underway in this program to provide 
standardized antigen materials. 

G erald Gleich, AI 07047, The Mayo Clinic , has successfully separated 
the two chains of Antigen E, the major allergen of ragweed pollen. Biologic 
activity of the individual chains was preserved, and studies are underway 
to determine the amino acid sequence of each chain, including especially 
those portions of the chains which are the determinants of the aller- 
genicity of the molecule. Presumably, if the ability to confer allergy 
resides in only a small portion of the molecule, then these studies could 
lead to a new and improved type of immunotherapy for ragweed pollen 
sensitivity. On the other hand, D avid Marsh, AI 09565, Johns Hopkins 
University , has focused his research on the highly basic proteins which are 
rapidly released from ragweed pollen grains, as contrasted with the acidic 
proteins, such as Antigen E, which are slowly released. Since allergenic 
reagents currently employed in immunotherapy contain a large propotion of 
acidic proteins, such as Antigen E, the addition of these rapidly released 
substances may lead to an improvement in immunotherapy. 

Several investigators, including Roy Patterson, AI 11403, Northwestern 
University , and P hilip Norman, AI 04866, Johns Hopkins University , have 

3-14 



pursued the development of chemically modified ragweed allergens termed 
allergoids. A variety of chemical agents (formaldehyde, urea, gluteralde- 
hyde, ethylene, glycol, etc.) have been employed, but the main allergen 
studied in immunotherapy is the more rapid induction of blocking IgG anti- 
bodies with greater safety than conventional materials now in use. Larger 
amounts of immunogenic material can be administered in a shorter period of 
time without the risk of generalized allergic reaction. Clinical trials 
are underway to assess the value of these materials and they should be 
licensed shortly for more widespread use. 

Other investigators such as Rebecca Buckley, AI 12016, AI 14314 , and 
AI 70830, Duke University , have utilized the purified ragweed antigens, 
such as Antigen E, which are supplied by our institute's allergenic 
reagents program. With these purified materials, both Drs. Buckley as well 
as Dr. Patterson have been able to demonstrate the synthesis of ragweed 
specific IgE from lymphocytes cultured in vitro, thus establishing a 
laboratory-based system for further in-depth studies of human allergic 
diseases. Because of the need for more widespread use of purified ragweed 
antigens, combined with the depletion of existing stock, it became 
necessary for the Allergy and Clinical Immunology Branch to acguire new 
materials. T. P. King, AI 82567 Rockefeller University , has produced RA 3, 
RA 5, Antigen K and Antigen E. These materials will be available to investi- 
gators and insure maintenance of a supply of highly purified and well- 
characterized allergens for further studies. Exciting recent advances have 
been further carried forward in the field of the sting of insects of the 
bee family called Hymenoptera, which induce an overwhelming and sometimes 
fatal reaction in sensitive individuals. While firm data is lacking, it is 
believed that about 2 million Americans may be sensitive to these stinging 
insects, and although the number of fatalities each year is not precisely 
known, it is believed to be less than 500. For individuals who develop an 
overwhelming reaction to a Hymenoptera sting, an injection of epinephrine 
may be lifesaving. Since exposure to these insects may occur in areas 
where prompt medical assistance is not available, it was important to 
develop an effective preventive measure. The Hymenoptera include honey 
bees, yellow jackets, hornets and wasps. Their venoms contain several 
vaso-active amines and other noxious simple chemicals which are responsible 
for the immediate irritation experienced by anyone stung by these insects. 
In addition, their venoms contain several proteins, and it is these 
proteins which are responsible for the induction of the hypersensitivity 
state and the subsequent development of anaphylaxis in very sensitive 
individuals . 

Until recently the only materials available for testing and treating 
allergic patients to insect stings were so-called whole body extracts which 
were prepared by grinding up insects. Through the collaborative efforts of 
L awrence M. Lichtenstein, AI 02870, Johns Hopkins University and Philip 
N orman, AI 10304, Johns Hopkins University , it was established that whole 
body extracts available to practicing allergists contain little if any 
active venom protein, and that their studies showed that injection 
desentization therapy with these extracts have virtually no utility in 
diagnosis or prevention of insect sting allergy. Subsequently, these 
investigators obtained venom itself, prepared solutions for clinical 

3-15 



evaluation. They found an excellent corrolation between results of venom 
skin testing with clinical history of Hymenoptera sensitivity. When these 
same venom materials were administered in a series of immunotherapy injec- 
tions to sensitive individuals, protection against serious reactions ensued 
in well over 95 percent of individuals. Other investigators have confirmed 
the efficacy of these materials, and in the spring of 1979, owing to their 
work, venom for desensitization was in fact made commercially available. 
Since this material has only been in the hands of practitioners for a short 
period of time, more data will be required to determine which individuals 
will require this form of treatment, further clarification of therapeutic 
regimens will have to occur, etc. What is clear, however, is that through 
clarification of the immunochemical properties of the active venom protein, 
efficacious immune therapy with this material has been possible, and the 
development of specific diagnostic procedures combined with skin testing, 
followed by immunotherapy in appropriate individuals, has resulted in 
protection in the vast majority of persons. 

6. Immunodeficiency 

The program on immunodeficiency is concerned with the etiology, 
ontogony, prevention, and treatment of structural and functional 
deficiencies of the immune system. Both naturally occurring and acquired 
disease states are included. Ongoing studies have focused on the 
congenital absence, failure of development, or other disorders affecting 
thymus or bone marrow cellular elements; abnormalities in production, 
inhibition or catabolism of immunoglobulin; deficiencies of specific 
complement components; and defective host defenses due to abnormalities of 
leukocytes. These naturally occurring or acquired defects of immunity 
provide a unique opportunity to expand the understanding of normal immune 
function. Only by its absence do we see a role for its presence in the 
sequence of steps which make up the normal immune response. Although these 
diseases are rare, the information gained is relevant to the general field 
of immunology and to many health problems including infectious diseases, 
allergy and arthritis. 

Certain immune deficient subjects have been shown to demonstrate high 
serum IgE levels. Rebecca Buckley, AI 12016, Duke University , has examined 
the function of accessory cells such as macrophages and polymorphonuclear 
white blood cells in these patients , and compared them with allergic 
individuals. Abnormalities have in fact been shown in both groups of 
persons. Richard Hong, AI 14354, University of Wisconsin at Madison , has 
suggested that the exaggerated response in these allergic individuals may 
be related to a defect in T lymphocyte regulation. Dr. Hong has also 
studied the role of thymus epithelial cells in reconstituting certain 
immuno-deficient patients. He is examining means to improve methods of 
culturing thymus tissue prior to transplantation, and learn by in vitro 
methods whether or not this is of benefit to such individuals. He has 
found that thymus glands may be preserved if kept properly in an incubator, 
and shown that thymic transplantation can provide restoration not only for 
immune function in classically immune deficient individuals, but has 
extended studies into work in cancer patients who have secondary immune 
deficiencies. Bone marrow transplantation has accomplished cures in 

3-16 



individuals with aplastic anemia, as demonstrated by Robert A. Good, AI 
11843, Sloan Kettering Institute for Cancer Research . In addition to 
generalized immune deficiencies, other important observations have been 
made which have permitted further extension in this exciting area of thera- 
peutics. For example, Ralph Wedgewood, AI 07073 , has shown that in the 
Wiscott-Aldrich Syndrome a combination of immune deficiencies may be 
observed, and further that a hereditary enzyme deficiency of adenosine 
deaminase could be reconstituted with use of bone marrow transplantation. 
Ronald C. Scott, AI 12617, University of Washington , by screening indivi- 
duals who are immunodeficient for adenosine deaminase and nucleoside 
phosphorylase, has provided further insight into biochemical causes of 
childhood immune deficiency states. 

Edward J. Moticka, AI 12191, University of Texas Health Science Center , 
Dallas , has studied the acquired immunologic incompetence which accompanies 
antigen- induced hypergammaglobulinemia. This interference with reactivity, 
an important regulatory mechanism for the immune system, may be responsible 
for the diminished immune capacity observed in patients with multiple 
myeloma. 

S andra S. Kaplan, AI 14218, University of Pittsburgh , studying the 
defect in polymorphonuclear leukocytes which occurs in the Chediak-Higashi 
Syndrome, which is almost uniformly fatal, has demonstrated that a 
defective blood platelet may be responsible for this susceptibility to 
infection because it fails to activate the white blood cell. Studies of 
drugs are underway to overcome this deficiency by activating leukocytes 
through biochemical changes similar to those mediated by normal platelets. 

Harvey J. Cohen, AI 00311, Childrens Hospital Medical Center, Boston , 
studying children with chronic grammulomatous disease, who are known to be 
missing an enzyme in their white cells, has preliminary evidence permitting 
the prenatal diagnosis of this disease by study of fetal granular sites 
obtained by amniocentesis. This work emphasizes how the study of basic 
mechanisms may have a broad range of potential diagnostic and therapeutic 
applications. 

Hugh Fudenberg, AI 13484, Medical University of South Carolina , has 
also been studying immune deficiency diseases and has described genetic 
markers on certain lymphocytes of patients with common variable agamma- 
globulinemia who develop a fatal kidney disease; and that IgA deficiency 
and X-linked hyper-IgM syndrome are heterogeneous diseases. Work on cystic 
fibrosis has shown a basic defect which may be related to a defect in 
alpha-2 macroglobulin in the serum. 

7 . Immune Complex Disease 

Immune complexes result from the binding of antigens by antibodies . 
Subsequently, components of the complement system also become involved. 
Such complexes, therefore, arise during the course of the development of 
the normal immune response. Under some circumstances, however, these 
entities appear within lesions and indeed are necessary for the development. 
The pathogenetic mechanisms by which immune complexes cause tissue damage, 



3-17 



particularly in the auto- mmune diseases with which they are increasingly 
associated are still poorly understood. The lack of sensitive, quantita- 
tive assays for all types of immune complexes has impeded work in this area 
to some extent. Recent studies by Vincent Agnelo, AI 70968, New England 
Medical Center Hospital , who has refined two such assays, one for rheuma- 
toid factor and anti-IgG antibody, and one for the ClQ component of 
complement, are helping to clarify these measures. An unexpected finding 
is that some rheumatoid factors crossreact with nuclear protein. A further 
advance by Douglas B. Cines, AI 05477, University of Pennsylvania was made 
by his utilization of a radio- immunoassay to quantitate IgG and the C3 
component of complement. This technique can be used also to estimate the 
affinity of an antibody for an antigen (R. M. Rothberg, AI 07854, Univer - 
sity of Chicago ) . 

W illiam P. Arend, AI 10975, University of Washington , has made an 
effort to determine what it is that renders an immune complex damaging. He 
reports that in rheumatoid arthritis, anti-IgG antibodies (rheumatoid factor) 
specific for the FC portion reacted with IgG to produce innocuous complexes, 
whereas anti-IgG antibodies specific for the FAB portion produced injurious 
complexes . 

Systemic Lupus Erythematosus is an auto- immune disease in which anti-DNA 
antibodies play a permanent pathogenetic role. The cause for the 
appearance of the auto-antibodies is being studied in a genetically defined 
mouse strain which develops an SLE-like syndrome spontaneously. Defective 
suppression of the immune response in these animals can break the self- 
tolerance usually seen (M. Eric Gershwin, AI 00193, University of 
California, Davis ) . Many people are studying the strain and have shown that 
the induction of tolerance to DNA can be accomplished by treatment with 
appropriately constructed antigen. Bernard Stollar, AI 14534, Tufts 
University has undertaken studies to define the chemical requirements for 
developing reagents that can specifically prevent the formation of anti- 
bodies to nucleic acid. In detailed studies of the specificity of 
tolerance, in studies done in collaboration with Yves Borel, AI 13867 , 
Boston Children's Hospital Medical Center , he has shown that tolerance to 
one nucleoside does not prevent the formation of antibodies to other 
nucleosides of DNA. This finding provides an approach to the study of the 
regulation of responsiveness and specific nonresponsiveness at the cellular 
level. The obvious hope in this regard is to develop information 
sufficient to permit appropriate manipulation of the immune response in 
order to effect therapeutic benefits. Thus, the development of specific 
immune tolerance would be a significant improvement over the use of non- 
specific immunosuppressive or anti- inflammatory therapy in the treatment of 
auto-immune disorders. 

Immune complexes which may be also associated with chronic infectious 
diseases and cause inflammation and tissue damage are also found to be 
involved in graft rejection. That they may have a beneficial effect is 
suggested by the evidence that such immune complexes can modulate the 
response to the antigen contained within them, and in fact prolong graft 
survival . Thus , Joseph P. Feldman, AI 07104, Scripps Clinic and Research 
Foundation , and associates have been studying circulating immune complexes 

3-18 



which appear after graft implantation and their effect on the host immune 
response. 

Many studies are now nationwide measuring serum immune complexes and 
trying to define their role in clinical disorders, whether they may be 
valuable in assessing diagnostic or other modalities. Continued emphasis 
by this program in developing expeditious ways whereby these measurements 
can be made standard and by which other investigators can compare data 
among several groups of individuals is of particular importance . 

8. Inflammation 

The process of inflammation is mediated by cells and by humoral 
factors. This process is essential for host defense against foreign sub- 
stances. However, in certain disease states, inflammation plays a major 
role in tissue damage. An understanding of what initiates and what 
modulates inflammation is, therefore, crucial to explaining and controlling 
these disease states. The noncellular mediators of inflammation can be 
divided into two general categories , those of small molecular weight and 
those of high molecular weight. The group of serum proteins derived from 
the complement system and the associated enzyme systems of coagulation, 
fibrinolysis, and kinin fall into the latter category. 

The initiation of the complement casade by either the classic or 
alternate pathway makes a major contribution to the development of the 
inflammatory response. Once activated, complement components have 
functions including opsonization, immune adherence, chemotaxis, cell lysis, 
and anaphylatoxin generation. Derangements of complement have been 
implicated in causing or contributing to a wide range of disorders 
including cancer, kidney diseases, collagen vasular diseases, infectious 
diseases, and inherited diseases. 

Understanding of the complement system depends on isolation and 
identification of its components. Through work done by our grantees, it is 
known that there are approximately 20 complement components. Paul A. 
Liberti (AI 12365, Thomas Jefferson University ) has devised a new isolation 
procedure for C12 which results in stable preparations retaining all native 
biological activities. He has used this procedure to measure the physical 
properties of Clq in order to determine its most likely molecular 
structure, thereby enabling them to relate its structure to function. Irma 
Gigli (AI 13809, New York University ) has continued studies examining the 
structural and functional proteins of the complement system, and has shown 
that Ch serves an important function in the initial steps of complement 
activation and that C4bp (previously reported) functions in the control of 
the formation of C42 convertase, confirming the presence of a control 
mechanisms in serum which regulates the classical pathway of convertase. 
S haun Ruddy, AI 13049, Virginia Commonwealth University , has shown that a 
newly discovered control protein, beta-1 H globulin, actually combine with 
one of the active complement proteins and blocks its function in producing 
inflammation. Understanding the mechanism of action should allow develop- 
ment of new methods to control inflammation induced by complement. 



3-19 



Particular interest has been placed on studies of the properdin or 
alternate complement pathway. The alternate pathway was, until recently, 
thought to have specific recognition proteins which bound to and thereby 
identified foreign particles for destruction. Hans Muller-Eberhard (AI 
07007, Scripps Clinic and Research Foundation) reports that the complement 
protein, C3 , has recognition function in the so-called alternate pathway. 
Antibody is not required for this reaction. A cell (fungal, bacterial, 
etc.) that is an activator of this pathway allows C3, from the plasma, to 
be deposited on its surface. The deposited C3 interacts with surface 
structures of activators such that a regulatory protein which ordinarily 
initiates inactivation of deposited C3 , cannot reach it. As a consequence, 
the orderly assembly of complement enzymes procedes without interference by 
regulators and the cell is killed by the membrane attack complex. 

Other studies of basic mechanisms have included those of Celso Bianco , 
AI 15221, S.U.N.Y. Downstate Medical College , who has examined the role of 
certain complement components in studies of macrophage motility. Prelim- 
inary experiments have shown that peritoneal macrophages respond to Bb, a 
cleavage product of factor B of the properdin pathway by spreading an 
inhibition of migration. Hans Muller-Eberhard, AI 13010, Scripps Clinic and 
Research Foundation , has also studied the responsiveness of human mono- 
nuclear phagocytes to activated factor B (Bb of the properdin system) 
showing that mononuclear phagocytes synthesize and secrete complement 
proteins, and that monocytes synthesize C5 which is intimately involved in 
processes which lead to activation of monocytes to spread in response to 
both Bb and anti-C5F (AB prime)2. Thus the Bb complement activation frag- 
ment influences monocytes, therefore creating a possible role for 
complement protein as mediators of cellular immunity. To whatever extent 
that it correct, it is of interest that this could have relevance in cancer 
study, and in fact, Alfred Esser, AI 14099, Scripps Clinic and Research 
Foundation, has shown that certain RNA tumor viruses can be lysed by human 
complement, supporting the proposition that complement can provide a major 
defense against infection with reto viruses (oncornaviruses, C-type viruses, 
mouse leukemia viruses which may be associated with the production of 
tumors in animals). 

These basic studies have been very rewarding because they are useful 
in explaining mechanisms for several clinical problems. Understanding of 
these mechanisms is important for possible prevention and treatment of 
these diseases. For example, K. Frank Austen (AI 07722, Robert B. Brigham 
Hospital ) reports that he has been able to work out the molecular structure 
and mechanisms of action of C3 nephritic factor, the abnormal protein 
present in patients with membranoproliferative glomerulonephritis. This 
protein causes the complement system to be fully activated in the serum of 
patients with various types of nephritis. This protein is a host antibody 
directed against protein of the alternate complement pathway and serves to 
stablize the interaction of the complement protein thereby circumventing 
all host regulatory mechanisms. This leads to virtually complete depletion 
of the complement system with resultant inflammatory reaction. He is 
currently working out a role of the alternate complement pathway in bullous 
pemphigoid, a disease in which the mast cell effector system also appears 
to be abnormal. 



3-20 



Other tantalizing clinical leads have come from basic research in 
complement. For example, Dr. Gigli states that recent studies have shown 
that the complement system may play an important role in the localization 
of myocardial infarction. Depletion of one of the components, C3, is 
accompanied by better outcome of animals in which experimental infarcts 
have been induced. Kenneth Mathews, AI 14950, University of Michigan, Ann 
Arbor , has described a patient with a deficiency of serum carboxy-peptidase 
B, which might predispose to radiographic contrast media reactions or 
chronic idiopathic urticaria or angiodema (worked on in collaboration with 
Tony Hugli, AI 15782, Scripps Clinic ) . The potential significance of this 
observation is that there is an implication that there is a subpopulation 
of patients who not only react adversely to radio contrast dyes, but who 
also may have the syndrome of chronic urticaria or angiodema, where serum 
carboxy-peptidase B deficiency may contribute to their clinical 
difficulties. 

J ohn P. Leddy (AI 12568, University of Rochester ) has been able to 
correlate the inordinate white cell depression described after donation of 
white cells to the activation and consumption of the complement system. 
This white cell activation is coincident with the formation of some white 
cell depressing factor in blood passing through the nylon filter used 
during this donation procedure (leukapheresis) . By determining the precise 
mechanisms for this problem it may in the future be eliminated. 

The genetics of the complement system have been studied by various 
investigators. Ha rvey R. Colten (AI 12791, Boston Children's Hospital 
Medical Center ) has been looking at congenital absences of complement 
protein in regard to increased susceptibility to infection, hereditary 
attacks of edema, and increased susceptibility to rheumatoid- like diseases. 
C hester A. Alper (AI 14157, Center for Blood Research ) has found that the 
second complement component and factor B but not the fourth component are 
controlled by genes in the region near the HLA genes. In a more basic way, 
John Goldman, AI 12792, Michael Reese , has continued studies investigating 
the relationship of early components of complement to the H2 complex in 
mice, finding that functional levels of CI, C4, and C2 are under the 
control of genes within, or closely linked to the S region of the H2 
complex. 

A second category, that of low molecular weight mediators of inflam- 
mation, was previously mentioned. A discussion of basophils (or mast 
cells) must accompany any discussion of these chemicals, because these are 
the cells which release them. The sequence of events leading to mediator 
secretion is as follows.- sensitization takes place; IgE attaches to the 
surface of mast cells and basophils,- on re-exposure, the cell-bound anti- 
bodies are bridged by the allergen signaling release of the mediators from 
the cells. 

Research in this area has been facilitated by several new advances: 
Herve Bazin (AI 12830, Catholic University of Louvain ) through the develop- 
ment of a colony of rats with IgE secreting plasmacytomas, has made this 
immunoglobulin readily available; and both Dr. Parker and Dr. Ishizaka have 
established rat basophilic leukemia cells in culture. 

3-21 



Both of the latter investigators are also studying basophil receptors. 
Dr. Ishizaka has been able to demonstrate histamine release from these 
cells via divalent anti-receptor antibody without participation of IgE. Also 
studying these receptors in F loyd J. Malveaux (AI 05383, Johns Hopkins 
University ) who has noted the relationship between serum IgE and the number 
of basophil IgE receptors. 

While Dr. Ishizaka has shown that mast cells can discharge allergic 
mediators without involvement of IgE, Lawrence M. Lichtenstein (AI 11334 , 
Johns Hopkins University ) has demonstrated release of mediators from other 
than mast cells. Namely polymorphonuclear (PMN) leukocytes contain and 
release slow reacting substance of anaphylaxis (SRS-A) and eosinophilic 
chemotactic factor (ECF) . 

P hilip S. Askenase (AI 12211, Yale University ) is studying so-called 
cutaneous basophil hypersensitivity (CBH) . Traditional views of delayed 
hypersensitivity and immediate hypersensitivity as being separate patterns 
of immune reactivity may no longer hold. For example, otherwise typical 
delayed reactions contain significant numbers of basophils. CBH can be 
transferred in animal models by either immune cells or serum. Similarly, 
the late IgE mediated reactions in man may be analogous to serum transfer 
of CBH. 

Despite this clouding of hypersensitivity reactions, some discussion 
of what is known classically as the type IV hypersensitivity reaction- 
cellular hypersensitivity, mediated by T cells is needed. These T cells 
also function as cytotoxic effector cells, have a role in secreting 
mediators of delayed hypersensitivity, regulate immune responses as helper 
or suppressor cells and act as memory cells. 

The first part of this discussion will be confined to studies of 
hypersensitivity relevant to clinical diseases. Contact dermatitis is a 
prototype for cell mediated immunity. Henry Claman, AI 12685, University 
of Colorado Medical Center ) has used a mouse model to study the development 
of delayed hypersensitivity to dinitrofluorobenzene . This experimental 
model is important because the complete antigen made after contact 
sensitization is DNP-coupled-to-self , and thought of in this way, contact 
allergy can be considered a form of autoimmunity where at least part of the 
antigen is self. V era Byers (AI 12947, University of California, San 
Francisco, is studying poison oak and poison ivy, two of the main causes of 
workman's compensation in California. She is developing systems to study 
the effects of these chemicals on human lymphocytes in vitro , in an effort 
to produce a state of unresponsiveness. Henry C. Maguire, AI 13337, Hahne - 
mann Medical College and Hospital of Philadelphia, is also studying contact 
dermatitis. He is trying to determine the mechanisms of an interesting 
clinical observation the use of cyclophosphamide under particular 
conditions will increase the intensity of allergic contact dermatitis 
reactions. Fred S. Kantor (AI 060706, Yale University ) is studying the 
opposite phenomenon--anergy . He has induced anergy in an animal model and 
found the effect to be mediated by a subset of T lymphocytes which adhere 
to nylon-wool columns . 



3-22 



When sensitized T lymphocytes are cultured in the presence of specific 
antigen, they release a variety of nonspecific mediators, or lymphokines, 
that act on other lymphocytes and macrophages among other cells. Manfred 
Mayer (AI 12371, Johns Hopkins University, has been doing basic research to 
determine what lymphokines are. In particular he has looked at lymphotoxin 
which plays a role in certain cytotoxic reactions mediated by lymphocytes, 
mitogenic factor, lymphocyte activating factor which is an amplifier of 
immune and allergic processes, and hydrophobic peptide. Louis W. Heck (AI 
05207, Robert Beck Brigham Hospital, has been studying migration inhibitor 
factor. He has shown that macrophages, pretreated with proteinase 
inhibitors demonstrated an augmented response to subthreshold doses of MIF. 

Eosinophils are known to be involved in inflammatory processes, 
particularly those allergic in nature. For this reason, some mention 
should be made of research in this area. Gerald Gleich (AI 09728, Mayo 
Clinic , has localized the basic protein of the eosinophil to the core of 
the granule. The clinical relevance of this finding is, as yet, unclear. 
T homas T. Hubscher (AI 15001, Georgetown University ) has discovered that 
eosinophils may serve to dampen the allergic response. 

Macrophages which are differentiated blood monocytes differ from the 
other phagocytes in that they are long lived, are capable of continuing 
lysosomal enzyme synthesis, and may further differentiate into epithelioid 
cells which, in turn, may fuse to form multinucleated giant cells. The 
technology for these studies has been advanced by the work of Steven 
D ouglas (AI 12478, University of Minnesota, who has developed a technigue 
of freeze-fracture in conjunction with electron microscopy making it 
possible to study macrophage membranes at high resolution. In particular, 
he is interested in developing technigues for the study of the genetic 
regulation of their plasma membrane receptors for immunoglobulin. Victor 
Najjar, AI 09116, Tufts University, is studying a peptide called tuftsin 
which stimulates phagocytosis. He is looking at the possible curative 
ability of this compound in animals, especially those that have had their 
spleens removed. 



3-23 



GENETICS AND TRANSPLANTATION BIOLOGY BRANCH 

A. Scope 

This Branch supports and manages research on the immunologic factors 
that determine the acceptance or rejection of grafts and on the genetic 
relationships that underlie them. 

The increasing prevalence of degenerative disease and of trauma that 
may impair the function of particular organs sufficiently to be incapac- 
itating or life-threatening identifies the perfection of a successful 
transplantation methodology as one of the objectives of highest priority 
in current medical science. The technical surgical aspects have proved 
quite manageable but immunological rejection of the graft is the obstacle 
that has yet to be surmounted. 

Research efforts supported by this Branch include: 

1. Studies of strategies designed to minimize the likelihood of 
rejection by assuring the best possible antigenic match between 
donor and recipient. 

2. Explorations of immunosuppressive regimens whose objective is to 
reduce the capacity of the host to reject the graft. 

3. Studies of treatments of the graft that will reduce its ability 
to provoke a response. 

Studies in animal models are supported that explore the genetic 
controls of the immune response, and, on a more practical level, test 
methodologies whose ultimate application is to man. 

Genetically conditioned associations between antigenic characteristics 
and susceptibility to various diseases have recently been brought to light 
and also fall within the purview of this Branch. 

Two important activities of the Branch are the management of the 
Kidney Transplant Histocompatibility Study which is now entering the 
analysis stage and the management of the HLA Serum Bank, a national cell 
typing resource for transplantation as well as basic research studies. 
Stocks of human B lymphocytes are being accumulated and eventually will be 
made available to the scientific community for use in immunological studies 
and histocompatibility typing. A comparable bank of mouse typing alloanti- 
sera is also maintained by the Branch. 

B. Awards and Support Levels 

The Genetics and Transplantation Biology Branch supports research 
activities through individual grants, research career development awards, 
fellowships, training grants, and very importantly, in conncection with its 
kidney transplant program and serum banks , through contracts . 



4-1 



Research Grants 




64 


Career and Career 






Development Awards 




9 




Subtotal 


73 


Fellowship Awards 




7 


Training Awards 




1 




Subtotal 


8 


Contracts 




39 




Total 


120 



The following shows the distribution of support for the activities of 
the Branch during FY 1979. 

Genetics and Transplantation Biology Branch 

FY '79 Awards 

Award Mechanism Number Amount 

$ 6,531,867 

300,783 
$ 6,832,650 

78,760 

12,298 

$ 91,058 

1,895,292 
$ 8,819,004 

Total costs, estimated using a 40% overhead for indirect costs. 

The majority of these awards, approximately 80%, are concerned directly 
with transplantation biology and the remainder support research on the 
genetics of the immune system and of disease associations. Approximately 
30% of these awards were for competing new or renewal applications; the 
remainder represents commitments to support awards made in prior years. 

C. Program Areas and Highlights 

1 . Immunogenetics 

An immune response is the result of a complex set of events involving 
recognition of the antigen and of interacting cells of the lymphoid system 
and of macrophages. Such recognition is in all instances determined by the 
genetic constitution of the responder. On it depend the ability to resist 
infection, the ability to accept or reject grafted tissue, and suscepti- 
bility to diseases of currently uncertain etiology, many of which appear to 
have an immune component. 

The pattern of recognition is predominantly, but not exclusively, 
determined by genes organized into a so-called Major Histocompatibility 
Complex (MHC) located, in all mammals studied to date, on a specific seg- 
ment of a specific chromosome. The organization of the MHC is best 
understood in the mouse and in man. It contains genes that code for cell 
surface proteins that act as strong transplantation antigens because, if 
the compositions of these proteins are disparate in the donor and recipient 
of a transplanted organ or tissue, the graft is usually rapidly rejected. 
On the other hand, certain foreign antigens such as viruses must become 
associated with these structures to elicit an immune response in the host. 



4-2 



These major transplantation antigens have been recognized to be highly 
polymorphic both in studies on rejection of grafts and by their ability to 
stimulate the production of specific antibodies. Approximately 70 speci- 
ficities have been identified at 3 loci in man on the basis of serologic 
reactivities, and the list continues to grow. 

There are other polymorphic loci within the MHC, also definable, by 
the ability of their gene products to stimulate antibody production on 
alloimmunization. These antigens have been designated la antigens in the 
mouse and DR antigens in man. The loci coding for them are clearly 
identical with or very closely linked to genes which control several of the 
components of the immune response in the mouse and probably also in man. 
Thus, the I-A, I-B and I-C control helper functions. The I-C subregion 
also controls suppressor functions related to the mixed lymphocyte reaction 
(see below), while the IE subregion codes for antigens on T cells capable 
of generating soluble suppressor factors. Helper and suppressor functions 
of T cells are also identifiable by means of the Ly cell surface proteins, 
which, however, appear to represent a separate system of differentiation 
antigens . 

Other regions within the MHC control the ability of lymphocytes to 
proliferate in response to exposure to other lymphocytes from a non- 
syngeneic donor, ultimately producing progeny capable of lysing the 
stimulator cell. The proliferative response is called the mixed lymphocyte 
reaction (MLR) and is associated with the leukocyte activating determinants 
(LAD), the principal one of which in man is designated the D locus. 

Because of their role as transplantation antigens, much effort has 
gone into attempts to assure their identity in the donor and recipient of a 
graft, be it a solid organ such as the kidney or tissue such as the bone 
marrow. Determination of the characters of these antigens is called tissue 
typing. The success of such efforts both in attaining good matches and in 
the prediction of the acceptance of the graft is limited by, on the one 
hand, the very extensive polymorphism of the genes and antigens, and, on 
the other hand, by the existence of very numerous other loci, as many as 
perhaps 500 in the mouse, coding for so called "minor" histocompatibility 
antigens, whose role in graft rejection becomes prominent when mismatch at 
the MHC is absent. 

Research on the MHC is currently focused on three areas. Genetic 
studies are being carried out on recombinant strains of mice (Schreffler) 
to characterize the topology of the MHC and the interdependence among the 
various gene products in the expression of their function. For example, it 
has become evident that the expression of the structural gene which codes 
for the I-A region antigen is controlled by a gene near the I-J region 
(McDevitt) . The appearance of the I-A incoded molecule on the cell surface, 
not its synthesis, requires the presence of the second gene either on the 
same (cis-complementation) or on the other (trans-complementation) 
chromosome 6 . 



4-3 



Such studies on recombinants make use of highly inbred colonies of 
mice. Such colonies are also necessary for the production of highly 
defined mutants at the MHC and other histocompatibility loci (Melvold) . 
Such mutants become an important resource for studies of structure- function 
and-antigenicity relations in the gene products when the mutations involve 
single-amino acid changes (Forman) or for the identification of loci when 
the mutations are deletions. It is with such a mutant line that a new 
mouse MHC locus H-2 L was identified very recently (Kohn and Melvold) . 

A third approach to the study of genetic regulation of the MHC employs 
wild mice (Klein). This permits estimation of the true degree of poly- 
morphism of the genes as well as of heterozygosity. Thus, the finding of 
very high levels of hetezygosity, about 100% at the K and D loci and 80% at 
the I-A locus, even in inbreeding colonies in the wild strongly implies the 
existence of selective pressures that may well be associated with the 
observed correlations of MHC types with disease (see below). Thus, homo- 
zygosity might accentuate the susceptibility to a disease associated with a 
particular antigen which would be present in a double dose. 

The second area in which much activity is evident is that of the 
functional roles of the histocompatibility antigens. The control 
mechanisms ascribed to the various loci or sub- regions of the I region of 
the MHC have already been discussed (David). Such a thorough dissection, 
analysis and correlation with function of a chromosomal region represents a 
most significant advance in mammalian genetics and begins to approach the 
level insight achieved in single celled organisms. 

The genetic control of the immune response has also been investigated 
through immunizations with well-defined antigens in animals, where control 
of the response to many antigens has been found to reside in the I region, 
or through associations with diseases which have an immunological component 
with MHC antigens in man (Amos, Duquesnoy, Fudenberg, Hsia, Marsh, Stastny, 
and Walford) . Sufficient progress has been made in determining the 
molecular basis for the involvement of MHC gene products in the recognition 
and control of the response to antigens in the mouse to permit the con- 
struction of well based hypothetical models. It appears that I-A and I-E 
region products must associate with the foreign antigen for it to be 
effectively recognized by the afferent limb of the immune system. This 
also seems to be the case in the recognition of certain viral antigens 
which must interact in some manner with the host's major transplantation 
antigens to elicit an immune response. 

The products of other I region genes are evidently involved in 
recognitions among the regulatory subsets of the lymphocyte population. 

The required associations of foreign antigens with MHC gene products 
have clear implications as to the mechanisms that underlie the observed 
statistical correlations between human MHC (HLA) types and various 
degenerative or infectious diseases. Direct investigations of these 
possible mechanisms are now much needed. 



4-4 



The third area of intense investigation of the MHC consists of 
physical and chemical studies of the surface antigens. Such studies employ 
sensitive separations of the cell surface proteins to identify the extent 
of the similarities and differences among the various gene products and 
among their various antigenic forms (Jones and McDevitt) , and of determina- 
tions of exact amino acid sequence to establish evolutionary patterns 
(Hood), relations to other products of the immune system such as antibodies, 
or the chemical basis of alloantigenicity. In this connection, it might be 
pointed out that the carbohydrate moiety of the HLA antigens is not 
recognized in alloimmunizations (Strominger) . 

2. Transplantation 

The major application of studies on the immunogenetics of man remains 
transplantation. The principal objective is to so reduce the antigenic 
disparity between donor and recipient as to minimize the tendency to reject 
the graft. Until recently efforts to match focused on two serologically 
identified loci of HLA, A and B (C is of minor importance), and at the 
locus identified by the mixed lymphocyte reaction (MLR), D. Matching is 
very clearly highly beneficial in transplants among related donors and 
recipients , as is matching at the D locus in renal and probably bone marrow 
transplantation. The inference that may be drawn is that matching a living- 
related donor and recipient at the two most accessible loci is effective 
because it also implies matching along the entire MHC and beyond. Its 
utility in the transplantation of organs and tissues from unrelated donors 
is much less clear and is the subject of continuing debate. As a result, 
the search continues for "the" transplantation antigen. A new candidate 
has appeared in the field of kidney transplantation: the product of the DR 
locus . This region is very closely linked to the D locus and appears to 
dominate the responses observed in the primed lymphocyte test (PLT) in 
which the proliferation of lymphocytes stimulated once by exposure to 
allogeneic cells is measured following a second exposure (Dausset). Matching 
at the DR locus has been found very promising in Europe, where matching at 
the A and B loci also prolongs graft survival, and is now being attempted 
in the U.S. The technical aspects of the procedure are difficult and a 
satisfactory level of consistency in DR typing is only now being approached 
(Third American Workshop, Fran Ward, Ed.). An extensive international 
study of the efficacy of DR matching in renal transplantation is being 
conducted under the auspices of the Eighth International Workshop on HLA 
Testing (Terasaki) . 

The limitations of the approach to matching at the MHC by typing 
lymphocytes must be kept clearly in mind, however. Thus, there are 
numerous other loci on various chromosomes that code for polymorphic cell 
surface structures which can, as a result, serve as transplantation 
antigens. The red blood cell antigens are an obvious example, but many 
other examples exist. The skin displays the effects of mismatching of 
these antigens most clearly: it is the most difficult tissue to transplant 
because it is at highest risk of rejection. It is in connection with these 
non-MHC antigens that the phenomenon of cyclic variation in the intensity of 
the response became apparent: alternate periods of stimulation and 



4-5 



suppression (Graff) . This variation is undoubtedly not restricted to 
responses to non-MHC alloantigens. A further complication is introduced by 
the existence of developmental or tissue-specific alloantigens, for example 
endothelial and skin antigens (Bailey and Sakai). These will be missed 
when only lymphocytes are typed and yet will cause rejection on transplan- 
tation. Clearly, additional knowledge of these alloantigens must be sought. 

Complete matching is possible only in identical twins. In all other 
cases, survival of the graft is totally dependent on immunosuppression of 
the recipient. The current mainstays of immunosuppression are adrenal 
cortical steroids and the cytotoxic agent azathioprine, used in rather 
standardized regimens. The philosophy of such treatment in renal trans- 
plantation is shifting towards minimization of drug doses with acceptance 
of increased risk of graft loss (Tilney) . Concurrently, there is a search 
for more specific immunosuppressants. One such agent, anti- lymphocyte or 
thymocyte globulin, has been under investigation for a number of years, 
with variable results. The reason may well be the highly variable potency 
of this biological material. This difficulty is being overcome by assessing 
the potency of the preparations in monkeys and by carefully monitoring the 
lymphocyte and T cell (E-rosetting cells) levels in the recipient (F. 
Thomas and J. Thomas). A further advance in immunological monitoring is 
impending as a result of the development of reagents (sera) capable of 
identifying the helper, suppressor and effector T cell subpopulations in 
man (Schlossman) . In addition to helping tailor drug doses, the sera 
should facilitate the elucidation of the regulatory mechanisms that 
determine the fate of the graft. 

An apparently still more highly selective immunosuppressant coming 
under investigation at this time is the drug cyclosporin A. Its mechanism 
of action is not yet clear but appears to involve suppression of the 
recipient's cells stimulated specifically by the graft. 

An alternative approach to immunosuppression depends on irradiation of 
the recipient. Studies in dogs employing supralethal irradiation followed 
by infusion of autologous bone marrow drawn earlier suggest that a state of 
high susceptibility to tolerance induction results, facilitating the 
acceptance of the graft (Rapaport). A similar state of susceptibility to 
tolerance induction follows the less drastic total lymphoid irradiation in 
which the marrow of the skull and long bones and the lungs are spared 
(Strober). This state applies not only to the recipient but also to the 
donor tissue in bone marrow transplantation and so avoids the development 
of graft-versus-host disease (GVHD). This approach clearly merits further 
development, and TLI is currently being attempted in renal transplantation 
at the University of Minnesota (Simmons). 

At present, the preparation of patients for bone marrow transplanta- 
tion with radiation and cytotoxic agents would be lethal were not donor 
marrow given. The recipient is thus at risk both from rejection of the 
marrow and from the development of GVHD. The two principal diseases 
treated with bone marrow transplantation are aplastic anemia and acute 
leukemia. In the former, survival rates of 85% are attained if prior blood 



4-6 



transfusions are avoided. The major danger is of rejection of donor cells. 
In acute leukemia, two year survivals of 55-60% have been achieved if the 
transplantation is performed prior to the end stages of the disease (E. D. 
Thomas). The major advances in bone marrow transplantation, other than 
those mentioned, have resulted from refinements in supportive care. 

3. Kidney Transplantation Histocompatibility Study 

The Kidney Transplantation Histocompatibility Study (KTHS) is a pro- 
spective study of the natural history renal transplantation carried out 
under contract by a consortium of over 40 U.S. and Canadian centers. At 
its inception, its principal objective was to determine the utility of 
matching at the A and B loci. However, comprehensive data were collected 
on all phases of renal transplantation, including characterizations of 
donors and recipients, selection procedures, intraoperative events, post 
operative management and outcome. A total of 2434 transplants were entered 
into the study in 1974-1976. Complete followup continued until the end of 
1978, with some additional information still to be acquired in 1979. The 
data to the end of 1978 have been reviewed and corrected and are currently 
being analyzed by the Naval Medical Research Institute's Data Analysis 
Office and GTBB. The salient conclusions evident at this point, some 
previously known, include the following: (a) Graft survival is substan- 
tially higher when the donor is related to the recipient. This advantage 
depends, however, on the adequacy of matching at the A and B loci. In 
transplants involving related donors in which a two haplotype match is 
achieved, two-year graft survival is about 85%, as against about 50% when 
the donor is unrelated. If only one haplotype match is attained, results 
are much inferior though still better (about 65%) than in the latter case. 
Matching has no significant effect on graft survival when the donor is an 
unrelated cadaver, (b) The age of the recipient has an important effect on 
survival of kidneys from cadaveric donors, significant deterioration 
setting in beyond age 30, but not when the donor is living and related, (c) 
The effect of blood transfusions prior to transplantation is very signifi- 
cantly beneficial. The improvement in graft survival, evident even after 
very few (less than 5) transfusions is not counterbalanced by rapid broad 
sensitization of the recipient with consequent increased difficulty in 
finding a cross-match negative donor. (d) The results of transplantation 
even of kidneys of unrelated cadaveric donors are very satisfactory from a 
personal and a societal point of view. Patient survival, in contrast to 
graft survival of cadaveric kidneys, is over 80% at one year after implan- 
tation. Moreover, of those who retain a functioning kidney, approximately 
70% are rehabilitated and return to fully normal activity. (e) Prior 
transplants do not reduce graft survival of unrelated cadaveric grafts. 

The second phase of analysis of KTHS data which will involve in-depth 
evaluation of specific issues in renal transplantation will be carried out 
under contract by the Harvard School of Public Health. 

It is also hoped that the formulation of additional multi-center 
trials and studies will be stimulated as a result of information generated 
by KTHS. One such trial, of immunological pre treatment of cadaveric kidneys 



4-7 



with cyclophosphamide, has been proposed, under the auspices of the 
American Society of Transplant Surgeons and will shortly undergo review for 
scientific merit. 

4. HLA Serum Bank 

The HLA Serum Bank is an international resource that supports all 
aspects of histocompatibility testing. Through a series of contracts it 
acguires, processes, stores and distributes human sera in bulk form and on 
trays. Sera are also acquired by direct purchase and by donation, the last 
accounting for approximately one- third of the current inventory. Bulk sera 
are made available predominantly for research while the principal use of 
the trays is in clinical applications, chiefly renal and bone marrow trans- 
plantation. Sera accepted by the bank are subjected to careful screening 
to characterize their behavior and validate their utility. They thus serve 
as standards for the research community. 

During the past year, the bank distributed 13 liters of human sera in 
1 ml freeze-dried samples to 150 U.S. and 94 foreign investigators. These 
sera were used in studies in genetics, immunology and anthropology, among 
others. A continuing application of great interest is study of the 
association of various diseases with HLA types. 

The Bank has also recently acquired homozygous typing cells which will 
also serve as reference standards. 

Approximately 35 thousand typing trays were distributed during the 
past year. Previously restricted to use in human transplantation, they have 
now also been made available for the characterization of panels of cells 
used to identify sera with useful specificities, as well as for selected 
research purposes. As a result of recent policy decisions, of the increas- 
ingly routine clinical use of sera and trays , and of our increasing 
understanding of where these materials can be used to greatest advantage, 
investigational applications will receive a higher priority and efforts 
will be made to have the routine clinical aspects provided for by more 
appropriate mechanisms. In this connection, the typing tray is being 
redesigned. A basic tray most directly useful in typing related graft 
donors and recipients and adequate in the majority of other applications 
will be produced and will be supported by a more limitedly available trays 
encompassing the rarer specificities and useful in studies of special 
problems and populations. 



Grants and Contracts Cited 

1. Amos, D. Bernard, Duke University; 5 K06 AI-18399-17 ; 5 ROl AI-08897-11 

2. Bailey, Donald W., Jackson Laboratory,- 5 ROl AI-13130-04. 



4-8 



3. Dausset, Jean, University of Paris; 2 R01 AI-09293-18. 

4. David, Chella S., Mayo Foundation; 5 R01 AI-14764-02. 

5. Duquesnoy, Rene J., Milwaukee Blood Center; 5 R01 AI-12507-05. 

6. Eighth International Histocompatibility Task Force and Workshop, 
Terasaki, Paul, Conference Chairman,- Contract Pending. 

7. Forman, James M. , University of Texas Health Science Center; 5 R01 AI- 
13111-03. 

8. Fudenberg, H. Hugh, Medical University of South Carolina; 1 T32 AI- 
07063-01A2. 

9. Graff, Ralph J., Jewish Hospital of St. Louis; 3 R01 AI-07437-llsl . 

10. Hood, Leroy E., California Institute of Technology,- 5 R01 AI-10781-08. 

11. Hsia, Shyuan, Medical College of Georgia; 1 ROl AI-16180-01. 

12. Jones, Patricia P., Stanford University; 1 ROl AI-15732-01. 

13. Klein, Jan, Max Planck Institute for Biology; 5 ROl AI-14736-02. 

14. Kohn, Henry, Harvard University,- 5 ROl AI-14039-02. 

15. Marsh, David G., Johns Hopkins University; 5 P01 AI-13370-03. 

16. McDevitt, Hugh 0., Stanford University,- 5 ROl AI-07757-13; 5 P01 AI- 
11313-07. 

17. Melvold, Roger W., New England Deaconess Hospital; 1 ROl AI/CA-15969- 
01. 

18. Naval Medical Research Institute, Kidney Transplant Histocompatibility 
Study; 2-Y01-AI-50001-06. 

19. Rapaport, Felix T., State University of New York Stony Brook; 5 ROl 
AI-14453-03. 

20. Sakai, Akiyoshi, Downstate Medical Center,- 5 ROl AI-12824-03. 

21. Schlossman, Stuart F., Sidney Farber Cancer Institute; 2 ROl AI-12069- 
06. 

22. Schreffler, Donald C, Washington University,- 5 ROl AI-12734-04; 1 T32 
AI-07163-01. 

23. Simmons, Richard L. , University of Minnesota at Minneapolis,- 5 ROl AI- 
12754-05; 5 ROl AI-14032-02. 



4-9 



24. Stastny, Peter, University of Texas Health Science Center Dallas; 2 
R01 AI-12563-04. 

25. Strober, Samuel, Stanford University; 2 R01 AI-10293-08 Al . 

26. Strominger, Jack L., Sidney Farber Cancer Institute; 1 R01 AI-15669-01; 
5 R01 AI-10736-05. 

27. Third American Workshop, Frances Ward, Ed.; NO1-TW-8-2110. 

28. Tilney, Nicholas L. , Harvard University; 5 R01 AI-12250-03. 

29. Thomas, Edward D., University of Washington; 4 K06 AI-02425-16. 

30. Thomas, Francis, Medical College of Virginia; 2 R01 AI-12822-04. 

31. Thomas, Judith, Medical College of Virginia; 2 R01 AI/CA- 12586-04. 

32. Walford, Roy L., University of California at Los Angeles; 5 R01 AI- 
10089-09. 



4-10 



IMMUNOBIOLOGY AND IMMUNOCHEMISTRY BRANCH 

A. Scope 

This Branch is concerned with the biology and chemistry of the immune 
system and its products. The fundamental studies which it supports on the 
structure and function of the immune system are directed toward acquiring a 
complete understanding of immune response mechanisms at their basic 
cellular and molecular levels as they function in health and disease. 

Activities in this broadly-based program area cross traditional disci- 
plinary lines of biology and chemistry and encompass anatomic, physiologic, 
pharmacologic, and microbial biology and organic, physical, and biological 
chemistry. Its scope includes studies of the origin, maturation, localiza- 
tion, and function of immunologically active cell populations, and the 
mechanisms involved in the induction, modulation, regulation, and expres- 
sion of immune reactivity, as well as physicochemical studies of antigens 
and their homologous antibodies and the mechanisms and kinetics of antigen- 
antibody reactions. 

Research activities supported within this program area are grouped in 
the general programs for Immunobiology and Immunochemistry and in a Program 
of Institute Emphasis (PIE) for Lymphocyte Biology. 

B. Awards and Support Levels 

Relevant activities in immunobiology and immunochemistry are supported 
through various mechanisms including contracts, individual post-doctoral 
fellowships, institutional pre- and post-doctoral training grants, career 
and career development awards, as well as investigator-initiated research 
grants . 

The following shows the distribution of support by award mechanism for 
the activities of the Branch during FY 1979. 

Immunobiology and Immunochemistry Branch 
FY 1979 Awards 



Award Mechanism 




Number 


Amount 


Research Grants 
Career and Career 


Development Awards 
Subtotal 


235 

28 

263 


$23,733,831 

660,146 

24,393,977 


Fellowship Awards 
Training Awards 


Subtotal 


19 

9 

28 


244,446 
687,721 
932,167 


Contracts 


Total 


5 
296 


25,609 
$25,351,753 



Total costs, estimated using a 40% overhead for indirect costs. 

5-1 



The distribution of these awards by discipline is approximately 3/4 
for immunobiology and 1/4 for immunochemistry . Approximately 35% of these 
awards were for competing new or renewal applications; the remainder repre- 
sents commitments to support awards made in prior years. 

Support for the Lymphocyte Biology PIE is included in the research 
grant category. During FY 1979, five program project awards, at a total 
cost of $4,280,855, were made to support this activity. 

C. Program Areas and Highlights 

1. Immunobiology 

This program is concerned with the processes leading to immunocyte 
differentiation, proliferation, and production of biologically-active sub- 
stances which mediate immune reactions. 

Research supported within this program includes studies on the origin, 
maturation, and localization of immunologically active cell populations, 
the interactions of lymphocyte subpopulations and their interrelationships 
with macrophages and other leukocytes, the cellular phenomena of antigen 
processing, immunologic tolerance and enhancement, and the mechanisms 
involved in the induction, modulation, and regulation of immune responses. 
The cellular mechanisms involved in the induction, maintenance, expression 
and pathophysiology of delayed-type hypersensitive immune reactivity are 
also included in this category. Also relevant are studies of the lympho- 
kines and other lymphocyte substances which activate macrophages and have 
pharmacologic effects on lymphocyte and other leukocytic cell functions 
such as chemotaxis, phagocytosis, blastogenesis , cytotoxicity, and 
microbial resistance. 

Definition of the origin, differentiative pathways, and functional 
roles of the various subpopulations of lymphocytes continues to be a focus 
of considerable investigative effort. Although it is recognized that one 
population of mature lymphocytes (T) is thymus dependent and another popu- 
lation (B) is thymus independent, their progenitors and their mechanisms of 
functional maturation are still not precisely identified. Studies on the 
ontogeny of lymphocytes being conducted by Irving Goldschneider (AI 09649 
and AI 14743, University of Connecticut Health Center) are centered on the 
identification and isolation of lymphohemopoietic stem and progenitor cells 
from rat bone marrow. Using specific antilymphocyte sera and the fluores- 
cence-activated cell sorter, he has enriched preparations of pluripotent 
hemopoietic stem cells and thymocyte progenitors and has identified two 
subsets of B lymphocytes which represent sequential stages in B cell 
development. He has demonstrated that the Thy-1 antigen, and T cell surface 
molecule, is present on the least mature members of the B cell series but 
is absent on mature B cells; for this reason, the Thy-1 antigen can serve as 
a useful marker of the early stages of B cell development. He also has 
shown that a subset of bone marrow cells which contain the enzyme terminal 
deoxynucleotidyl transferase (TdT) is composed almost exclusively of thymo- 
cyte progenitors. Other data indicate that nucleotide metabolizing enzymes 



5-2 



such as adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) 
play important roles at different stages of T lymphocyte development. Of 
interest are the results of related studies in man by Rochelle Hirschhorn 
(AI 10343, New York University) who has found that approximately one-half of 
the patients with severe combined immunodeficiency disease are deficient in 
ADA and PNP. She believes that these enzymes are crucial for normal 
differentiation and function of immunocompetent cells and suggests that the 
pathophysiology of this disease involves a primary inhibition of T cell 
maturation and a toxic effect on cells differentiating after antigenic 
stimulation. She has found that some of the immunodeficient and enzyme 
deficient patients can be immunologically reconstituted by infusion of 
irradiated erythrocytes which contain ADA and has proposed that deoxyadeno- 
sine triphosphate is the mediator of the toxic effect in ADA deficiency. 
Defects in human B lymphocyte development also have clinical expressions. 
Alexander R. Lawton, III (AI 11502, University of Alabama in Birmingham) 
has found that a defect in differentiation of B lymphocytes from their pre-B 
cell progenitors occurs in one form of congenital agammaglobulinemia, while 
failure of pre-B cell development occurs in a type of hypogammaglobulinemia 
associated with a tumor of the thymus gland. 

The results of other ontogenic studies similarly have led to recogni- 
tion of new developmental markers. On immunization of mice with 
teratocarcinoma cells, Barbara Knowles, recipient of a Career Development 
Award (AI 00053, Wistar Institute), has obtained an antibody which reacts 
with embryonic carcinoma cells of both mouse and human origin and with some 
preimplantation stage mouse embryos. She believes that this stage-specific 
antigen on the cell surface is a hallmark at the undifferentiated state; it 
is first detected on cells of the 8-cell stage embryos and is lost by 
embryonic carcinoma cells when they differentiate. Using a histochemical 
stain for viable DNA, Michael R. Loken (AI 14872, University of Chicago) 
has developed a method to quantitatively discriminate between viable B and 
T lymphocytes in both mouse and man. He believes that this histochemical 
stain will serve as an important marker to differentiate lymphocyte subpop- 
ulations based on their cytochemical composition rather than on their cell 
surface molecules. The results of preliminary studies of mouse bone marrow 
cells suggest that differentiating lymphocytes can be discriminated on this 
basis. 

Although the functional distinction of lymphocyte populations is 
convincingly documented, the purpose and mechanisms of their interactions 
with each other and with macrophages is still being examined. It is clear 
that B lymphocytes differentiate to become antibody- secreting plasma cells 
and that T lymphocyte subsets can exert helper or suppressor effects on B 
lymphocytes as well as effector functions in transplantation reactions and 
in cell-mediated immune reactions to various microbial agents. The coopera- 
tive and effector functions of T lymphocytes have been found to correlate 
well with the presence of a cell surface molecule, the Ly antigen. It also 
is known that these functions are controlled and regulated by other cell 
surface antigens which are products of the genes in the major histocompati- 
bility complex (MHC). The regulatory role of MHC products and the 
functional role of other cell surface antigens in the immune response have 
been defined most extensively in the mouse although systems analogous to 

5-3 



the H-2 component of the murine MHC have been identified in other animals 
and in man. 

On recognition of the regulatory role which the MHC exerts on the 
immune system, efforts to clarify the responsible mechanisms have intensi- 
fied. To facilitate research in this area, the program has contractually 
supported the acquisition and distribution of antisera with specificities 
against various mouse cell surface antigens. A supply of antiserum against 
many of the H-2 gene products has been available and antisera specific for 
antigens of the I region of the H-2 complex have recently been prepared by 
Donald C. Shreffler (AI 6-2502, Washington University) for distribution to 
investigators. 

Evidence obtained in several experimental systems has convincingly 
demonstrated that efficient physiological interactions among macrophages, T 
cells, and B cells require that these cells share membrane molecules 
encoded for by the major histocompatibility complex of the species. David 
H. Katz (AI 13874, Scripps Clinic and Research Foundation) has provided 
substantial evidence that the genes controlling interactions between T and 
B lymphocytes are located in the I region of the mouse H-2 complex. He has 
found that T lymphocytes will not exert effective helper functions for B 
cells when these cells differ at the relevant I region locus. He believes 
that self-recognition is the critical mechanism by which cell-cell communi- 
cation takes place in the immune system. In a related project (AI 13781), 
he has obtained evidence indicating that the process of differentiation of 
stem cells and their progeny also may be critically regulated by MHC gene 
products. Using bone marrow chimeras, he has demonstrated that lymphocyte 
differentiation may be "adaptive" to the environment in which it takes 
place. He believes such an "adaptive differentiation" results in 
preferential interactions among cells that have undergone their differen- 
tiative processes in the same genotypic environment; "adaptive 
differentiation" thus may represent a manipulative approach to define 
genetic and molecular mechanisms responsible for cell-cell interactions. 

Genetic restrictions imposed by products of the I region of the H-2 
complex on interactions between macrophages and immune T cells are being 
investigated by Carl W. Pierce (AI 13915, Jewish Hospital of St. Louis). 
Using antigen pulsed macrophages, he has found that naive lymphocytes will 
respond to antigen even if it is presented on an histoincompatibile macro- 
phage. However, the secondary immune response is genetically restricted; 
the immune T cell must be stimulated with antigen-bearing macrophages which 
are genetically identical to the macrophage that presented the antigen 
during the primary immunization. He has demonstrated that such genetic 
restrictions are controlled by products of the I-A subregion of the H-2 
complex and believes that these restrictions involve active suppressive 
phenomena when antigen is presented on an inappropriate macrophage. In a 
related study, Judith A. Kapp-Pierce (AI 13987, Jewish Hospital of St. 
Louis) has found that an antigenspecific suppressor T cell factor is 
activated when nonresponder lymphocytes are stimulated by antigen; suppres- 
sion by this factor is mediated by a molecule encoded by the I-J subregion 
of the H-2 complex. She also has shown that this suppressor factor does not 
require participation of macrophages for effective suppression; she 

5-4 



suggests that the suppressor factor acts directly on primed B cells. The 
results of similar studies by Donal B. Murphy (AI 14349, Yale University) 
suggest that the different functional subpopulations of immunocompetent 
lymphocytes express different products of the I region on their surface. 
He has demonstrated that suppressor T cells bear an I region antigen not 
found on suppressor T lymphocytes. Of interest is his observation that, 
although different T cell subpopulations bear distinct I region products, 
all or many of these products appear to be expressed on the surface of the 
macrophage so that its interaction with these lymphocytes would not be 
encumbered by this type of genetic restriction. 

The mechanisms by which antigens are presented to T cells by macro- 
phages to initiate the immune response are being examined by David W. 
Thomas (AI 14226, Jewish Hospital of St. Louis). He is using macrophages 
modified with trinitrophenyl (TNP) as a model to study the "immunologically 
relevant" processing of antigen by macrophages which is necessary for its 
subsequent recognition by T cells and which is different from the degrada- 
tive and metabolic breakdown of antigen by macrophages. His results 
indicate that T cell recognition of TNP modified macrophages does not 
simply involve a surface display of TNP but that macrophages somehow 
process TNP to create an efficient immunogen. He has concluded that T 
cells recognize and respond to macrophage-bound antigen only when it is 
associated with surface products of the I region of the MHC. His physioco- 
chemical studies, however, have provided evidence that TNP is not directly 
conjugated to macrophage I region products. On this basis, he believes 
that antigen processing by macrophages involves a physical association of 
antigen with I region products rather than an alteration of "self" by 
chemical linkage of antigen with I region products. 

Although it is recognized that physical contact between macrophages 
and lymphocytes is necessary in the immune response, there is considerable 
evidence that each of these cell types produce factors which also are 
involved in the initiation of the immune response. Byron H. Waksman (AI 
06455, Yale University), for example, has demonstrated that the lymphocyte 
activating factor which stimulates helper T cells is a macrophage enzyme. 
He has provided evidence to indicate that this enzyme alters lymphocyte 
surfaces so that calcium incorporation is stimulated and cyclic nucleotide 
activity is consequently enhanced. In a reciprocal fashion, Michael P. 
Rabinovitch (AI 10969, New York University), has demonstrated that 
interferon, an antiviral agent produced by lymphocytes, controls the 
phagocytic functions of macrophages. He has demonstrated that macrophages 
obtained from animals treated with interferon inducers exhibit enhanced 
phagocytosis while macrophages obtained from mice treated with an antibody 
which neutralized interferon had diminished phagocytic activity. Interferon 
also has been shown to have immunoregulatory effects on lymphocytes; Barry 
R. Bloom (AI 09807, Albert Einstein College of Medicine) has found that 
interferon was produced and generated suppressor T cells when lymphocytes 
of normal individuals were cultured with measles virus. Of interest is his 
observation that lymphocytes from patients with multiple sclerosis failed 
to produce interferon when similarly exposed to measles virus. He believes 
this selective defect in response to measles virus in patients with 



5-5 



multiple sclerosis may play a significant role in the persistence of this 
disease. 

Cell-cell interactions are required not only to initiate the immune 
response but also to regulate it. It is now recognized that the help and 
suppression provided by T cell subsets is carefully balanced within the 
responding host. As demonstrated by Richard K. Gershon (AI 10497, Yale 
University), helper T cells, on stimulating B cells to produce antibody, 
also become susceptible to regulation through a feedback inhibition 
mechanism by inducing suppressor T cells. In collaboration with Harvey 
Cantor (AI 13600, Sidney Farber Cancer Institute), the functional subsets 
have been identified on the basis of differentiation molecules, termed Ly 
antigens, present on their surfaces. Thus, helper cells have been shown to 
be Ly 1+ while suppressor cells are Ly 2 , 3+. Cantor recently has provided 
convincing evidence that the Ly 1 helper cell can induce an uncommitted Ly 
1, 2, 3+ subset to participate in specific suppressor activity. In this 
context, Gershon believes from his current studies on the cellular feedback 
inhibition mechanism that suppressor dysfunction is a cause of at least 
some autoimmune diseases. He acknowledges, however, that, in some auto- 
immune diseases, the aberrant activity of suppressor T cell is not due to 
inherent dysfunction of that cell but due to defects in other cells that 
regulate it or are regulated by it. 

Although research on functional subsets of T lymphocytes is proceeding 
at an accelerated pace, it is recognized that this work is technically 
limited by the need to separate the subsets in adequate numbers for study. 
This difficulty may be obviated by the recent work of Frank W. Fitch (AI 
04197, University of Chicago) who has developed a method to isolate and 
culture cloned lines of reactive T cells , including separate lines with 
distinct helper and suppressor activities. In a similar fashion, research 
in this area should be facilitated with the Program's acquisition of a 
supply of antisera against Ly antigens which now are available for distri- 
bution to qualified investigators. These sera were prepared under contract 
through the efforts of Jeffrey A. Frelinger (AI 7-2528, University of 
Southern California). Development of methods and materials for preparing 
antisera against newly- recognized Ly antigens and other immunologically- 
relevant cell surface molecules also is being supported through a contract 
with Edward A. Boyse (AI 8-2541, Sloan Kettering Institute for Cancer 
Research) . 

Approaches to obtain helper and suppressor factors from T cell subsets 
also are being explored. Sirkka K. Kontiainen (AI 13145, University of 
Helsinki) has developed methods to obtain active suppressor factor from 
cultured mouse suppressor T cells. Using anti-Ly antiserum, she has demon- 
strated that the target for suppression is the Ly 1+ helper T cell and has 
characterized the suppressor factor as having both an antigen combining 
site and a determinant encoded by the I region of the H-2 complex. Using a 
similar approach. Marc Feldmann (AI 15653, University College, London) has 
obtained a helper factor from cultured human peripheral blood lymphocytes 
and has developed an assay for its activity using mouse spleen cells. This 
technology will permit analysis of human lymphocyte immunoregulatory 



5-6 



function in disease states and should facilitate attempts to selectively 
manipulate the immune response in immunologically impaired individuals. 

In addition to the factors produced by T cells which have immuno- 
regulatory functions, another group of lymphocytes products, termed 
lymphokines, is receiving considerable investigative attention. Lympho- 
kines have been recognized as being the mediators of a variety of 
immunologic reactions including inflammation, graft rejection, tumor and 
other cell killing, and delayed hypersensitive reactions. The cellular and 
molecular mechanisms of action of lymphotoxin, a cell lytic effector, are 
being investigated by Gale A. Granger (AI 09460, University of California 
at Irvine). He recently has found that human lymphotoxin molecules form an 
interrelated system of cell toxins which is composed of noncovalently 
associated subunits. He has identified three separate types of molecules 
in this system; it contains lytic subunits, condensing molecules which 
facilitate association of the subunits, and antigen binding receptors. He 
has found all three types of molecules are released predominantly from T 
lymphocytes and that the lytic action of the molecular complex is directed 
by the component containing the antigen binding receptors. In similar 
studies of another human lymphokine, macrophage migration inhibition factor, 
Stanley Cohen (AI 13258, University of Connecticut Health Center) has 
obtained evidence that it also may consist of subunits, noncovalently 
associated into an active complex. In a related study (AI 12477), he is 
exploring ways to therapeutically alter lymphokine-mediated reactions in 
the intact host. He has found that various monosaccharides which are 
capable of inhibiting lymphokine activity in vitro also are effective in 
suppressing in vivo manifestations of cell-mediated immunity. For example, 
L-fucose was demonstrated to inhibit the delayed hypersensitive skin 
reaction induced by antigen in actively immunized guinea pigs. Furthermore, 
he and Takeshi Yoshida, recipient of a Career Development Award (AI 00082, 
University of Connecticut Health Center), have been able to induce mediator 
production in vivo and abolish delayed hypersensitive skin reactivity in 
actively immunized guinea pigs. They found that desensitization could be 
passivley transferred using mediator-containing serum from desensitized 
animals; the results of physicochemical studies of such sera excluded the 
participation of antibodies or immune complexes and suggested that the 
desensitizing substance may represent another type of endogenous 
lymphokine . 

2. Immunochemistry 

Research grouped in this program category centers on the defined 
molecular components of the immune system and utilizes chemical and physico- 
chemical approaches for study of immunologic reactants and reactions. 
Studies on the chemical composition and structure of humoral and secretory 
immunoglobulins and of natural and synthetic antigens, as well as of the 
mechanisms and kinetics of antigen-antibody reactions and of the mechanisms 
of immunoglobulin biosynthesis and its regulation, are included within this 
program area. Also relevant are studies at the molecular level of cell 
surface components related to immunologic activity and of subsurface cyto- 
skeletal and contractile assemblies believed to play a role in cellular 
activation. In addition, the newly-developing field of immunopharmacology , 



5-7 



which is concerned with the identification, characterization, isolation, or 
synthesis of chemical regulators of immune function and the elucidation of 
their mechanisms of action is supported in this program area. 

A major aim of studies supported by this program is to elucidate the 
primary structure of antibodies and to correlate their structure with their 
antigen binding, specificity, and other biological functions. The results 
of structural studies have established that an immunoglobulin basically 
consists of two identical light (L) and two identical heavy (H) chains, 
linked by disulfide bridges to form a two-fold symmetrical molecule, each 
side of which consists of an L and an H chain. Two clases of L chains 
(kappa and lambda) and five H chains (gamma, alpha, mu, episolon and delta), 
which determine the immunoglobulin class (IgG, IgA, IgM, IgE, and IgD, 
respectively) have been recognized. Limited proteolysis of a typical IgG 
molecule results in three fragments of nearly egual size, 2 Fab fragments 
and an Fc piece. It is now clear that the Fab components contain the 
antigen binding (combining) site and that the Fc fragment plays an impor- 
tant role in complement fixation. Amino acid sequence studies of 
immunoglobulins have demonstrated the existence of three domains with 
constant sequences on the heavy chain (C ) and one domain of variable 
sequence (V ) ,- each light chain consists or two domains, a constant region 
(C ) and a variable domain (V.). The close association of the V and V 
domains in the Fab fragment, containing the combining site, produces a 
continuous hypervariable surface which can be readily altered by amino acid 
substitution, insertions or deletions, and provides an extremely large 
number of structural combinations which are believed to confer immunologic 
specificity. 

Considerable effort now is being expended to gain an understanding of 
the mechanisms controlling and regulating immunoglobulin biosynthesis and 
of the basis for antibody diversity. Attempts to explain the diversity of 
variable region sequences have centered on a multiple germ- line gene theory 
which presupposes that all information necessary for antibody production is 
encoded in the germ line and is inherited or on a theory of somatic 
diversification which assumes that a limited number of germ-line genes are 
diversified during an individual's lifetime by somatic processes, such as 
point mutations, recombinations or introduction of errors into the gene 
sequence, followed by repair to yield a new set of encoding genes. 

Amino acid sequence analysis and structural studies provide one 
approach to understanding immunoglobulin diversity. The results of studies 
by Fred Karush (AI 09492, University of Pennsylvania) support the view that 
the V genes expressed in murine IgG antibody are encoded in the germ line. 
A contract project under the direction of Elvin A. Kabat (AI 8-2158, 
Columbia University) in collaboration with Tai T. Wu, recipient of a Career 
Development Award (AI 70497, Northwestern University), supports the tabu- 
lation and analysis of immunoglobulin sequence data reported in the world's 
scientific literature. Using the PROPHET Computer System, this project 
provides the concerned scientific community with a unique and invaluable 
data resource and serves as a source of data for molecular model building 
and statistical predictions of immunoglobulin molecular structures. The 
accumulated data base on immunoglobulin variable region sequences has been 

5-8 



published and a series of computer-assisted analyses of the data base has 
been completed. Evidence has been obtained to suggest that the amino acid 
sequence characteristic of segments in the variable region of an immuno- 
globulin is uniquely associated with antibody specificity. Computer- 
generated inferrences concerning amino acids at key positions in light and 
heavy chains agreed well with structural conclusions obtained by X-ray 
crystallography. Additional predictions of the three dimensional structure 
of the combining site have led to the suggestion that the structural gene 
for the variable region of an antibody may be assembled from "mini genes" 
which have been conserved in the germ-line. Support for the germline theory 
also has been provided from studies conducted by Joseph M. Davie (AI 11635, 
Washington University) who interprets the uniformity of isoelectric 
focusing patterns of rat antibodies as being consistent with the view that 
these antibodies are products of germ-line genes. 

In contrast, using antibodies prepared against the combining site, or 
idiotype, of an immunoglobulin, Alfred Nisonoff (AI 12895, Brandeis 
University) believes that it is not necessary for a mouse to inherit V 
structural genes. He feels that antibody diversity can be generated through 
a random process of somatic mutation, rather than through a programmed, 
genetically-controlled series of mutations. The results of his related 
studies (AI 12907 and AI 12908) are providing strong evidence for the 
importance of an idiotype-antiidiotype network as a mechanism regulating the 
immune response. He believes that the idiotypespecific regulation by 
antiidiotype antibody and by suppressor T cells is an important physio- 
logical mechanism determining the magnitude of a given immune response. 
The results of research by Matthew D. Scharff (AI 05231, Albert Einstein 
College of Medicine) and Malcolm L. Gefter (AI 13357, Massachusetts 
Institute of Technology) support the view that immunoglobulin diversity is 
generated by somatic diversification. Studies of immunoglobulin synthesis 
at the level of the nucleus of a cell also has served as an approach to 
evaluate theories of immunoglobulin diversity. The results of sequence 
studies of DNA and messenger RNA and of their function in immunoglobulin 
synthesis conducted by several investigators including Ursula B. Storb (AI 
10685, University of Washington), Randolph Wall (AI 13410, University of 
California at Los Angeles), and Janet M. Stavnezer (14617, Sloan-Kettering 
Institute for Cancer Research) are difficult to reconcile with a strict 
germ-line theory and argue for a somatic process of immunoglobulin 
diversification. 

Efforts are now being made to extend such studies to man although they 
are still in their preliminary stages. Moyra Smith-Wright (AI 14287, Mt. 
Sinai School of Medicine), using a somatic cell hybridization technique, 
has demonstrated that the structural genes encoding IgG heavy chains are 
located on human chromosome 6. Using a serologic approach to examine the 
diversity and inheritance of immunoglobulin genes, An Chuan Wang (AI 13388, 
Medical University of South Carolina) has discovered a new genetic marker 
which is located in the V region of human immunoglobulins G, M, and A. 
This observation represents the first description of a human immunoglobulin 
variable-region genetic marker; the results of pedigree and population 
analyses suggests that it has a dominant mode of inheritance. 



5-9 



Although sequence and serologic studies of serum immunoglobulins are 
not generally restricted by the availability of material for study, similar 
studies of cell-bound immunoglobulins and other cell surface receptor 
molecules have indeed been limited by the small amounts of material 
available for study. Recent technical advances may obviate this problem, 
however. Gerald M. Edelman (AI 09273, Rockefeller University) had developed 
a system to synthesize authentic mouse S --microglobulin and its precursor 
in rabbit reticulocytes using messenger RNA from murine tumor cells; its 
authenticity has been verified by radiochemical-sequence analysis. The 
same system is now being used to synthesize H-2 antigens, TL antigens, and 
other cell surface proteins found on lymphoid cells. Similarly, Sheldon 
Dray (AI 04073, University of Illinois Medical Center) has been employing 
liposomes, synthetic micellar structures which mimic cell surface lipid 
structures, to selectively insert functional messenger RNA into cultured 
cells. He recently has obtained evidence which indicates that cultured 
human epithelial carcinoma cells treated with liposomally-encapsulated 
rabbit globin messenger RNA are stimulated to produce a globlin-like 
protein. Matthew D Scharff (AI 05231, Albert Einstein College of Medicine) 
is employing somatic cell genetic technology to refine the Kohler-Milstein 
technique for production of hybridomas, the product of fusing malignant 
plasma cells and normal antibody- forming cells. He has developed an exten- 
sive library of drug-marked myeloma cells for use in fusion experiments and 
has adapted the fusion technology to significantly improve the frequency of 
functional hybridomas. Further application of the hybridoma technology, as 
well as the insertion and translation of messenger RNA in cultured cells, 
has the potential to provide an unlimited source of homogeneous antibody 
and other immunologically- important molecules for study. Application of 
recombinant DNA technology to this problem can similarly be expected to 
expand the supply of study material. Randolph Wall (AI 13410, University 
of California at Los Angeles) already has successfully inserted mouse 
myeloma immunoglobulin DNA into a bacterial plasmid and has isolated 
recombinant clones which contained all of the light chain constant and 
variable region coding sequences. 

Efforts to define and understand the molecular basis of antigenicity 
and of the antigenic or determinant site similarly has progressed by appli- 
cation of amino acid sequence analysis. For example, M. Zouhair Atassi (AI 
13181, Mayo Foundation) and Ahmed F. Habeeb (AI 14791, University of Puerto 
Rico) have employed hen egg white lysozyme as a model antigen and have 
systematically sequenced it. They have identified three peptide fragments 
which contain the antigenic sites of this protein. Using a method of 
surface simulation, they have synthesized peptides containing the component 
amino acid residues in appropriate structural order and have been able to 
accurately define the voundary, residues, and conformational structure of 
the three determinants of the native protein. Their results clearly demon- 
strated that these three sites quantitatively accounted for the total 
antigenic reactivity of the native protein. Thus, the entire antigenic 
structure of lysozyme has been precisely defined. In a related study, Eli 
E. Sercarz (AI 11183, University of California at Los Angeles has demon- 
strated that peptide fragments containing these determinants can be 
distinguished by their different immunologic functions. The L, peptide, 
containing amino acid residues 1-12, induced a suppressor cell response ,- 

5-10 



the L„ peptide containing amino acids 13-105 induced helper cells while the 
L_ peptide had no effect. This demonstration of helper and suppressor 
sites on the same molecule provides a powerful tool to investigate the 
cellular mechanisms of immune recognition. 

A somewhat different approach to the problem of antigenicity is being 
employed by Conrad Scheurch (AI 12509, State University of New York at 
Syracuse) who has prepared a variety of synthetic polysaccharides, modeled 
after the naturally occurring bacterial polysaccharide dextran as well as 
yeast and fungal mannans. In addition to providing synthetic antigens with 
well defined chemical structure for immunologic study, this work is 
purposely designed to identify clinically useful plasma expanders, like 
dextran, which would be safer for use because they do not possess dextran' s 
allergenic properties. These synthetic polysaccharides are now being 
examined for allergenic potential. 

3. Program of Institute Emphasis for Lymphocyte Biology 

This Program currently supports five program projects, each of which 
is a multidisciplinary research effort integrating the technical expertise 
of cell biologists, geneticists, cellular immunologists and immunochemists 
and is directed by an acknowledged leader in the field. Their combined 
studies are designed to expand knowledge of the morphologic and functional 
heterogeneity of lymphocyte populations and to develop the capability for 
identification and selection of lymphocyte subpopulations with specific 
immune reactivity or antigenic composition, for hybridization of such 
populations and for selective production of specific lymphocyte products. 
The objective of this Program is to acquire as complete a knowledge as 
possible of the life history of immunocompetent cells and of the physio- 
logic and external factors that determine their fate and function in vivo 
and in vitro. 

This program was initiated approximately seven years ago with the 
award of four program project grants, each directed toward the goal of 
understanding the biology of the lymphocyte and having different but com- 
plementary approaches. Three of the original projects have successfully 
undergone competitive peer review and have received renewal of support for 
a five-year period. In addition, support for two new program projects has 
recently been provided, one each in FY 1978 and FY 1979. 

The program project directed by Matthew D. Scharff (AI 10702, Albert 
Einstein College of Medicine) has focused its approaches on the molecular 
biology and somatic cell genetics of the immune system to define the 
genetic and molecular control mechanisms of immunoglobulin synthesis, the 
structure and synthesis of the antigens of the major histocompatability 
complex (MHC), and the functions and interactions of T and B lymphocyte 
populations and macrophages. In many of these studies, continuous cloned 
cell lines and variants derived from these cell lines are being used to 
study the role of the cells and their products in the immune response. For 
example, Dr. Scharff and his associates have devoted considerable time and 
effort to generate immunologically functional hybrids between mouse myeloma 
cells and spleen cells from immunized mice. They have developed a technology 

5-11 



for fusing meyloma and spleen cells at a high freguency and some of the 
drug marked cells which are now widely used to prepare hybridomas 
origniated in their laboratory. They have provided cell lines and 
assistance to well over a hundred investigators around the world to 
establish this technology in their own laboratories. Studies within the 
program project have been productive and important contributions have been 
made. Cultured mouse myeloma cell lines have been used to examine the 
genetic control of the expression of immunoglobulin genes. Analysis of 
antigen-binding variants of these cell lines indicate that the variants have 
small structural changes in the V region, arise spontaneously at a very 
high freguency, and may be relatea to the normal process which regulates 
the generation of antibody diversity. Primary seguence analysis of 
constant region variants has established that they arise as a result of 
crossing over between two closely related constant region genes. Studies 
of the biochemical properties and primary structure of the H-2 MHC prodcuts 
in an attempt to relate functional and genetic properties with molecular 
structure have suggested that discrete amino acid changes in the H-2 glyco- 
proteins are directly related to their immunologic specificity. In order 
to pursue the mechanisms underlying the complex and coordinated functions 
of macrophages and to identify the molecular basis for modulation of these 
functions by products of activated lymphocytes, physiologic and functional 
studies of cloned macrophage- like cell lines have been initiated. An 
unusual macrophage-like cell line has been established which can migrate, 
suppress antibody production by hybridoma cell lines and suppress mitogenic 
responses of T and B cells. These differentiated functions have not been 
previously observed in continuous cell lines. Insulin was found to depress 
phagocytosis in these cells but this effect could be overcome by cyclic 
nucleotides, establishing for the first time that cyclic nucleotides play 
an important regulatory role on the phagocytic process. The functional 
status of immunoregulatory mechanisms in chronic infections also is being 
investigated. When stimulated with specific antigen, T lymphocytes from 
patients with lepromatous leprosy have been found to suppress mitogenic 
responses; this observation may explain the anergic state freguently seen 
in these patients. In contrast, patients with multiple sclerosis exhibit 
defective T suppressor and cytotoxic functions which may contribute to the 
persistence of the disease. 

The approach directed by Gerald M. Edelman (AI 11378, Rockefeller 
University) centers on the analysis of molecules and molecular assemblies 
involved in the control and recognition of antigenic stimuli at the surface 
and within the cells of the immune system. The results of his studies have 
provided several insights into the nature of recognition and control events 
in lymphocyte function. Of special interest is his evidence for the 
existence of a surface modulating assembly (SMA) which regulates cell 
surface receptors and mediates transmembrane signalling for functional 
cellular responses. Dr. Edelman and his associates picture the SMA as a 
three-component system consisting of cell surface receptors that penetrate 
the cell membrane, submembranous microfilaments which coordinate receptor 
movement, and microtubules which provide anchorage for the receptors and 
propogate signals to and from the cell surface. To study the mobility of 
the cell surface receptors and the molecular basis for transmembrane 
control of receptor mobility, the fluorescence photobleaching recovery 

5-12 



method has been adapted to directly measure surface receptor mobility on 
lymphocytes. Using this technique, different classes of receptors have 
been found to be under separate control mechanisms. A viral gene protein 
has been established as a valid probe to dissect the stimulatory sequence 
leading from the cell membrane to the cytoplasm and the nucleus and to 
study the enzymes involved in cellular DNA replication. Evidence has been 
obtained which indicates that the microtubules and microfilaments which 
form the cytoskeleton act as signal regulators in controlling cell growth 
and division. Lectins also are being employed to probe the mechanisms of 
mitogenesis; favin, like concanavalin A, is mitogenic but has been found to 
have a subunit structure very different from that of concanavalin A and, 
consequently, a different capacity to bind cell surface glycoprotein 
receptors. Favin thus offers an alternative probe for examining cell 
surface events. The results of structural studies of cell surface glyco- 
proteins provide evidence that these surface receptors from aggregates with 
each other and with antigenic components through formation of a transient 
disulfide bond. To further understand the nature of molecular interactions 
at the cell surface, X-ray crystallography is being applied to study the 
structural organization of mitogenic stimuli, particularly concanavalin A. 

Evidence has been obtained to suggest that the normal geometry of 
concanavalin A is dramatically altered in the presence of calcium and 
manganese ions. These changes in structural geometry are associated with 
mitogenic activity and are believed to be involved in the binding of con- 
cavalin A to the carbohydrate components of surface glycoproteins. 
Although Dr. Edelman's research is centered on the role of the SMA in the 
immune response, his results suggest that the SMA has a key role in 
controlling cell movement, growth, and differentiation in general. 

The program project directed by Jonathan W. Uhr (AI 11851, University 
of Texas Health Science Center at Dallas) is centered on the study of the 
biochemistry and biology of lymphocyte surface molecules. The structure of 
immunoglobulins on the membrane of B cells is being studied in order to 
define its attachment to the plasma membrane and to clarify the molecular 
interactions that occur after surface immunoglobulins react with specific 
antigens. The broader purpose of such studies is to obtain an under- 
standing of the molecular mechanisms which underlie signalling of B 
lymphocytes and control their differentiation. Studies of T cells 
emphasize the process of immune recognition; efforts are being made to 
biochemically characterize the T cell receptor and to define the role of la 
antigens in the interaction of antigen-pulsed macrophages with T cells. 
Relevant studies include the role of major histocompatibility complex 
products in recognition of viral infected cells and structural analysis of 
these products to develop insights into their evolution genetic control and 
function. The major concern and principal contributions of Dr. Uhr's 
project have been in the area of the ontogeny, regulation, and function of 
B cell surface immunoglobulins. An immunoglobulin D (IgD)-like molecule was 
found on the surface of circulating murine lymphocytes ; ontogenic studies 
showed that it appeared after immunoglobulin M (IgM) when mice were 
approximately one week of age. The results of additional studies have led 
to the acceptance of a model which assigns an important role for IgD in the 
development and function of B cells. In this model, immature B cells first 

5-13 



develop surface IgM, then acquire surface IgD to become "double bearers"; 
some cells subsequently lose surface IgM to become cells that bear only 
surface IgD. Dr. Uhr and his associates have demonstrated that cells which 
bear only surface IgM become tolerized after encounter with antigens. They 
also have shown that surface IgD acts as a triggering receptor so that 
cells that bear both surface IgM and IgD give a primary IgM response after 
encounter with antigen; cells that bear only surface IgD give an IgG 
response after encounter with antigen. On stimulation with lipopoly- 
saccharide, cells with surface IgM enlarge and produce a polyclonal IgM 
response; cells with surface IgD do not exhibit an IgM polyclonal response 
but instead undergo blastogenesis and proliferation. Similar results were 
obtained using antigenic stimulation. It was concluded from such studies 
that cells bearing surface IgD are the major progenitors of IgG antibody 
forming cells in the secondary antibody response. Long-term memory cells 
bear small amounts of both surface IgM and IgG. Studies of surface 
receptors on T cells have similarly led to important conceptual advances. 
Evidence has been obtained to indicate that specific binding of antigens 
coupled to autologous macrophages by T cells represents the initial event 
in immune stimulation. The macrophage plays a key role in this stage 
because T cell receptors do not specifically bind to native exogenous 
antigens. Structural analyses of T cell surface receptors have 
demonstrated a marked homology between the products of the major histocom- 
patibility complex of humans, mice, and guinea pigs, implying analogies in 
function. 

The program project directed by Baruj Benacerraf (AI 14732, Harvard 
Medical School) is designed to gain understanding of the genetic controls 
of the immune system and of the regulation of functional expressions of 
immunocompetent cells. Component projects are concerned with the identifi- 
cation and function of immune response genes, the induction, regulation, 
and function of helper and suppressor T cells , and the identification and 
function of surface receptors on T and B cells. Using genetically- 
determined responder and nonresponder strains of mice, Dr. Benacerraf and 
his associates have found that antigen-specific suppressor factor derived 
from T cells nonresponder mice. They also have found that specific 
suppression can be eliminated by administration of antisera specific for I-J 
gene-controlled determinants; immunopotentiation of nonresponder mice with 
this antiserum was demonstrated to reflect a reduction of specific 
suppressor responses. In other studies, macrophages of responder mice were 
shown to be able to present antigen in an immunogenic form and to play a 
central role in regulating the balance of activated helper and suppressor T 
cells,- these findings suggested that suggested that an Ir gene defect at 
the macrophage level any account for the predominant suppressor T cell 
responses in nonresponder mice. The studies of murine B cells have led to 
the detection of monovalent and divalent IgD on the cell surface; evidence 
has been obtained to suggest that both forms of IgD are native to the cell 
surface and may represent different forms of attachment of these molecules 
to the membrane of the cell. A detailed analysis of the aggregation or 
"capping" of cell surface molecules has led to the demonstration of two 
functionally different mechanisms. The first type which occurs 
spontaneously appears to be an active process involving a direct link 
between the surface molecules and the cytoplasmic contractile apparatus. 

5-14 



This type appears to be related to the process of locomotion; myosin was 
found to be concentrated in the cytoplasm directly beneath the capped area. 
The other type occurred when B cells were treated with antigens, antibodies 
against cell surface receptors, or mitogens and appeared to result simply 
from aggregation of crosslinked molecules in the plane of the membrane; 
this type of capping did not result in a redistribution of cytoplasmic 
myosin or stimulation of cell locomotion. It is implied that the inter- 
action of surface immunoglobulins, at the molecular level, with other 
components of the cell membrane and cytoplasm represents the earliest step 
of specific antigen recognition and subsequent triggering of B cells. In 
other studies, the IgG-bearing B cell subpopulation from human spleen has 
been isolated and characterized; these cells have IgM or IgD on their 
surface and give rise to nearly all of the IgG- and a significant franction 
of the IgM-secreting cells after stimulation by mitogen. The recognition of 
an antigenic determinant found both on murine macrophages and on human 
monocytes using hybridoma antibody has made it possible to use the fluores- 
cence-activated cell sorter to prepare pure populations of human monocytes 
from peripheral blood for subsequent functional studies. Also, by using a 
cytofluorometric technique, an approach has been developed to recognize 
small numbers of malignant B lymphocytes in a population of normal lympho- 
cytes. This method may facilitate the planning and monitoring of therapy 
in patients with lymphoid malignancies. 

The program project directed by Dr. Carl W. Pierce (AI 15353, Jewish 
Hospital of St. Louis) was awarded in FY 1979 and has the immunobiology of 
the major histocompatibility gene complex as its central theme. Dr. Pierce 
is an outstanding cellular immunologist who has made important contribu- 
tions to this area; in recognition of his accomplishments, he was the 1979 
recipient of the Parke-Davis award presented by the American Society for 
Experimental Pathology. His co-principal investigators at Washington 
University, Dr. Donald Shreffler and Dr. Joseph Davie, are equally out- 
standing in their fields. Their combined efforts represent a multidis- 
ciplinary approach to several problems of fundamental importance to an 
understanding of the mechanisms and functions of the major histocompat- 
ability complex. Areas to be examined include the nature of the 
interactions between histocompatability gene products and conventional 
antigens in the generation of effector lymphocytes, the structure of 
genetically restricted suppressor factors, the structure and genetic 
relationships of components of the complement system that are produced by 
genes of the major histocompatability complex, the identification and 
structure of gene products on T cells and macrophages, and the 
identification and characterization of receptor molecules on the surface of 
T lymphocytes . 

This Program of Institute Emphasis represents a major investment of 
the IAIDP and NIAID in the concept that advances in the understanding of 
the behavior of lymphocytes can be effectively achieved through carefully 
organized efforts by large, highly interactive groups led by well- 
established and highly productive senior scientists. To date, there is 
every indication that this program has been successful and productive and 
that it will continue so in the future. 



5-15 



MICROBIOLOGY AND INFECTIOUS DISEASES PROGRAM 

N I A I D 
TABLE OF CONTENTS 

REPORT OF THE DIRECTOR , 6-1 

Bacteriology 6-1 

Mycology 6-2 

Parasitology 6-3 

Virology „ 6-3 

International Heal th 6-8 

I. BACTERIOLOGY AND VIROLOGY BRANCH 7-1 

Program Summary 7-1 

Bacteriology 7-2 

Sexual ly Transmitted Diseases 7-2 

Hospital Associated Infections 7-3 

Streptococcal Diseases and Sequelae 7-5 

Other Disease Problems 7-7 

Leprosy 7-8 

Tuberculosis 7-9 

Mycology 7-10 

Virology 7-12 

II. CLINICAL STUDIES BRANCH 8-1 

Closed Vol unteer Facil ities 8-1 

Collaborative Clinical Trials with Antibiotic Therapy 8-2 

Rapid Viral Diagnosis 8-3 

Nutrition, Infection and Immunity 8-4 

Other Activities 8-5 

III. DEVELOPMENT AND APPLICATIONS BRANCH 9-1 

Introduction 9-1 

Program Summary 9-2 

Research Highl ights 9-4 

Influenza 9-4 

Respiratory Diseases 9-5 

Vi ral Hepati ti s 9-6 

Anti vi ral Substances 9-8 

Bacterial Vaccines 9-10 

Enteric Diseases 9-13 

Viral Vaccine Program 9-15 



MICROBIOLOGY AND INFECTIOUS DISEASES PROGRAM 
N I A I D 
TABLE OF CONTENTS 

Continued 

IV. EPIDEMIOLOGY AND BIOMETRY BRANCH 10-1 

HLA and Infectious Disease Epidemiology 10-1 

Influenza 10-1 

CI inical Trials 10-2 

Impact of Infections 10-3 

Epidemiology of Nosocomial Infections 10-3 

Trans-NIH 10-3 

V. MOLECULAR MICROBIOLOGY AND PARASITOLOGY BRANCH 11-1 

Introduction 11-1 

Molecular Microbiology 11-2 

Program Summary 11-3 

Research Highl ights 11-3 

Recombinant DNA Molecular Research 11-5 

Research Highl i ghts 11-6 

Parasitology 11-7 

Program Summary 11-8 

Research Highl ights 11-8 

Contract Activity 11-11 



MICROBIOLOGY AND INFECTIOUS DISEASES PROGRAM 

Annual Report, 1978-1979 

Director's Report 



The accomplishments of the five branches of the Microbiology and Infectious 
Diseases Program (MIDP) are summarized in the following sections; activities 
in international health are reviewed at the end of this section. This report 
addresses the implications of these accomplishments and activities for the 
future, with particular emphasis on virology. 

Humans are infected by an astounding number and variety of organisms, ranging 
in size and complexity from tiny viruses which are little more than nucleic 
acids to large multi-cellular parasites. These infectious agents and their 
progeny have been living together with man and his progeny for millenia, and 
both have changed as a result. Concurrently, the physical and socio-economic 
environments shared by man and his microbial companions have changed markedly, 
at least in some parts of the world, and chemicals have been developed which 
may be introduced into man or his environment to inhibit the growth of microbes 
and parasites or to limit their transmission. And, in the last two hundred 
years, man has tamed certain of these agents, or their toxins, so as to pro- 
duce attenuated vaccines (smallpox, yellow fever, poliomyelitis, measles, 
mumps, rubella) or toxoids (diphtheria, tetanus) which, if properly used, 
provide virtually complete protection against "wild" or natural strains. The 
net result of all this has been that, in the developed world, many serious 
life-threatening infectious diseases, particularly those of childhood, have 
been prevented with a consequent increase in life expectancy. A major victory 
for man! 

But this is no time to be complacent. Infectious diseases are still respon- 
sible for nearly 25% of all visits to physicians in the U.S. each year, and 
they account for 65% of all such visits for children under 16 years. In some 
developing countries, up to 40% of children still die before the age of five 
years because of infectious diseases. Microbes and their vectors have fought 
back around the world. Malarial parasites have become resistant to once 
curative drugs; mosquitoes which transmit encephalitis viruses and malarial 
parasites have become resistant to insecticides; bacteria have developed 
resistance to antibiotics and the ability to transfer this resistance to other 
bacteria. A potential victory for microbes! 

Bacteriology 

It is appropriate, then, that NIAID encourage additional studies on mechanisms 
of antibiotic resistance. In doing so it will not only improve the care of 
patients with bacterial infections, but it will also contribute basic infor- 
mation regarding man and microbes, for current knowledge of molecular genetics 
derives largely from studies of bacteria and viruses. 



6-1 



One of the major recent discoveries in this field is that a number of specific 
DNA sequences existing at multiple locations on prokaryotic genomes can 
transpose and translocate spontaneously to new sites by a mechanism that is 
independent of the host's normal recombination system. Progress in the 
identification, mapping and characterization of these insertion sequences has 
been made possible by the development of techniques for the analysis of DNA 
structure. These techniques have shown that multiple copies of insertion 
sequences are normal constituents of the bacterial chromosome and also of 
extra-chromosomal genomes; their location on bacterial plasmids has suggested 
that they play a key role in chromosome evolution. Insertion sequences have 
been identified among the repititious DNA sequences which flank drug-resistance 
genes on bacterial plasmids, strengthening earlier evidence that the spread 
of multiple drug resistance among bacterial pathogens is a manifestation of 
genetic plasticity made possible by DNA insertions. 

There are encouraging signs that studies of other properties of bacteria will 
benefit from the application of new technologies. Already, there is new in- 
formation about mechanisms of attachment to mucosal surfaces, and the role of 
pili in such attachment. Studies, of chemotaxis, of enzymes, of membrane 
biogenesis, and of the structural dynamics and functions of outer membranes, 
are providing insights regarding bacterial virulence and the prospects for 
new bacterial vaccines. Work continued during the year on such vaccines. A 
polysaccharide vaccine has been developed and licensed for Groups A and C 
meningococci, but attempts to produce an immunogenic and protective Group B 
polysaccharide vaccine have failed. Groups B, C, and Y meningococci share 
an outer membrane protein antigen, type 2, and a type 2 protein vaccine is 
being developed for the prophylaxis of Group B meningococcal infections. Two 
proteins for the gonococcus -- the principal outer membrance protein and pili 
protein -- have been shown to be single proteins with only a small amount of 
1 ipopolysaccharide (endotoxin), and are ready for Phase I trials. Of several 
protein antigens from toxigenic E. coli, one -- described as "colonizing 
factor antigen" -- when used as a sub-cutaneous vaccine protected against 
illness and reduced intestinal colonization in initial volunteer challenge 
studies. Another antigen related to attachment -- pili -- will soon be tested 
in volunteers. Work is progressing on characterization of the antigens of 
Groups B streptococci as potential vaccine components. 

Mycology 

Systemic fungal infections have long been recognized as important causes of 
severe, sometimes life-threatening disease. Primary infections with two 
fungi -- Coccidioides immites and Histoplasma capsulatum -- are particularly 
common in the southwestern and central U.S., respectively. Other fungi are 
opportunistic pathogens, and infections with such organisms are being recog- 
nized with increasing frequency as the use of immunosuppressive therapies 
increases in the management of patients with malignant diseases or organ 
transplants. There are few effective drugs for the treatment of mycotic 
infections in patients with impaired immune defenses. 

Consideration of these problems by an MIDP sponsored workshop led to the 
recommendation that several national mycology centers be established to assemble 



6-2 



a critical concentration of scientists knowledgeable in biochemistry, molecular 
biology and genetics, as well as in the clinical aspects of medical mycology, 
to provide a focus for contemporary research. Two institutions, the University 
of Washington, St. Louis, Missouri, and the University of California at Los 
Angeles, successfully competed for program project grants for the support of 
such mycology research units. It is believed that augmented research at these 
two strategically located units will revitalize the field of mycology and lead 
to more effective prevention and treatment of fungal infections. 

Concurrently, multi-center clinical trials of antifungal agents in selected 
infections such as cryptococcal meningitis have gotten underway. In prospect 
is a controlled trial of a new drug, ketoconazole, a synthetic oral antifungal 
compound which inhibits the biosynthesis of ergosterol , the major sterol in 
most yeast and fungi. The drug apparently can be used without impairing 
cholesterol synthesis in man. It should be possible to design other such 
targeted approaches as more is learned about the molecular structure and 
dimorphism of the pathogenic fungi. 

Parasitology 

The new and expanded programs in international health and tropical medicine 
implemented this year through the support of collaborative research overseas 
(International Collaboration in Infectious Diseases Research - ICIDRs) and of 
multidisciplinary programs in the U.S. (Tropical Disease Research Units - 
TDRUs) will augment research on parasitic diseases, particularly those target- 
ed by the World Health Organization: malaria, schistosomiasis, trypanosomiasis, 
leishmaniasis, and filariasis. This effort was facilitated by the many ties 
that University scientists have maintained with foreign investigators, and by 
the already existing studies of the Parasitic Diseases Panel of the U.S. -Japan 
Cooperative Medical Science Program. Four of six ICIDRs will be funded in 
FY 79; two ICIDRs and at least two TDRUs are projected for FY 80. Details 
are provided at the end of the Director's Report and in the report of the 
Molecular Microbiology and Parasitology Branch. 

This increased effort comes at an opportune time. In preparation for a 
"Conference on Pharmaceuticals for Developing Countries" held in January, 
1979, by the Institute of Medicine, National Academy of Sciences, a survey 
was made of current programs in U.S. government and academic laboratories 
for the development of preventive, prophylactic, diagnostic and therapeutic 
agents for the five parasitic diseases listed above plus leprosy and enteritis. 
In 1978, of the $36,013,000 spent by federal agencies for these purposes, 
$11,181,000 was invested by NIAID, $9,135,000 in behalf of extramural research. 
Considering the billions of world citizens infected with these and related 
diseases, an increasing number of whom are being offered refuge in the U.S., 
this is a sensible investment indeed. 

Virology 

One of the outstanding accomplishments of the year was the publication of the 
six volume report of the Virology Task Force, culminating two years of review 
by distinguished scientists for all parts of the U.S. who assessed the state 
of the art in virology and identified opportunities for fostering the under- 

6-3 



standing, prevention and control of viral diseases. Each of the five Task 
Force panels made a number of recommendations which were combined into a total 
of 25 in the final volume of the Report. These, in turn, were consolidated 
by the National Advisory Allergy and Infectious Diseases Council into ten 
research areas which, the Council recommended, should be emphasized and 
expanded by more than doubling the Institute's investment in research on 
virology in the next five years. That such an investment will yield significant 
dividends is buttressed by the Task Force's accounting of recent accomplishments 
and its projection of what lies ahead. As illustrated by paraphrased or ex- 
cerpted sections of the report, the future is bright. 

The history of virus research mirrors the progression from descriptive 
approaches to the era of molecular genetics. The development of improved 
biochemical and biophysical techniques for studying the structure and assembly 
of viruses has led to increased knowledge of the biosynthesis of viral compon- 
ents. This, coupled with the isolation of suitable mutants, has opened the 
way for a concentrated attack on the precise mechanisms involved in assembly 
processes and their regulation. Further efforts should lead to greater know- 
ledge of important pathogens on the one hand, and of normal macromolecular and 
cellular organization and function on the other. 

The fact that viruses, as bearers of unique genetic information encoded in 
their DNA or RNA, bring into cells conveniently small probes of essential 
life processes has made viral research the cornerstone of molecular biology. 
Viruses occur in all living creatures, and progress toward an understanding 
of those which cause disease in man has derived from concepts and techniques 
first applied to viruses of plants and bacteria. Viruses which infect bacteria 
are known as bacteriophages. Historically, the ideas of messenger RNA, of the 
triplet code of stop and start codons , even the very definition of a gene 
arose from their study. Future research is likely to yield results of impor- 
tance to the medical community in many ways. 

Temperate phages, along with plasmids, are members of a class of important 
genetic elements known collectively as "episomes." A recent finding in the 
field of episomal genetics has been that the genes which confer resistance to 
the antibiotics are carried on translocatable DNA elements capable of trans- 
position from one bacterial genome to another. Thus, phage research is 
applicable directly to the mechanisms of pathogenesis in bacteria since many 
bacteria are dangerous only when they contain particular plasmids or phage 
genomes. Understanding the mechanisms of maintenance , immunity, incompatibil- 
ity, gene expression, and the evolution of episomes will be of direct benefit 
just as it will be in the case of infectious drug resistance. One might even 
imagine devising competitor plasmids and phages which could effectively compete 
with undesirable plasmids or phages from the bacterial cell. 

Ideas about the fundamental mechanisms of gene expression, recombination, 
replication, and evolution, as well as related technology derived from phage 
research, are often directly applicable to the study of animal viruses or 
other organisms. Genetic analysis of viruses will seek to determine the 
number of viral genes, the function of each gene, and the order or position 
of these genes on the nucleic acid chromosome of the virus. Studies involving 
the process of recombination will then position the genes relative to each 

6-4 



other, yielding a genetic map of the virus. Once this has been accomplished, 
several steps may be taken with viral genes of known function to elucidate 
the mechanisms of viral virulence or to produce candidate viruses or antigens 
for vaccine production. Mutations in viral genes can be sought which alter 
pathogenicity, e.g., by affecting temperature of growth, to produce live, 
attenuated vaccine strains. These viruses can then be used to induce immunity 
similar to that following natural disease, but without illness. Alternatively, 
the gene for an immunizing antigen, e.g., influenza virus hemagglutinin, may 
be transferred to an E_. col i host via recombinant DNA technology, permitting 
replication of the gene and synthesis of the desired antigen as the bacterium 
grows in culture. Hepatitis and influenza genes are now being tested in such 
systems. Successful production and purification of specific antigens would 
have a great impact on vaccine manufacture and immunization practices for the 
prevention of acute viral infections. 

Selected Acute Infections : Infections are the most common cause of illness; 
viruses are the most common cause of infections; respiratory viruses and 
enteric viruses are the most common causes of viral infections, ranking first 
and second, respectively, as causes of illness. Although considerable progress 
as been made in identifying the viruses responsible for these illnesses, they 
persist as major public health problems because of the inadequacy of knowledge 
essential to the development of effective preventive measures and therapy. 

Acute respiratory infections are caused by a multiplicity of viruses, the most 
dramatic of which is influenza A, the cause of periodic world-wide-epidemics 
associated with high attack rates, significant morbidity and excess mortality. 
The most recent influenza A variant to appear -- in this instance, to reappear 
-- is the Russian strain (H-jN-]) which caused outbreaks in children and young 
adults in 1978 and 1979. In 1978, MIDP coordinated clinical trials of A/USSR 
influenza vaccine, combined with A/Texas and B/Hong Kong antigens, in approxi- 
mately 2,100 subjects. Reaction and serologic data from these trials provided 
the basis for the formulation of vaccines and recommendations as to dosage 
schedules for the 1978-79 season. At a series of technical meetings during 
the past year, culminating with the Surgeon General's Meeting on February 12, 
and the Secretary's Conference on March 6, 1979, these data were updated for 
those developing immunization recommendations for 1979-80. These reviews were 
greatly facilitated by the availability of NIAID's own computer competence in 
the Epidemiology and Biometry Branch, and by the close working relationship of 
the three agencies responsible for surveillance (CDC), control of vaccine 
production (BoB/FDA), and research (NIAID). The Director's office emphasized 
this collaboration when it elected to display DHEW research on influenza 
according to Science Base, Application, Transfer, and Training, the SATT model. 
The Director, NIH, then requested that this model be used as a basis for 
drafting a DHEW trans-agency five year research plan for influenza as an 
example of health research planning. 

The most important respiratory disease of children is that caused by respiratory 
syncytial (RS) virus. In infants and young children RS produces severe, 
sometimes fatal bronchiolitis. Older children and adults experience less 
serious illness during reinfection than do infants undergoing primary infection. 
Since serious RS virus disease occurs most often during the first few months 
of life, when infants possess passively transferred serum antibody, it is clear 

6-5 



that such neutralizing antibody in serum does not provide effective protection 
against the most serious effects of the virus. Nor, as noted, does it protect 
adults; reports during this year have documented the occurrence of moderately 
severe upper respiratory illnesses in adults exposed to children during the 
epidemics which occur every year in urban centers. Clearly, vaccines cannot 
be expected to prevent RS infection, so the current approach is to seek 
amelioration of the first infant illness by inducing local antibody with 
attenuated virus prior to infection with wild virus. Volunteer studies now 
in progress may permit this hypothesis to be tested in the coming year. 

Acute viral gastroenteritis affects a broad segment of the population through- 
out the world. In the developed countries it is a major cause of morbidity 
in infants and young children, whereas in the developing countries it is a 
major cause of both morbidity and mortality in this same age group. Consider- 
able progress has been made in elucidating the etiologic agents of viral 
gastroenteritis. Of note is the fact that these advances have been made 
without the use of classical cell culture systems which have been the keystone 
in the progress of modern virology but which are of no value in detecting 
these fastidious gastroenteritis agents in clinical specimens. 

Two groups of etiologic agents of viral gastroenteritis -- the parvovirus-! ike 
group of which the 27 nm Norwalk particle is the prototype, and the 70 nm 
rotavirus group -- have been identified. Studies of rotaviruses have depended 
heavily on the use of the electron microscope (EM) and studies of the Norwalk 
group of viruses have relied exclusively on the electron microscope, utilizing 
almost always the technique of immune electron microscopy (IEM) -- a method 
which might be defined as the direct observation of antigen-antibody interaction 

It is evident now that a 70 nm rotavirus is a major etiologic agent of sporadic 
infantile gastroenteritis. This virus has been associated etiologically with 
up to 50 percent of the hospitalized cases of diarrheal illness in many 
developed countries in temperate climates. Viruses of the Norwalk group are 
being related to more and more local outbreaks. Currently, second generation 
tests which are being or have been developed offer great promise for further 
unravelling the natural history of these agents. Future studies will have to 
address (i) the efficient propagation of these agents in cell culture, (ii) 
a comprehensive definition of their overall importance over a sustained period 
in the etiology of gastroenteritis in various populations, (iii) elucidation 
of the immune mechanisms involved in host defense, (iv) the development of 
effective methods to prevent or treat illnesses due to these agents, and (v) 
the continuing search for other etiologic agents of actue gastroenteritis. 

Similarly, the ability to recognize infections due to hepatitis A or B viruses 
has disclosed the need to research for other etiologic agents of hepatitis. 
The existence of "non-A, non-B" hepatitis viruses is inferred from the 
identification of hepatitis cases that lack serologic evidence of infection 
by hepatitis A or B viruses, cytomegalovirus or Epstein-Barr virus. Non-A, 
non-B hepatitis cases have been detected throughout the world. At present 
approximately 90 percent of post-transfusion hepatitis in the U.S. is type 
non-A, non-B. Recent studies of patients with repeated cases of apparently 
acute hepatitis suggested that at least two agents not related to type A or 
type B viruses exist, and recent transmission studies in chimpanzees have 

6-6 



provided electron microscopic evidence of two different viruses. Thus, with 
the prospects of an effective vaccine for hepatitis B, and the recent culti- 
vation of hepatitis A virus, it is important to determine what proportion of 
hepatitis cases occurring annually in the U.S. is caused by as yet unidentified 
agents. 

Influenza and other respiratory viruses are being studied at the Influenza 
Center and at several Vaccine Evaluation Centers; viral gastroenteritis is 
being studied at three Enteric Diseases Centers; viral hepatitis is being 
studied in several populations. There are, of course, many other viruses 
which infect man. A number of these infections can be related etiologically 
to specific agents, but, unfortunately, viruses have yet to be isolated from 
many presumed viral illnesses. The time is ripe for expanded epidemiologic 
studies of appropriate populations to define the natural history of these 
infections. In the view of the Virology Task Force this will best be done 
for the many widely prevalent viruses by continuing surveillance of family 
units. Although costly, relatively difficult, and requiring a long period 
of time to be productive, such family studies have two important advantages: 
(1 ) a given study can serve to describe the behavior of a large number of 
known viruses; and (2) it will yield a valuable "library" of specimens and 
illness information that can be exploited anew when new methods become 
available for studying infections with important new viruses or with currently 
known viruses which, for lack of readily applicable methods, have not yet 
been extensively studied. MIDP wishes to initiate one or more "Family Studies" 
as recommended by the Task Force. 

Chronic Disease : It has long been suggested that viruses may cause chronic 
as well as acute diseases. It is now known that one or more of the viruses 
that cause actue hepatitis may persist and cause chronic liver disease. There 
are other viruses which induce no acute symptoms, but which as a result of 
"slow", progressive persistent infections, are responsible for such rare CNS 
syndromes as Kuru, Creutzfel d- Jakob, and Familial Alzheimer Diseases. Obser- 
vations in the past year have contributed new knowledge regarding the role of 
viral infections in a major chronic disease, diabetes. These studies support 
the hypothesis that acute destruction of specialized tissue cells may lead to 
chronic loss of function of those cells, specifically the beta cells of the 
islets of Langerhans of the pancreas. 

The common cold and influenza, along with measles, chicken-pox, and mumps, 
are examples of acute viral diseases from which, barring complications, patients 
recover. In rare instances, measles virus may cause a severe and fatal brain 
disease called subacute sclerosing pan encephalitis (SSPE). In other instances, 
not so rare, the chicken-pox virus may persist in ganglion cells of the nervous 
system and be reactivated to produce the painful lesions of herpes zoster. 
Mumps virus, which sometimes produces meningitis and pancreatitis, as well as 
parotitis, has been questioned as a cause of diabetes. While the association 
of mumps with diabetes has yet to be established, at least one virus now has 
been incriminated as a cause of juvenile onset diabetes in humans. This is 
a coxsackie B4 virus isolated by investigators at the National Institute of 
Dental Research from the pancreas of a previously healthy ten-year-old boy 
who died in diabetic coma ten days after the onset of symptoms of an acute 
febrile illness. Previous studies in animals had shown that Venezuelan equine 

6-7 



encephalomyelitis virus, encephalomyocarditis virus, reo virus type 1, and 
coxsackie B4 virus could induce diabetes in certain strains of mice. They 
do so by destroying the beta cells. Since it is these cells which produce 
insulin, their malfunction or destruction lead to a lack of insulin, and thus, 
to insulin dependent, or juvenile, diabetes mellitus. The coxsackie B4 virus 
isolated from the young boy produced diabetes in susceptible mice. 

Many questions remain to be answered in FY 80 and beyond. Is human suscepti- 
bility genetically determined as is the case in mice? Antibodies to coxsackie 
B4 are present in about half the population; even more common are antibodies 
to mumps. Diabetes may occur in children who lack antibody to either virus. 
Perhaps initiation of juvenile-onset diabetes requires more than a simple 
virus infection in genetically predisposed individuals. Why do damaged beta 
cells not regenerate? Clearly, additional epidemiological studies are needed 
to examine these questions. Such prospective, longitudinal, mul tidiscipl inary 
studies will be costly and demanding, but the answers they seek would form the 
basis of new approaches to prevention of a particularly devastating chronic 
disease. 

International Health 

International health programs are administered by Dr. Earl Beck, Special 
Assistant to the Director, MIDP, in conjunction with individual program officers 
and coordinators. 

The International Centers for Medical Research (ICMR) that have been operative 
for nearly 20 years will be phased-out on or about May 31, 1980. The four 
institutions participating in the ICMR program are the Johns Hopkins University 
with an overseas base presently at the Gorgas Memorial Laboratories, Balboa 
Heights, Canal Zone; Tulane University with a research unit in Cali, Colombia; 
the University of Maryland with a base in Lahore, Pakistan; and the University 
of California with a unit in Kuala Lumpur, Malaysia. 

ICMR Evaluation : The plan to evaluate the ICMR program with 1% set-aside 
funds was dropped because of prohibitive cost projections and timing. Since 
the ICMRs will be in their phase-out year, it was decided to make plans now 
to evaluate the ICIDR program in about three years. 

International Collaboration in Infectious Diseases Research (ICIDR) is a new 
initiative in the Microbiology and Infectious Diseases Program. It is divided 
into Part A, International Program Project Grants, and Part B, International 
Exploratory/Developmental Research Grants. The research emphasis of the new 
ICIDR program is tropical infectious diseases and the immunology of these 
diseases. Special attention is given to the six diseases of the WHO Special 
Program for Research and Training in Tropical Diseases. 

Part A, International Program Project Grants, embraces broadly based mul ti- 
discipl inary research programs that have a well-defined central focus or 
objective, with major portions of the research being conducted overseas with 
an acceptable foreign affiliate. Fourteen applications were received and 
reviewed; nine were approved, and five disapproved. Of the nine approved, 
six were within the fundable range. The Institute plans to fund four of the 

6-8 



program project grants in late FY 79, and two in FY 80, depending on the 
availability of funds. The grantees will be collaborating with scientists 
in Brazil, Thailand, Sudan, Colombia, and Pakistan. 

Part B, International Exploratory/Developmental Research Grants, encourages 
an individual investigator to develop a biomedical research program with an 
overseas affiliate, with most of the work being done in the host country. 
Thirty-nine proposals were received and reviewed; sixteen were approved, and 
twenty-three disapproved. Of the sixteen approved grants, five were within 
the fundable range. These grantees will be collaborating with scientists in 
Mexico, Brazil, India, and Nigeria. Based upon the Institute's Advisory 
Council's recommendation that the Part B portion of the ICIDR program be 
readvertized, consideration is now being given to this recommendation so as 
to provide applicants as much time as possible for the preparation of appli- 
cations. Funding of these grants is projected for FY 81. 

Diplomatic initiatives by successive U.S. administrations have involved NIH 
in a growing number of bilateral health agreements, with MI DP being assigned 
responsibility for coordinating participation of NIAID investigators in 
collaboration in infectious diseases research. The oldest and most productive 
of these bilateral programs is that with Japan, now completing its fifteenth 
year. 

The U.S. -Japan Cooperative Medical Science Program provides the mechansim for 
scientists of the United States and Japan to collaborate on the following 
diseases or disease categories of importance to the health of the people of 
Asia: cholera, environmental mutagenesis and carcinogenesis, leprosy, mal- 
nutrition, parasitic diseases, tuberculosis, and viral diseases. 

The Joint Subcommittee on Program Review and Planning met in Honolulu on 
February 8 and 9, 1979, and again on July 25, 1979, at NIH, just prior to the 
Fifteenth Joint Committee meeting. Major topics discussed and acted upon at 
the latter meeting included the following: 

1. The formal review of the Joint U.S. -Japan Panels on (Methods for 
Evaluating) Environmental Mutagenesis and Carcinogenesis, initiated in 1978, 
was completed. The Committee accepted the report with revised guidelines as 
recommended by the Subcommittee. The future emphasis of this program will 
change from developmental methodology to population monitoring. 

2. The formation of a Hepatitis Panel was formally approved along with 
appropriate guidelines and Panel members. The first joint meeting of the new 
panels will be held in Japan in 1980. 

3. Revised guidelines for the Tuberculosis Panels were considered and 
approved. The guidelines were sharpened to reflect the heavy emphasis on 
research relating to the pathogenesis and immunological aspects of the disease. 

4. Preliminary discussions were held to explore the possibility of 
introducing discipline oriented panels into the program, with the first such 
Panel to be one on Immunology. The matter is to be considered further at the 
next meeting of the Subcommittee on Program Review and Planning in February, 

6-9 



1980. The Subcommittee will make these recommendations to the next Joint 
Committee meeting in Tokyo in 1980. 

5. A schedule was developed for publishing a Third Five Year Report, 
1975-1980. A rough draft is to be ready for Subcommittee review at its 
February, 1980 meeting. 

U.S.-U.S.S.R. Health Cooperation : The Institute's responsibility under this 
program consists of its participation in the U.S.-U.S.S.R. Collaborative 
Agreement on Influenza and Acute Respiratory Diseases. For the period October 
8 through November 5, 1978, Dr. Fred Hayden, University of Virginia, partici- 
pated in clinical trials of anti-infl uenzal drugs at the All Union Institute 
for Influenza, Leningrad. As part of a month-long visit to the U.S., Dr. Yuri 
Ivannikov of that Institute spent a week at NIH in March, 1979, discussing 
epidemiological studies. 

As U.S. Coordinator of Problem Area IV - Chemotherapy and Chemoprophylaxis - 
Dr. George Galasso organized a meeting on this subject at the Influenza Center 
in Houston, Texas, on February 1-2, 1979. Five Soviet scientists participated, 
with three remaining for visits to U.S. laboratories. It is expected that 
several Soviet investigators will review the Russian experience with amantadine 
at the Consensus Conference on the use of this anti-infl uenzal drug scheduled 
for mid-October, 1979. Two days later, a joint meeting in Bethesda will address 
Problem Area II - Immunoprophyl axis - and its two topic areas. Dr. W. S. Jordan, 
Jr. is U.S. Coordinator for Topic 1, Development of killed and live influenza 
vaccines; Dr. Frank Ennis (BoB/FDA) is U.S. Coordinator for Topic 2, Standardiz- 
ation and control of influenza vaccines. 

U.S. -China Cooperation in the Science and Technology of Medicine and Health : 
On June 22, 1979, representatives of the Ministry of Public Health of the 
People's Republic of China and of the Department of Health, Education, and 
Welfare of the U.S. signed a Protocol of agreement, and later participated in 
the first meeting of the U.S.-P.R.C. Joint Committee for Cooperation in Medicine 
and Public Health. One of the subjects designated for cooperative research is 
to be Infectious and Parasitic Diseases, with initial cooperation in four areas: 
1) viral hepatitis; 2) schistosomiasis; 3) influenza; 4) malaria. Plans are 
now being made to implement this new bilateral agreement. 

Cholera Research Laboratory : For nearly twenty years the NIH/NIAID has operated 
the Cholera Research Laboratory (CRL) in Dacca for the U.S. Agency for Inter- 
national Development (AID) as a field station for studying cholera and related 
diarrheas. The CRL was launched under the Southeast Asia Treaty Organization 
in 1960, and became the International Center for Diarrheal Disease Research/ 
Bangladesh this year. This change in name reflects a change in administration 
negotiated by the CRL with its various sponsors. The final steps in the trans- 
formation of CRL to ICDDR/B occurred during the last week of June, 1979, when a 
new Board of Trustees met in Dacca and assumed responsibility for operating the 
Center. Dr. Carl Miller has served the U.S. administrative needs of the CRL for 
many years, and was particularly helpful during the recent period of transition. 
NIH will now relate to the ICDDR/B in its new status as an autonomous institu- 
tion. It is anticipated that the laboratory will continue to make important con- 
tributions to the treatment and control of cholera and other diarrheal diseases. 



6-10 



BACTERIOLOGY AND VIROLOGY BRANCH 

The Bacteriology and Virology Branch is responsible for the administration 
of a broad-ranging program of biomedical research; it supports program 
project grants, individual research grants, training grants, career develop- 
ment awards, individual postdoctoral fellowships, and contract programs of 
targeted research in bacteriology, virology, and mycology. Dr. Milton Puziss 
administers the Branch and the Bacteriology Program. Dr. William P. Allen 
administers the Virology Program, and also serves as Executive Secretary of 
the Virology Panel, U.S. -Japan Cooperative Medical Science Program. One of 
his recent activities, for which he received the NIH Director's Award, was 
bringing to a successful conclusion the work of the Virology Task Force. 
Dr. Darrel D. Gwinn administers the Mycology and Mycobacterial ogy Programs, 
and also serves as Executive Secretary of the Leprosy Panel and of the Tuber- 
culosis Panel, U.S. -Japan Cooperative Medical Science Program. 

Approximate Level of Support 

Bacteriology and Mycology 

Activity Number Amount 

Research Grants 

Research Program Projects 

Career Awards 

Training Grants 

Fellowships 

Research Contracts 

Total 

Virology 

Research Grants 

Research Program Projects 

Career Awards 

Training Grants 

Fellowships 

Research Contracts 

Total 
Branch 

Total 
Program Summary 

The program of this Branch focuses upon projects in both fundamental and applied 
biomedical research, with the ultimate objective being the translation of the 
knowledge gained into more practical methods for the diagnosis, prevention 

7-1 



178 


$12,821,120 


7 


2,167,800 


11 


399,169 


19 


1,563,342 


10 


142,200 


12 


1,003,379 


237 


$18,097,010 


194 


$16,016,949 


2 


464,087 


21 


699,908 


13 


1,177,476 


17 


211,150 


1 


22,000 


248 


$18,591,570 


485 


$36,688,580 



and therapy of bacterial, fungal and viral diseases. Certain research areas 
have been considered as Programs of Institute Emphasis; research in these 
targeted endeavors is continuing and expanding where possible. These ongoing 
Institute Emphasis Programs are in Sexually Transmitted Diseases, Hospital 
Associated Infections, Streptococcal Diseases and Sequelae, and Mycology, 
plus several in Virology. Viral infections have a significant and continuing 
impact on the health of the public; control of these infections is a vital 
part of the Virology Program where studies in Persistent Infections and 
Chronic Viral Diseases, Clinical Virology, and Biology of Viruses have been 
highlighted for increased attention. Targeted research on rabies, dengue and 
other arthropod transmitted diseases under the auspices of the U.S. -Japan CMSP 
is also a part of the thrust toward expanding knowledge of viral diseases 
commonly found in the developing countries of the world. 

Bacteriology 

Sexually Transmitted Diseases (STD) 

Sexually Transmitted Diseases continue as one of the most pressing problems 
in public health today, with little indication of any significant diminution 
in their prevalence. Gonorrhea is still the most important of these diseases, 
although many experts now consider that this has been superseded by the rapid 
increases in nongonococcal urethritis (NGU) and in herpes simplex genital 
infections (HSV-2). There is also increasing concern regarding other 
diseases, such as hepatitis, amebic dysentery, and shigellosis that are now 
being seen with increasing frequency, particularly among homosexual groups. 
The Institute's overall research program in support of the STD problem 
includes program projects (STD Centers), grants, contracts, and training. 
An important new thrust in FY 1979 was the initiation of a contract to 
develop candidate vaccine materials for the eventual control of gonorrhea. 

Research Highlights 

P01 AI 12192-04 K. K. Holmes (University of Washington) : Dr. Holmes, the 
director of this project, has reported that chlamydial infections in females 
have a strong association with neonatal death, in addition to fetal wastage 
and low birth weights. He also found that these infections may be a cause 
of peri -hepatitis (liver surface infection), as detected by a rise in anti- 
body titers. Dr. Falkow of this group has found that Chlamydia trachomatis 
and C_. psittaci have a group plasmid of apparently the same size, 4.5 daltons. 
This plasmid was also found in Lymphogranuloma venereum strains of Chlamydia ; 
there is, therefore, a very close genetic relationship between these chlamy- 
dial organisms. The chlamydial plasmid DNA has been cloned into an E_. coli 
plasmid; the role of this plasmid as a virulence factor in disease, however, 
is not yet clear. Dr. Eschenbach has undertaken a study of vaginitis as 
an STD problem. He has reported that Hemophilus vaginalis infections can 
be successfully treated with metronidazole (Flagyl]"? In a study of male 
partners of infected females, he isolated H_. vaginalis from both the urethra 
and semen. He further noted that vaginitis may be a mixed infection with 
H_. vaginalis together with an anaerobic flora. By DNA homology studies 
H_. vaginalis was determined to be unrelated to Corynebacteria species or 
to any other similar organism -- it may be a new genus. 

7-2 



AI 13233-03 J. A. Yorke (University of Maryland) : This investigator is 
studying the epidemiology of gonorrheal infections by developing a mathemati- 
cal model of the existing and potential effectiveness of various control 
programs. His study indicates that screening programs for gonorrhea 
discover only one-tenth of all infected women, usually in the asymptomatic 
stage. He also emphasizes the importance of relatively small "core" groups 
who are repeatedly reinfected and are efficient disease transmitters. In 
a model based on use of a hypothetical gonorrheal vaccine administered to 
this "core" group, he reported that there would be a substantial reduction 
in gonorrhea prevalence, even with immunity of short duration. Dr. Yorke 
emphasizes that it is roughly three to five times as important to identify 
and treat the source of the primary infection as it is to treat a secondary 
infection. This concept of a "core" group of efficient disease transmitters 
can be valuable in understanding the transmission dynamics of other diseases. 

P01 AI 12618-04 J. M. Knox (Baylor College of Medicine) : Dr. G. Dreesman, 
an investigator in this program project, is attempting to develop efficient 
techniques to purify native, immunologically active herpes virus type 2 
(HSV-2) glycoproteins. Such a viral subunit would have significant potential 
as a candidate vaccine. Dr. Dreesman demonstrated that HSV-2 glycoproteins 
of 119,000 MW will induce virus neutralizing antibodies in immunized rabbits 
and guinea pigs. A new technique was developed for fractionation of viruses; 
distinct peaks were isolated, with MW ranging from 20,000-200,000. These 
were tested for HSV type-specific antibody by a microsolid phase radio- 
immunometric assay. The peak containing protein with 100,000-140,000 MW 
reacted preferentially with an HSV-2 antibody positive human serum, but not 
with an HSV-1 (oral herpes) serum. This material is now being evaluated for 
induction of protective antibody in guinea pigs. Other studies by this 
group show that 10% of females with prior HSV-2 episodes shed virus during 
their asymptomatic periods long after their overt disease phase, a finding 
of significant epidemiological importance relative to spread of the viral 
infection. 

AI 14648-02 A. G. Plaut (New England Medical Center, Boston) : Dr. Plaut 
previously reported the discovery of a protease enzyme in the gonococcus that 
destroys IgA immunoglobulin. Purification kinetics of this enzyme are under 
study. Antibody to the protease is inhibitory to the enzyme; patients with 
active gonococcal infection have a high titer of antibody to this protease. 
He has also found that this IgA protease occurs in Streptococcus pneumoniae 
and Hemophilus influenzae , in addition to the pathogenic Neisseria species. 

Hospital Associated Infections (HAI) 

Nosocomial, or Hospital Associated, Infections are an extremely troubling 
national health problem, resulting in significant mortality and excessive 
costs. As the immunocompromised patient population increases, the HAI 
problem tends to increase as well. The gram negative bacteria that are 
most commonly involved in these infections show a disturbing trend to 
antibiotic resistance, causing ever great difficulty in patient therapy. 



7-3 



Research Highlights 

AI 10108-09 A. I. Braude (University of California, S.D.) : Dr. Braude 
reported earlier on the efficacy of his J5 antiserum, obtained from human 
volunteers immunized with the "core" glycol ipid from an E. coli strain, in 
lowering the death rate from gram negative bacteremia. Newer data with 
50 of 90 additional patients (the study is still incomplete) indicate that 
the trend is very favorable, consistent with the life saving therapeutic 
effectiveness reported earlier. In these newer studies the prominence of 
bowel, lung, and urinary tract sites for gram negative infections was 
significant. The bacteremias were caused primarily by normal aerobic 
bowel inhabitants (E_. coli and Klebsiella species ) and antibiotic resistant 
opportunistic microorganisms ( Pseudomonas and Serratia ) ; Pseudomonas was the 
organism most frequently isolated from the blood. The prophylactic value 
of J5 antiserum was studied in agranulocytopenic rabbits to determine if 
it could prevent bacteremia. Results indicate that the intravenous injection 
of J5 antiserum protects rabbits against lethal Pseudomonas bacteremia; this 
protection lasts for at least six weeks. This study is being extended to 
determine the potential prophylactic efficacy of the J5 serum in burn 
patients. 

AI 14533-01 R. Garibaldi (University of Utah) : This project's goal is to 
establish cost-effective approaches to prevent post-operative nosocomial 
infections. A micropore membrane filter contact technique for quantitating 
bacterial contamination of wound surfaces was developed. This proved to be 
a sensitive and reproducible method for recovering bacteria; it may be an 
accurate predictor of wound colonization. In a retrospective comparison 
of hospitalization duration following post-operative infections, the data 
showed that wound infection rates varied with the type of surgery and likeli- 
hood of intra-operative bacterial contamination. The highest infection rate 
and prolonged hospitalization occurred following colon surgery (3.8%, with 
excess hospitalization of 23.8 days). This represents a significant addition 
to the costs of medical treatment. It was also shown that administration of 
antibiotics prophylactically prior to surgery was frequently misused. Such 
misuse probably predisposes to colonization and infection with antibiotic 
resistant bacteria. The routine use of an in-line micropore bacterial gas 
filter in anesthesia equipment had no effect on the incidence of post- 
operative pulmonary infection. Use of these devices is not cost effective 
and probably should be discontinued. 

AI 13120-03 C. D. Cox (University of Iowa) : Dr. Cox previously reported 
investigations on the iron-binding siderophore, pyochelin, of pathogenic 
Pseudomonas . He showed that all clinical isolates apparently possess this 
iron-binding pigment. Knowledge of the pyochelin structure is important 
for understanding iron metabolism and how to interfere with this effect 
during an infection. Most of the pyochelin structure has now been 
elucidated -- it is about 650 MW, composed of two identical subunits 
each containing a salicylic acid and sulphur. The compound is also very 
labile. The chelated iron is adsorbed rapidly to the bacterial surface; 
the pyochelin is exceptionally effective in stimulating iron uptake by the 
bacteria at extremely low concentrations. Pseudomonas bacteria passed 
through mice infected intraperitoneal^ are most responsive to host iron 

7-4 



for increased lethality; organisms harvested from lung tissue (intrathoracic 
infections), however, suppress phagocytosis and cause increased lethality. 
This mouse model describes differences in ecological niches in the host 
as well as mechanisms of Pseudomonas virulence. 

AI 12936-03 J. W. Alexander (University of Cincinnati) : Dr. Alexander has 
been studying immune responses in surgical infections. Previous data 
indicated that abnormal neutrophil function was clearly associated as a 
predisposing factor in post-surgical infections. This group is now studying 
the close association between nosocomial infection and nutritional status. 
Further data indicate that well-nourished burn patients supplemented with 
plasma did very little better than controls, but patients with a "high 
protein" regime showed a significant improvement in neutrophil function. 
In a mouse model system, administration of Corynebacterium parvum vaccine 
resulted in increased macrophage activation. The C_. parvum vaccine also 
resulted in increased survival following challenge by Staphylococcus aureus 
and Listeria monocytogenes . Additional study in guinea pigs showed that 
skin grafts in cortisone-treated immunosuppressed animals had enhanced 
survival times when C. parvum was administered. These observations are 
of clinical significance in establishing a rationale for use of substances 
like £. parvum in treating infections in certain immunosuppressed conditions. 
The clinical aspects of these studies are now being investigated. 

AI 11271-06 L. S. Young (University of California, L.A. ) : This investigator 
has focused on host interaction to Pseudomonas infection. He has reported 
that patients with advanced Cystic Fibrosis (CF) showed a high level of 
circulating immune complexes against Ps_. aeuruginosa . Further these com- 
plexes had "enriched" antibody titers against lipopolysaccaride antigens 
of the organism. No significant antibody level against Pseudomonas exotoxin 
A in the CF patient sera was observed; preliminary data indicated, however, 
that the immune complexes contained endotoxin-like activity. 

Conference 

A workshop on Pseudomonas infections in Cystic Fibrosis (CF) was held on 
January 9, 1979, to explore the magnitude of this infectious disease 
problem. These infections are responsible, in large part, for the extreme 
disability and early mortality of the young CF patient. Recommendations 
for expanded research in selected areas of this disease problem were 
developed as a result of this meeting; publication of the summary pro- 
ceedings will be in the Journal of Infectious Diseases . 

Streptococcal Diseases and Sequelae (STP) 

Group B streptococcal disease (GBS) persists as a significant cause of 
neonatal mortality or serious sequelae. Infants of mothers with low 
maternal antibody levels are at high risk of developing GBS infections. 
Research leading to a better understanding of the disease problem and to 
development of a candidate vaccine is of high priority. 



7-5 



Research Highlights 

NO! AI 72538 D. L. Kasper (Peter Bent Brigham Hospital, Boston) : This 
contract is for study of the antigens of the GBS and development of a 
candidate vaccine to protect neonates at risk. The objective is to 
vaccinate pregnant women with low maternal antibody levels so that the 
neonate at birth will have a high protective maternal antibody. Research 
is focused on the capsular polysaccharide of the type III GBS. This 
purified polysaccharide elicited a significant antibody rise in immunized 
human volunteers, correlated with a rise in the opsonic index. The antigen 
has a sialic acid side chain attached to a carbohydrate "core"; the side 
chain is important in eliciting the protective antibodies. Studies of GBS 
types la and II polysaccharide antigens have also been initiated. It is 
expected that eventually a tri-valent GBS vaccine will be developed, incor- 
porating these three antigenic polysaccharides. Investigation has also 
begun on plasmapheresis procedures to prepare high titer human immune 
globulins (IgG) from human volunteers. If successful this material will 
be used for passive immunization to protect high risk infants against the 
late-onset type of GBS disease. 

AI 13150-03 H. R. Hill (University of Utah) : Host-defense mechanisms 
against GBS infections are not fully defined. Dr. Hill has developed a 
promising rat model for study of GBS pneumonia. Newborn rats (under 16 
hours old), inoculated with GBS type III intranasally (LD50 of 5x106 
organisms) developed fatal pneumonia and sepsis. Adult animals with 
similar challenge showed no bacteremia or mortality. Human serum contain- 
ing GBS opsonic activity, when given intraperitoneal^, protected challenged 
neonatal animals. The routes of infection, rapid death, neutrophil changes, 
and neonatal susceptibility are important parallels to human infection. 
When whole blood containing heat-stable antibody to the infecting GBS was 
given to human neonates, a rise in neonatal opsonic activity resulted. 
Nine of nine infants receiving this transfusion survived their septic 
episodes, whereas three of six infants receiving blood lacking antibody 
to their infecting strain died. 

AI 14361-02 J. B. Zabriskie (Rockefeller University) : This project is a 
study of the streptococcal and host factors that are of pathogenic importance 
in the sequelae of streptococcal infection. A unique population in Trinidad 
is under study, where both rheumatic fever (RF) and acute glomerulonephritis 
(AGN) are common sequelae of the streptococcal infections. Results show that 
patients with RF respond primarily to antigens present in the protoplast 
membrane of streptococcal strains associated with RF and do not react 
to antigens found in strains associated with nephritis. Those strains 
associated with AGN secrete a protein antigen found in the streptococcal 
cell membrane; this antigen is also detected in patients' glomeruli. 
Each patient group thus reacts specifically to those antigens present 
in streptococcal strains associated with a particular disease. This 
patient reactivity may be genetically inherited. 



7-6 



Other Disease Problems 

Staphylococcus aureus is an organism that can cause a number of distinct 
disease syndromes within the same host; the organism also produces an 
impressive array of biologically active toxins. 

AI 14998-02 M. E. Melish (Kapiolani-Children' s Medical Center, Honolulu) : 
Dr. Melish is investigating the clinical-pathological correlates of infection 
with staphylococcal exfoliatin, or epidermolytic toxin (ET), the cause of 
staphylococcal "scalded skin syndrome" (SSS) in infants. A newly-developed 
antibody radioimmunoassay was 1,000 times more sensitive than earlier methods 
for detection of ET in blood tissues; this method detected ET in acute sera 
from all SSS patients tested. No free ET was detected, however, in patient 
sera after antibiotic therapy was begun. In an adult mouse model, epider- 
molysis by ET could be induced regularly; this developed 10-16 hours after 
peak serum levels of ET were established. Patients with generalized SSS 
showed no evidence of pre-existing ET neutralizing antibody. In contrast to 
generalized toxemic SSS, localized bullous impetigo can develop and progress 
despite high levels of circulating antitoxin. Screening of normal adults 
revealed that over 85% of those over age 20 have ET antitoxin, whereas only 
24% of children under age two years do so. Thus, lack of this age-related 
antitoxin appears to be associated with the prevalence of SSS in children. 

AI 07826-10 F. A. Kapral (Ohio State University) : In this study on SSS, 
Dr. Kapral showed that the syndrome is the result of the intercellular 
separation among cells of the granular layer; it does not involve actual 
cell destruction. In a neonatal mouse model, he showed that a positive 
Nikolsky sign (loss of skin elicited by gentle stroking) cannot serve as 
the sole criterion for exfoliatin action. Recent data show that certain 
S_. aureus strains produce a substance causing a positive Nikolsky sign 
distinct from exfoliatin. This substance caused destructive lesions 
beneath the granular layer; it also differed in antigenic specificity from 
ET. S_. aureus strains isolated from patients produced this new exfoliatin- 
1 ike material. Additionally, an extractable lipid-like material found 
within abscesses exhibited marked bactericidal activity against certain 
S. aureus strains. 

AI 03772-19 P. A. Murphy (Johns Hopkins University) : The pathogenesis of 
fever is only vaguely understood. Dr. Murphy has shown, in a goat model, 
that purified macrophages make at least four distinct pyrogens; these are 
in response to stimulation by labeled endotoxin, by labeled staphylococcal 
organisms or by influenza viruses. The purified pyrogens all gave double- 
labeled peaks regardless of the stimulating source. One of these macrophage 
pyrogens fs identical to that from neutrophils; the other three are clearly 
different. 

AI 09366-09 T. A. Stamey (Stanford University) : Urinary tract infections 
in women represent a costly public health problem. Dr. Stamey has found 
that cervicovaginal antibody in female volunteers was directed against a 
variety of E_. coli strains but not against the volunteers' indigenous flora. 
Antibody was uniformly found in salivary fluid, eliminating the possibility 

7-7 



of an immunodeficient reaction at mucous surfaces; bacterial pili, however, 
played a significant role in attachment to vaginal epithelial cells. The 
defect allowing E_. coli attachment and disease initiation is most probably 
a local immunologic one in vaginal secretions. 

Leprosy 

Leprosy is found in almost every country in the world; it is a significant 
health problem in the developing countries of the world. The majority of 
the estimated 12 to 15 million cases of leprosy (10 million cases are 
recorded at WHO) are found in Asia, Africa and South America; as many as 
2,000 cases are in the United States. Approximately 150 new cases of 
leprosy are reported in the United States yearly, with about 25% of these 
occurring in native-born Americans. More significant than the actual 
number of leprosy cases is the evidence that not more than 20% of the 
estimated cases in the developing countries are under regular treatment. 
Also, children make up roughly one third of the total number of those people 
infected with leprosy. Because of its worldwide importance, leprosy research 
continues to be supported by NIAID under the auspices of the U.S. -Japan 
Cooperative Medical Science Program. 

Research Highlights 

AI 10094-09 W. E. Bullock (University of Kentucky) : Dr. Bullock has been 
concerned with further studies on the nature and function of suppressor cell 
populations generated within spleens of mice experimentally infected with 
Mycobacterium lepraemurium . Most recently, he has investigated the nature 
of splenic suppressor cells from infected mice that suppress cell mediated 
lymphocytotoxic activity by normal spleen cells. Through these studies it 
has been demonstrated that two such suppressor cell populations are generated 
within the spleens of M. lepraemurium infected mice. One suppressor population 
is composed of T lymphocytes and the other of macrophages or, possibly, B cells, 
The activity of the two populations acting together in whole spleen cell pre- 
parations usually is greater than the activity of either cell population 
alone. 

AI 08417-10 A. H. Fieldsteel (Stanford Research Institute) : Dr. Fieldsteel 
has continued his studies utilizing the neonatally thymectomized Lewis rat 
(NTLR) as a model for detecting persistent M. leprae . These experiments 
have demonstrated the superiority of the NTLR over mice for the detection of 
small numbers of viable M. leprae . Normal mice are routinely utilized to 
monitor the efficacy of leprosy chemotherapy. However, the maximum number of 
M_. leprae that can be inoculated into mouse foot pads to demonstrate multi- 
plication is 10^, and the ceiling of multiplication is approximately 10° 
On the other hand, the NTLR can be inoculated with up to 10' M_. leprae and 
the ceiling of multiplication is 10^ to 10 9 organisms. These experiments demon- 
monstrate that the NTLR can be used as a model of M. leprae persistence. 

AI 07801-12 J. L. Krahenbuhl (U.S. Public Health Service Hospital, San 
Francisco]" : Triatoma barberi (a blood sucking bedbug) was evaluated to 
determine the period of viability of M. leprae following ingestion of approxi- 
mately 5 x 10 7 organisms suspended in rabbit blood. Inocula of 5000 M. leprae 

7-8 



organisms recovered from the Triatoma between one hour and four days after 
ingestion were viable and found to multiply in mouse foot pads to a maximum 
of 1 x 106 organisms; M. leprae recovered seven days after ingestion by 
Triatoma multiplied to only 3 x 104 organisms, and after 14 days no multi- 
plication was observed. 

Tuberculosis 



In 1978, 28,521 cases of tuberculosis were reported to CDC. This represents 
a decrease, since 1977, of 5.4% in the number of cases reported. Since 
antibacterial drug therapy for tuberculosis was initiated, a shift in 
tuberculosis victims from young adults to older people has taken place. 
The atypical mycobacteria are related, but not identical, to Mycobacterium 
tuberculosis ; although diseases caused by atypical mycobacteria have symptoms 
similar to tuberculosis, the infections are not transmitted from man to man. 
Also, the atypical mycobacteria are frequently resistant to the antibacterial 
therapy employed in treating ordinary tuberculosis. The number of cases of 
tuberculosis and atypical tuberculosis in the U.S. is small in comparison to 
the millions of cases found in the developing nations of the world. Tuber- 
culosis in these countries remains one of the major causes of morbidity and 
mortality in all age groups. Tuberculosis research continues to be supported 
under the auspices of the U.S. -Japan Cooperative Medical Science Program. 

Research Highlights 

AI 13813-03 H. Gruft (New York State Department of Health) : Dr. Gruft is 
surveying the estuaries in the eastern U.S. to establish a basis for under- 
standing the epidemiology of atypical mycobacteria in relation to their 
geographical distribution. He has found a higher concentration of 
Mycobacteri urn avi um-i ntracel 1 ul are-scrof ul aceum (MAIS) strains along the 
Gulf Coast and in the waters of the Carol inas and Georgia. The highest 
concentration of MAIS strains are found in waters of 1-2% salinity, i.e., 
estuaries and mouths of rivers. MAIS strains are found in high concentration 
in waters from regions where most of the cases of positive skin tests have 
been found. They can also be found in the air in droplets small enough to 
enter the alveoli . 

AI 11807-04 B. M. Sultzer (SUNY Downstate Medical Center) : Since Dr. Sultzer's 
original discovery that PPD- tuberculin is a polyclonal activator of B-lympho- 
cytes from uninfected or unimmunized mice, other laboratories have found PPD 
to be a useful probe for studying a variety of basic immunological problems. 
Further studies on the nonspecific activation of lymphocytes have shown that 
PPD can act as a polyclonal activator (PCA) of human peripheral blood lympho- 
cytes; that PPD can act as an adjuvant and as a PCA in vitro and in vivo ; and 
that PPD stimulates B-cell hyperplasia when directly injected into mice. 
Continuing studies have shown that this intrinsic biological property of PPD 
to activate B-cell s is due to tuberculoprotein and not to extraneous materials. 
Fractionation studies indicated that the antigenic and mitogenic activities 
reside together in several molecular species of tuberculin proteins. Antigens 
without mitogenic activity can be separated from PPD; however, mitogens with- 
out antigenic activity have not been separated from PPD as yet. 

7-9 



NOT AI 02079 J. K. McClatchy (National Jewish Hospital) : Laboratories 
throughout the world have sent Dr. McClatchy in the last year alone almost 
1,400 isolates of atypical mycobacteria for classification and identification. 
In addition to his work with these cultures under the contract, he has sup- 
plied a number of investigators in the U.S. and abroad with reagents to 
perform their own serological identification. He has also investigated the 
chemical structure of the antigens which are used to classify the various 
atypical mycobacteria into different species and subspecies. The development 
of this methodology has been very helpful; Dr. McClatchy can now biochemically 
identify certain members of the atypical mycobacteria which could not be 
taxonomically classified using serologic methodology. Current work is being 
directed toward the identification of atypical mycobacteria found in patho- 
logic specimens from diseased individuals. 

Mycology 

Primary fungal pathogens that are directly involved in pulmonary diseases, 
such as Coccidioides immitis and Histoplasma capsulatum , are well established 
as significant causes of morbidity and mortality, especially in well defined 
geographic areas of the U.S. Other fungal pathogens, such as Candida and 
Cryptococcus , are important as opportunistic microorganisms, particularly in 
patients with impaired host defense mechanisms. Treatment of these fungal 
infections requires prolonged administration of relatively toxic drugs and 
the therapy may not always be effective. The Institute is presently funding 
research in which these pathogenic fungi and others, including Blastomyces 
and Aspergillus species, are being investigated. The Institute has established 
a special program in Mycology to intensify research efforts in this area. 

Research High! ights 

TO! AI 00459-06 G. Medoff (Washington University) : Dr. Medoff's group has 
been studying the control of dimorphic transition in fungi. H_. capsulatum , 
an important human pathogen, is one of these dimorphic fungi. Its saprophytic 
form is mycelial and is found in nature, whereas the unicellular yeast is the 
parasitic phase found in infected tissues. One phase can be induced to trans- 
form to the other in culture when the temperature of incubation is adjusted 
to an appropriate level. An understanding of the biology and genetic regula- 
tion of the process, it is believed, will provide insights into the pathogen- 
esis of disease caused by dimorphic pathogens and may help in the control of 
these infections. The major differences found between the two forms of this 
fungus were: 

(a) Discovery and purification of a cystine reductase present only 
in the yeast phase of H_. capsulatum . This enzyme and a cystine 
permease appear when the temperature is switched from 25C to 37C. 
Both enzymes may be involved in providing the sulfhydryl groups 
(in the form of cysteine) that are necessary to initiate and 
maintain the yeast phase. 

(b) The level of cyclic AMP is approximately five times higher in the 
mycelial phase than in the yeast phase of H. capsulatum . Further- 
more, addition of either cyclic AMP or inhibitors of cyclic AMP 

7-10 



phosphodiesterase induces transformation of yeast to the mycelial 
phase even at 37C, the nonpermissive temperature for mycelial 
growth in nature. 

(c) The yeast and mycelial phase each have three RNA polymerases, 
similar to those in other eukaryotic cells. In H_. capsulatum , 
the enzymes in one phase are clearly different, functionally and 
structurally, from the enzymes in the other. This represents the 
first case in which a clear difference in RNA polymerases has been 
shown to exist between morphologic phases of the same eukaryotic 
organism. 

Dr. Medoff is now testing, with several mutants of H_. capsulatum , a working 
hypothesis related to the sequence of events and control mechanisms in this 
transition. 

K04 AI 00325-05 R. D. Diamond (Boston University School of Medicine) : 
Previous studies demonstrated that neutrophils could attach to, damage, and 
probably kill Candida albicans hyphae by a nonphagocytic mechanism. In con- 
trast to most phagocytic systems, attachment and spreading of neutrophils 
over live hyphal surfaces with activation of microbial mechanisms occurred 
in the absence of serum. Addition of IgG from human serum augmented the 
process. In the absence of serum, neutrophils did not attach to killed 
hyphae. However, supernatants from actively growing hyphae or hyphae killed 
with ultraviolet light inhibited attachment of neutrophils to live hyphae. 
Evidence from Dr. Diamond's current work suggests that the soluble inhibitor 
acts directly on neutrophils; however, the neutrophils are not significantly 
damaged by exposure to the inhibitory substance. 

AI 13770-03 M. H. Weiner (University of Texas) : The objective of Dr. Weiner's 
project is to develop antigen immunoassays to diagnose systemic fungal infec- 
tion. Dr. Weiner has developed radio labeled antigen-binding inhibition 
assays (RIAs) to detect Aspergillus and Candida antigenemia. He has now 
analyzed the effectiveness of the RIAs with sera obtained from hospital 
patients with systemic bacterial and fungal infections and from normal donors. 
Using the RIAs he was able to detect accurately Aspergillus and Candida anti- 
genemia in the sera from patients suffering from these fungal infections. 
This is the first demonstration of Aspergil lus antigenemia detected in human 
sera obtained antemortem. 

AI 05022-15 G. S. Bulmer (University of Oklahoma) : Cryptococcosis, a fre- 
quently fatal meningeal fungus disease of man, is caused by the heavily 
encapsulated yeast Cryptococcus neoformans . Dr. Bulmer's goal is to deter- 
mine why and how certain individuals contract this disease. His studies, 
with the intragastric inoculation (mice) of C_. neoformans , indicate that 
medical personnel should be alerted to the possibility that cryptococcosis 
may begin in the gastrointestinal tract; i.e., cryptococcosis may not always 
originate in the lungs. Additionally, these studies indicate that the mouse, 
and possibly other rodents, may act as vehicles for dissemination of the 
pathogenic yeast in nature. Heretofore, pigeons have been considered to be 
the sole natural source for this organism. In another study Dr. Bulmer has 
shown that an aqueous extract of garlic is a potent killing agent of C_. 

7-1] 



m 



neoformans . This material should be investigated further as a possible 
therapeutic agent against cryptococcosis and other mycoses. 

Virology 

Program Summary 

Viruses continue to be a major cause of human illness in nearly all parts of 
the world, but modalities for control and treatment of these pathogens remain 
relatively limited. The large number and diversity of viruses that impact on 
human health add to the complexity of the problem of their control. Signifi- 
cant advances have been made in unraveling the components of viral structure, 
replication and interactions with the afflicted host, but much remains to be 
learned before a conquest of viral diseases can be attained. The Virology 
program within the BV Branch encourages and supports multidisciplinary 
approaches to research on viruses and viral diseases through its subprograms 
on Clinical Virology, Persistent Infections and Chronic Viral Diseases, and 
Biology of Viruses. 

Clinical Virology 

Clinical Virology is a program of Institute emphasis focused on pathogenesis 
and immunopathogenetic mechanisms of viral diseases of man. Clinically 
relevant research on pathogenetic viruses is encouraged to bridge a widen- 
ing gulf between molecular-oriented virologists and clinical investigators 
specializing in infectious diseases. Research projects on herpesviruses are 
especially prominent, a reflection of the high incidence and severity of 
human diseases caused by herpes simplex viruses, varicella-zoster virus, 
cytomegalovirus and Epstein-Barr virus. 

Research Highlights 

AI 14341-02 L. Aurelian (The Johns Hopkins University) : It is well estab- 
lished that human infections with herpes simplex virus (HSV) elicit both 
specific humoral and cellular responses. Dr. Aurelian has obtained pre- 
liminary data supporting the hypothesis that patients with a history of 
recurrent lesions suffer from an impaired generation of effector lymphoid 
functions (differentiated lymphoid cells that react specifically in response 
to exposure to viral antigens). The hypothesis further suggests that various 
HSV antigens may induce different responses, that such responses are affected 
by clinical status (vis., quiescence versus recrudescence), and that various 
in vitro assays measure the expression of different populations of lymphoid 
cells. In support of the hypothesis, recent results indicate that maximal 
effector levels are reached shortly after onset of recrudescence, i.e., 
during convalescence. They begin to wane thereafter, with borderline to 
negative levels being maintained during quiescence. New recrudescences 
occur on this background of low (or negative) effector functions. The 
phenomenon is virus specific. 

AI 14564-02 R. R. McKendall (University of California, San Francisco) : In the 
presence of specific antibodies, herpes simplex virus (HSV) becomes local i zed 
to specific sites in the body and is eventually eliminated or reduced to a 

7-12 



state of latency. Dr. McKendall has been investigating the characteristics 
of specific antibody molecules responsible for this defense against infection 
in a murine model. He has found that the neutralizing fragment, Fab, of the 
antibody failed to avert disease, which indicates that neutralization alone 
is not the major effect of antibody defense against herpetic infections. The 
results further suggest that effective control of viral spread within tissues 
is dependent upon the Fc fragment of the antibody molecule. The Fc fragment 
is not antigen specific. Moreover, if destruction of the blood-brain barrier 
is a late event in the course of herpes encephalitis or herpes myelitis, then 
serum neutralizing antibody would not be expected to be much of a host advant- 
age. This suggests a new therapeutic approach through methods which would 
enhance both antibody and cellular penetration into the central nervous system 
early in the course of disease. 

AI 14373-02 J. A. Zaia (Sidney Farber Cancer Institute) : Infections with 
varicella-zoster (VZV) exert deleterious effects on the clinical course of 
immunosuppressed patients, particularly those with lymphoproliferative dis- 
orders, causing significant morbidity and occasional death as well as inter- 
rupting scheduled chemotherapy. Dr. Zaia's research is aimed at understanding 
the pathogenesis of VZV infection in these patients, including an understand- 
ing of the elements of immunity which may be defective during cancer therapy. 
He has developed the methodology for efficient identification of human plasmas 
containing VZV-immune globulin by means of screening large amounts of recovered 
plasma for specific antibody to VZV. In recent studies he has demonstrated 
that this immunoglobulin (VZIG) prevents chickenpox in immunosuppressed 
children. The VZIG was as effective as standard zoster immune globulin (ZIG) 
in preventing severe complications due to chickenpox. The advantage of VZIG 
is that it can be produced easier and made more available than ZIG. He has 
also developed a new laboratory method for the rapid detection of VZV viremia 
during zoster infection. The method utilized the Fab component of VZIG for 
the detection of VZV membrane antigen in peripheral blood mononuclear cells. 
These results also indicate that specific antiviral antibodies constitute 
the major defense against VZV infections in man. 

AI 01475-22 E. H. Lennette (State of California Department of Health) : Anti- 
body against varicella-zoster virus (VZV) is routinely detected by compl ement- 
fixation (CF), or immune adherence hemagglutination (IAHA) or neutralization. 
Although neutralization is the most meaningful test in terms of protection 
against chickenpox or zoster, it is extremely cumbersome and expensive. 
Investigators in Dr. Lennette 1 s laboratory compared the newer techniques 
of enzyme immunoassay (EIA) with neutralizing, IAHA, and CF and found that 
EIA offers a rapid, sensitive, and specific method of antibody assay that 
is applicable in a clinical setting. The EIA test had a 94% correlation 
with neutralization and could reasonably replace the neutralization test 
and reduce costs for serodiagnosis of varicella or zoster. 

AI 11788-09 P. M. Horstmann (Yale University) : In general , the presence of 
antibody to rubella virus detected by hemagglutination-inhibition (HI) tests 
indicates that the person has protective immunity. However, recent evidence 
obtained by Dr. Horstmann demonstrated that, in a few individuals naturally 
infected or vaccinated, the presence of HI antibodies in the absence of 
detectable neutralizing antibodies (NT) did not prevent reinfection. 

7-13 



Therefore, the NT test is the one of choice for assay of protective levels of 
antibody in vaccinees. The study showed that NT responses to the new RA 27/3 
vaccine were higher titered and persisted longer at high levels than with the 
currently used HPV77DE5 and Cendehill vaccines. NT titers at 3-5 years post- 
infection were lower in vaccinees than in natural immunes. At nine years 
post-vaccination, 11% of 290 children who received the HPV77DE5 vaccine were 
found to be antibody negative by HI, and all of these had NT titers below 
protective levels. The results point to the superiority of the RA 27/3 
vaccine, which will soon become available in the United States. 

Workshop on Cytomegalovirus Infections during Organ Transplantation 

As the import of cytomegalovirus (CMV) infection has become evident, improved 
techniques to study the molecular biology of this agent have been developed, 
and a variety of possible therapeutic interventions has been proposed that 
might be useful in preventing and/or treating this infection. In an effort 
to coordinate research efforts aimed at the control of CMV infection during 
transplantation and to evaluate those therapeutic and preventive modalities 
currently available, a workshop was convened in June 1978 by NIAID comprised 
of a small group of experts in the field of CMV who were intimately involved 
and concerned with clinical transplantation. The goal of the workshop was to 
summarize presently available information, and to prepare an agenda which would 
lead to the systematic application and evaluation of each of the therapeutic 
modalities discussed. A summary of the proceedings has been published in the 
Journal of Infectious Diseases . 

Persistent Infections and Chronic Viral Diseases 

The mechanisms are little understood by which some viruses can infect a host 
and then remain within the host in a quiescent state for years or slowly 
proliferate, eventually to cause chronic disease. It is speculated that 
many chronic diseases of undetermined etiology may be caused by viruses with 
unique capabilities of escaping the host's defenses. The Virology program 
has encouraged investigations on persistent and slow virus research in efforts 
to identify and characterize the putative agents. Current emphasis in this 
program has shifted from the search for additional agents to more complete 
characterization of known persistent and slow viruses and their pathogenetic 
mechanisms. 

Research Highlights 

AI 06246-15 J. G. Stevens (University of California, L.A.) : Dr. Stevens has 
been studying the pathogenesis of herpes simplex virus (HSV) in model labora- 
tory and animal systems. Until recently, he was not able to establish a 
latent infection in mice similar to that which occurs naturally in man. 
Dr. Stevens has now characterized 15 mutants of HSV with respect to their 
ability to become latent in mice. Seven of the mutants did become latent 
whereas eight did not. Latency did not depend on the ability of the mutant 
to replicate DNA within infected cells, but did appear to be dependent upon 
viral protein syntheses beyond the immediate-early polypeptide. The discovery 
of these latent mutants promises to make studies of latency and reactivation 
at the molecular level in model HSV systems more feasible than previously 

7-14 



possible. It is also significant that latency can be determined by the viral 
genome. It should now be possible to identify the locus and defect in the 
viral genome which regulates latency and, ultimately, to explain the mechanism. 

AI 12438-05 R. M. Welsh (Scripps Clinic and Research Foundation) : One of the 
prime models for study of persistent infections is lymphocyte choriomeningitis 
virus (LCMV) in mice and cell culture. Dr. Welsh has been investigating the 
relationships between the immune response and defective interfering (DI) 
particles in the maintenance of chronic virus disease. Particular emphasis 
has been on the role of natural killer (NK) cells in response to infections 
with LCMV. It has been shown that cells infected with DI synthesize viral 
proteins at a wery slow rate. The decrease in expression of viral antigens 
could help to explain how these persistently infected cells escape immuno- 
logic attack mechanisms of the host. Dr. Welsh has also shown that acute 
infection with LCMV induces an augmented NK cell activity that is correlated 
with the synthesis of interferon type I (antiviral), and that NK cells were 
activated in vivo by interferon injections. Thus, these NK cells may be of 
great importance in controlling virus infections and contributing to immuno- 
pathology in acute and chronic infections. DI particles may be of signifi- 
cance in chronic infections by regulating viral antigenic expression, thereby 
limiting the severity of immunopathologic disease. 

Biology of Viruses 

Acquisition of knowledge concerning virus structure, function and interaction 
with the host has required technologies developed by several scientific dis- 
ciplines including microbiology, biochemistry, biophysics, genetics, physical 
chemistry and immunology. The field of biology of viruses is very fluid, and 
is often found moving in the direction of a new technology. Current trends 
reveal a substantial movement toward such technologies as recombinant DNA, 
recombinant RNA, oligonucleotide fingerprinting, and nucleotide and peptide 
sequencing to approach questions yet to be answered about the composition of 
viruses and how they infect cells, replicate and cause disease. 

Research Highlights 

AI 12717-04 E-S. Huang (University of North Carolina) : Dr. Huang, in col- 
laboration with Dr. C. Alford, University of Alabama, has been analyzing by 
restriction enzyme cleavage strains of cytomegalovirus (CMV) isolated from 
patients presenting various clinical manifestations such as recurrent infection, 
congenital defect and mononucleosis. The objectives of the study are to under- 
stand the mode of viral transmission, to classify viral strains, and to identify 
the origin of CMV isolates. In comparing viruses isolated from five sets of 
mothers and their offspring the restriction enzyme-fragment patterns were found 
identical even though some of the offspring viruses were isolated several years 
after virus was isolated from the mothers. In one recurrent case the fragment 
patterns of original and recurrent isolates were identical, indicating that 
the two isolates were derived from the same parental strain. In another recur- 
rent case, the recurrent isolate was distinctly different from the original, 
suggesting that the recurrent infection was caused by a new virus strain. 
In other collaborative studies, Dr. Huang has demonstrated that CMV virus 
isolated from an immunosuppressed renal transplant patient is different from 

7-15 



the CMV virus used to immunize that patient before the transplantation. This 
is an important observation because it indicates that the CMV used for vaccin- 
ation was not reactivated after transplantation and during immunosuppression. 
Rather it suggests that the post-transplantation infection was probably 
derived from the donor kidney. 

AI 14049-02 L. C. Norkin (University of Massachusetts) : SV 40 virus is an 
agent found to persistently infect kidney cells of rhesus monkeys. First 
discovered as a contaminant of early polio vaccines, it has become an excel- 
lent model for study by molecular virologists because of its simplicity, its 
ease of propagation and its oncogenic properties. Dr. Norkin has been inves- 
tigating variants of this virus that arise during persistent infections with 
respect to properties that might affect the course of infection. He has 
shown that stable persistent infections of rhesus monkey kidney cells can be 
established with little, if any, visible damage to cells. Only about half 
of the kidney cells are initially susceptible to infection, but after three 
to 11 weeks all cells contain and express virus genetic information. Only 
a small percentage of persistently infected cells produce infectious virus 
and these eventually die. The virus-producing cells are eventually killed 
and the productive infection is perpetuated by the emergence of new virus- 
producing cells from the population of nonproducing cells. Defective viral 
particles eventually emerge which interfere with the replication of infec- 
tious virus. Dr. Norkin has demonstrated that this interference is not 
associated with interferon. 

AI 12458-05 F. B. Bang (The Johns Hopkins University) : Dr. Bang's laboratory 
has been investigating the effect of diet on susceptibility to viral infec- 
tions. They have focused their studies on vitamin A deficiences in chickens 
infected with Newcastle disease virus (NDV), and on low protein diet in 
genetically susceptible and resistant strains of mice infected with mouse 
hepatitis virus (MHV). Results have demonstrated a synergistic effect of 
NDV and vitamin A deficiency in producing massive destruction of the bursa 
and thymus (two major organs of the immune system) in the chicken. Similar 
effects on the immune system were found with low protein diets and MHV in 
susceptible mice. The mouse system proved more amenable than the chicken 
for study at the cellular level in vitro . In this context, recent work has 
shown that in terms of susceptibility to MHV, the behavior of the macrophages 
in genetically resistant and susceptible mice mirrors both the genetic and 
phenotypic susceptibility of the intact mouse. These systems in the chicken 
and mouse promise to provide models for a better understanding of the effects 
of diet deficiencies on susceptibility to infectious agents. 

AI 09706-09 H. Koprowski (The Wistar Institute) : The new technology of using 
hybridoma cells to produce monoclonal antibodies is opening new approaches to 
the study of viral proteins. In this laboratory the technique was applied to 
the study of proteins of rabies virus. Cultures of hybridoma cells prepared 
by fusing mouse myeloma cells with spleen cells from rabies-immunized mice 
resulted in numerous clones producing highly specific antibodies. The anti- 
bodies produced by over 100 clones were analyzed and divided into the cate- 
gories of specificity corresponding to the known rabies virus antigens. 
Because of the highly specific properties of these monoclonal antibodies, the 
investigators were able to differentiate strains of rabies virus antigenically. 

7-15 



Previous to these observations, it was generally believed that all strains of 
rabies were antigenically yery closely related. The monoclonal antibodies have 
also substantiated that antibodies against the glycoprotein of rabies virus are 
responsible for neutralization of viral infectivity and for immune lysis of 
infected cells. 

Virology Research under the Auspices of the U.S. -Japan Cooperative Medical 
Science Program 

Goals for this program include the encouragement and support of research that 
will advance our knowledge of and lead to eventual control of rabies, dengue 
and other arthropod diseases and viral gastroenteritis. The program is moni- 
tored by the Panel on Viral Diseases, an advisory group to the U.S. -Japan CMSP. 

Research Highlights 

Rabies 

AI 09706-09 H. Koprowski (The Wistar Institute) : The new WI-38 human diploid 
cell rabies vaccine (HDCV) developed at the Wistar Institute is now produced in 
both France and West Germany and is licensed for pre- and post-exposure human 
prophylaxis in several European and Asian countries. The licensing by Wyeth 
Laboratories, Philadelphia, of HDCV produced in the U.S. is still in progress. 
The necessary clinical investigations have been completed; production will be 
initiated immediately in newly constructed facilities. The license is expected 
to be granted after completion of the first four lots of vaccine. In the mean- 
time, the HDCV is widely used as an Investigational New Drug (IND) with the 
authority for distribution in the Center for Disease Control, Atlanta. Over 
the last year, several hundred people in the U.S. were successfully treated 
with HDCV. 

Conferences 

Workshop on St. Louis Encephalitis 

A workshop convened in June 1979, under the auspices of the U.S. -Japan Coopera- 
tive Medical Science Program, was held at the Rocky Mountain Laboratory in 
Hamilton, Montana. The state of the science of St. Louis encephalitis (SLE) 
virus was reviewed and potential areas for future research were discussed. 
It was the consensus of the participants that substantial new information has 
been obtained on the biology and biochemistry of SLE virus, and exciting new 
research on flavi viruses is in progress. Research leading toward an SLE 
vaccine was encouraged, but reservations were expressed as to the advisability 
of developing a vaccine now before there is sufficient information on the pro- 
tective response to SLE in man. Potential control of SLE through a weak link 
in its mosquito vector transmission was discussed, i.e., the emerging female 
mosquito after overwintering. More complete information on the ecology of 
overwintering vectors is needed to locate sites where control measures would 
be most effective. A summary of the workshop will be published in the Journal 
of Infectious Diseases. 



7-17 



2 


$90,578 


6 


$390,795 















CLINICAL STUDIES BRANCH 



The Clinical Studies Branch (CSB) serves as the Institute's focus for the 
development and processing of Investigational New Drug Applications, and main- 
tains liaison with the NIH Human Research Review Panel and the NIAID-Clinical 
Research Subpanel . It provides and monitors a closed clinical facility for 
Institute volunteer studies, and conducts, promotes and supports the develop- 
ment and evaluation of procedures for the diagnosis, prevention and treatment 
of infectious diseases, particularly those procedures leading to improved 
patient care. 

Approximate Level of Support 

Activity Number Amount 

Research Grants 
Research Contracts* 
Training Grants 
Fellowships 

Total 8 $481 ,373 

* Two contracts have been forward-funded with FY 1978 MIDP 
funds and those funds do not show here. 

Program Areas 

Closed Volunteer Facilities 

The CSB currently supports one research contract with the University of Mary- 
land. This contract maintains and supports The Center for Vaccine Development 
(CVD) where volunteers are studied before and after they are challenged with 
potentially transmissible infectious agents and candidate vaccines. 

During the past year, the contractor performed 24 volunteer studies which 
were components of six more comprehensive research protocols approved by the 
Institutional Review Boards of the University of Maryland and NIAID. The 
challenge studies were distributed as follows: enteropathogenic E_. col i , 
seven studies, two protocols; cholera, five studies, one protocol; parvo-like 
gastrointestinal virus, three studies, one protocol; H3N2 influenza, four 
studies, one protocol; H-j N-j influenza, five studies, one protocol. Highlights 
of the studies in volunteers are summarized below. 

A volunteer model of El Tor cholera was established to study mechanisms of 
immunity to El Tor and to evaluate cholera vaccines. The clinical severity 
of El Tor cholera, Inaba and Ogawa serotypes, was distinctly less than that 
previously seen with classical cholera strains of the same serotypes. Further 
studies also suggest that although V. cholerae in endemic areas may be water- 
borne, the contaminated water must be ingested with food in order for clinical 
disease to occur in the normochlorhydric host. A naturally occurring, non- 



toxigenic El Tor strain isolated from sewage in Brazil was shown to have most 
characteristics demanded of an oral, attenuated vaccine strain; it will be 
tried as a vaccine in the near future. Preliminary trials with an oral, killed 
Inaba cholera vaccine suggested that the vaccine was efficacious; further 
studies are planned. A highly purified E. col i type 1 somatic pilus vaccine 
prepared by Dr. C.C. Brinton, Jr., University of Pittsburgh, was shown in pre- 
liminary studies to have low local reactogenicity, absent systemic toxicity, 
excellent immunogenicity, and efficacy (P = .04) in protecting immunized 
persons against challenge with the virulent homologous E_. coli strain. The 
CVD found the ELISA for measurement of IgG cholera antitoxin in human sera 
to be a simple, sensitive, and reproducible test to carry out sero-epidemio- 
logic studies. In collaboration with the Center for Disease Control they 
found that long-lived cholera antitoxin is stimulated by natural infection 
with V. cholerae or enterotoxigenic E. col i . In collaboration with the NIAID 
Laboratory of Infectious Diseases, studies were carried out with small parti- 
cle (27 nm) agents of gastroenteritis to measure serum and intestinal antibody 
and resistance to rechallenge. Stools containing the 27 nm Norwalk agent and 
Hawaii agent were collected as sources of virus for biophysical studies; 
results are pending. One of twenty-one volunteers inoculated intranasally 
with 1 07.0 TCID 50 of A/Alaska/6/77-ts-l-A 2 (H 3 N 2 ) attenuated influenza vac- 
cine strain developed systemic illness. No reactions occurred in 18 volunteers 
inoculated with A/Alaska/6/77 CR29 (H 3 N 2 ) cold-adapted attenuated vaccine 
strain. Two of 12 volunteers developed systemic illness after A/Hong Kong/ 
123/77 ts-A 2 (H-| N-j ) attenuated influenza vaccine, while one of 12 volunteers 
inoculated with the cold-adapted strain developed coryza but no systemic ill- 
ness . 

Collaborative Clinical Trials With Antibiotic Therapy 

The major aim of this program is to sponsor collaborative clinical trials 
designed to improve medical care of patients with bacterial and mycotic 
infections. Approved antibiotics, or promising new drugs that lack the eco- 
nomic potential to justify testing by the pharmaceutical industry, are evalu- 
ated in patients with selected infections. 

During FY ' 79 a contract was initiated with the University of Alabama to study 
ways to improve therapy of cryptococcal meningitis. The first part of this 
three part protocol includes a controlled clinical trial by 14 collaborating 
institutions to determine the shortest treatment regimen utilizing Amphoter- 
icin B (AMB) and 5-fl uorocytosine (5-FC) in combination. Four and six week 
treatment regimens are being compared. The Collaborative Cryptococcal Study 
Group during the first year has made limited progress in patient enrollment, 
but has nevertheless achieved balanced randomization into the two treatment 
groups. Progress was slower than projected because of the unexpectedly low 
occurrence of the disease this year. It is anticipated this can be offset in 
future by more aggressive publicity and a recruitment campaign among the 
medical community. The original plan for part 2 of the protocol was designed 
to evaluate the toxicology of Amphotericin B methyl ester (AME) in patients 
with selected disseminated mycotic infections. Preliminary clinical and lab- 
oratory data from another open trial suggested that the long-term administra- 
tion of AME may be associated with the induction of neurological abberrations 
and various gradations of a diffuse leucoencephalopathy . Pending a thorough 



review and determination by the FDA, an indefinite moratorium has been placed 
on the clinical administration of this antimycotic agent. Before the mora- 
torium, the University of Alabama subcontracted with the Kresge Hearing Insti- 
tute, University of Oregon to conduct ototoxicity studies with AME in labor- 
atory animals. This study may help predict the ototoxicity and neurotoxicity 
of AME and other methylated polyene-! ike antifungal agents now under develop- 
ment. 

Following a contractor's meeting of the Mycotic Disease Collaborative Group 
in June, 1979, we decided to replace AME and conduct phase II studies with 
ketoconazole (R41,400), a new, oral imidazole derivative with broad-spectrum 
antimycotic activity and low toxicity developed by Janssen Pharmaceutical of 
Belgium. The Collaborative Group will evaluate oral ketoconazole for safety 
and effectiveness in the treatment of the following group of systemic mycotic 
diseases: chronic cavitary histoplasmosis, disseminated or localized blasto- 
mycosis, disseminated coccidioidomycosis without meningitis, disseminated 
sporotrichosis, and non-meningeal cryptococcosis. Efficacy studies of keto- 
conazole in animal models of cryptococcal meningitis will also be done. 

In response to an RFP, issued during FY '79, a new contract was awarded to the 
University of Illinois to perform a placebo-controlled, double-blind evaluation 
of the efficacy of penicillin in the prevention of early onset Group B Strept- 
ococcal ( GBS) sepsis in the newborn. In addition, the study is designed to 
determine if penicillin prophylaxis influences the rate of late onset GBS 
disease, increases or decreases the likelihood of later penicillin hypersen- 
sitivity, and causes the emergence of penicillin resistant GBS or other peni- 
cillin resistant organisms. It is anticipated that this study will require 
at least three years and 20,000 mother-infant pairs before conclusive data 
will be available to demonstrate efficacy of the prophylactic regimen. 

Based on recommendations from the NIAID Symposium on the Impact of Infections 
on Medical Care in the U.S., held May 30-31, 1978, Staff prepared and issued 
an RFP during FY '79 entitled "Improved Therapy of Endocarditis Due to Viri- 
dans Streptococci." Response to this RFP was limited to one technical pro- 
posal. During the primary review process it was determined that a controlled 
clinical trial was not feasible, because a properly designed trial would take 
too long (seven-ten years) and the resulting costs would be excessive. With 
this information, a determination was made by Staff to cancel the RFP and not 
award a contract for the trial. Subsequently, plans were developed to hold 
a Consensus Development Conference on the optimum therapy of penicillin- 
sensitive, non-enterococcal endocarditis. The conference will be co-sponsored 
by the NIAID, NHLBI, the NIH Office of the Medical Applications of Research, 
and the American Heart Association. 

Rapid Viral Diagnosis 

The purpose of this program is to place greater emphasis on the rapid diagnosis 
of viral infections. The ultimate goal is to provide the physician with reli- 
able tests so that a specific viral diagnosis can be made in time to help in 
the management of the patient. To do this, the methods must be practical, 
precise, reproducible and inexpensive, and simple enough to be conducted safely 
in the physicians' offices or in small hospital laboratories. Three contracts 



8-3 



were awarded for the development of such tests. A contractor at the Univer- 
sity of California, San Diego, will further develop an immunological technique 
that incorporates two important features. The first is the immobilization of 
the virus on filter paper discs and washing using an immunofiltration manifold. 
This technique eliminates the need for primary capture antibody, and effi- 
ciently immobilizes infected cells and viral antigens. The second important 
feature is the use of purified, radiolabeled or enzyme labelled Staphylococcal 
protein A, which binds to the Fc portion of certain mammalian immunoglobulins 
thereby eliminating need for anti species detection sera. The investigators 
will adapt this methodology to detect influenza viruses, respiratory syncytial 
virus, parainfluenza viruses, and cytomegalovirus in less than one hour. A 
contractor at the Harvard School of Public Health will use sensitive, solid- 
phase immunoassays utilizing viral antibodies covalently coupled to nylon balls 
or nylon powder in an enzyme-linked immunoassay. This increases the amount of 
antibody that can be immobilized, and lessens the problem of antibody desorp- 
tion. The test is now sensitive enough to permit rapid identification of 
influenza virus in clinical specimens, and will be applied next to other respir- 
atory viruses. A contractor at Johns Hopkins Hospital will develop two tests 
for rapid viral diagnosis. The first is an enzyme-multiplied radioimmunoassay 
(EMRIA) which is the standard enzyme immunoassay but uses a tritium labelled 
substrate. This test has been shown to detect small quantities of non-replica- 
ting cytomegalovirus antigen in clinical specimens. The enzyme-linked 
fluorescent assay (ELFA) using a fluorescent substrate is somewhat less sensi- 
tive than EMRIA, but it is more rapid and simpler to use. These two tests will 
be adapted to detect other respiratory viruses. Coordination of activities 
within the NIH and with the Pan American and European groups on Rapid Viral 
Diagnosis continues to prevent duplication of effort. 

Nutrition, Infection and Immunity 

The goal of this program is to promote research on the interaction of malnutri- 
tion, infection and immunity in American hospitals and in overseas populations. 
Since the malnourished patient is at high risk for infection, this goal is con- 
sistent with the overall mission of the CSB which is to encourage research 
leading to improved patient care. 

In FY '79 the NIAID program in Nutrition, Infection and Immunity consisted of 
12 grants which were monitored by at least four project officers in the IAID 
and MID programs. The research focused on four different areas; the modulating 
effect of specific nutrients on immune function, mechanisms of food allergies 
and immune response to ingested antigens, the interaction of nutrition and 
infection in the tropical environment and in American hospitals, and the modu- 
lating effect of specific nutrients on microbial virulence. 

During FY '79, the Director of NIH appointed Dr. Edelman vice-chairman of the 
NIH Nutrition Coordinating Committee (NCC). Through this committee and in an 
effort to stimulate research in nutrition, Dr. Edelman helped design and initi- 
ate several new nutrition research programs. One of these new initiatives was 
the establishment of clinical nutrition research units (CNRU) sponsored by 
NIAMDD, NIA, and NCI. Infection and immunity research appeared in several 
approved proposals that were submitted in response to the RFA on CNRU. These 
units should help stimulate research in clinical nutrition, and will serve as 

8-4 



models of excellence for many American academic centers. Another initiative 
involved the establishment of institutional and individual fellowship training 
grants in nutrition. Seven Institutes have agreed to fund these National 
Research Service Awards. NIAID has agreed to co-sponsor Research Career Devel- 
opment Awards in clinical nutrition when the NCC develops a joint NIH 
announcement for these in the near future. Dr. Edelman served on ad hoc study 
sections to critique applications submitted to the Fogarty International Center 
for the support of national and international nutrition meetings. 

Dr. Edelman served as chairman of an international workshop sponsored by the 
NCC entitled "Nutritional Support of the Patient: Research Directions for the 
1980' s." The participants in this two day workshop held in September, 1979, 
developed a set of research priorities that will be used for program planning 
by eight Institutes of the NIH. The workshop focused on the feeding of the 
sick patient by means of enteral and parenteral nutrition support. One of the 
six workshop panels was relevant to NIAID programs, and delt with trauma and 
infection. The workshop proceedings will be published by the American Journal 
of Clinical Nutrition . 

Dr. Edelman delivered three invited lectures on "Nutrition, Infection and 
Immunity" at the Walter Reed General Hospital and Walter Reed Army Institute 
of Research. Staff of the CSB continued their collaboration with the Diet, 
Nutrition and Cancer program of the National Cancer Institute by helping assess 
the effect of hyperalimentation and improved nutrition on infections and the 
immune response in selected cancer patients. The final results of this study 
will be known early in FY 1980. 

Other Activities 



Mrs. Mattheis collects and reviews the necessary documentation required to file 
and maintain Investigational New Drug Applications (INDAs) with the Food and 
Drug Administration (FDA) for the Microbiology and Infectious Diseases Program. 
Currently 18 active INDAs are filed with the Bureau of Biologies, FDA, and four 
with the Bureau of Drugs, FDA. During the past year new INDAs were filed for 
the study of "Meningococcal Group B Type 2 Outer Membrane Protein Vaccine", 
"Live, Attenuated Respiratory Syncytial Virus Vaccine", "Amphotericin B Methyl 
Ester Aspartate", and "Topical 9-(2-hydroxyethoxymethyl ) guanine." Staff sup- 
port is provided for the NIAID-Clinical Research Subpanel (CRS) which reviews 
both intramural and contract supported clinical studies. All contract suppor- 
ted clinical studies are reviewed by the CRS initially and every three years 
thereafter, and by the NIAID-Clinical Director, in the intervening years. 

The CSB and the Bureau of Biologies, FDA, co-sponsored an International Sympo- 
sium on Potentiation of Immune Response to Vaccines, held at the NIH on 
February 20 - 21, 1979. The symposium participants discussed the influence of 
molecular structure on the adjuvant! city of muramyl dipeptide and other synthe- 
tic immunological adjuvants, including liposomes and polynucleotides. The 
mechanisms of immunoregulation by these synthetic substances, and their use as 
immunological probes and as potential vaccine potentiators in man was reviewed. 
The possibility was discussed of certain adverse side effects attending their 
use in man. Nevertheless, synthetic adjuvants, including 800 variants of the 
muramyl dipeptide molecule alone, are emerging as exciting new substances with 



8-5 



many basic and applied applications. 

Dr. Edelman served on the following committees of other Federal agencies: 1) 
NIH liaison member on the Infection Control Committee of the Veteran's Admini- 
stration, 2) the Human Ethics Committees (Institutional Review Boards) of the 
Walter Reed Army Institute of Research and 3) the Frederick Cancer Research 
Center. As ex-officio voting member of the National Commission on Digestive 
Diseases, Dr. Edelman participated in the natural demise of the Commission 
when it officially submitted its Report to Congress on January 31, 1979. 

Dr. Horton served as liaison member of the NIH Coordinating Committee on Cystic 
Fibrosis and participated at the National Scientific Working Conference on 
Cystic Fibrosis, March 4-7, 1979, South Padre Island, Texas. This working 
conference, co-sponsored by the Cystic Fibrosis Foundation, NIAID, NIAMDD,and 
NICHD was highly productive, and provided for an excellent exchange of scien- 
tific information among multiple disciplines. It identified future research 
opportunities with guidelines for implementing these activities. A report on 
this conference will be prepared and distributed by the Cystic Fibrosis 
Foundation. 



8-6 



DEVELOPMENT AND APPLICATIONS BRANCH 

It is the primary objective of the Development and Applications Branch to 
translate new information derived from basic research into methodologies 
appropriate for the control or prevention of designated infectious diseases 
in humans. Fundamental and applied research activities supported by the 
Branch include: identification of important infectious disease problems with 
potential for control or prevention through immunization or utilization of 
antivirals, development of appropriate vaccines, antivirals, and other control 
measures, design and support of appropriate clinical trials for evaluation of 
control measures, and basic research in related areas. 

Approximate Level of Support 

Activity Number Amount 

Vaccine Evaluation Centers 

Research Contracts 

Influenza 

Research Contracts 

Interagency Agreements 

Research Grants 

Fellowships 

Young Investigator Awards 

Respiratory Diseases 

Research Contracts 
Interinstitute Agreements 
Research Grants 

Hepatitis 

Research Contracts 

Interagency Agreements 

Research Grants 

Career Awards 

Young Investigator Awards 

Antiviral Substances 

Research Contracts 
Research Grants 
Career Awards 
Fellowships 

Training Grants 



5 


$ 1,218,351 


11 
2 

24 
2 
2 


2,103,956 
51,138 

2,192,305 
25,237 
83,812 


1 
1 
4 




47,872 

264,492 


7 
2 
8 
2 
1 


1,160,711 

157,497 

877,193 

86,665 

36,195 


13 

29 

2 

2 

1 


2,659,248 

2,337,501 

71 ,432 

20,877 

85,688 



9-1 



Approximate Level of Support (cont. 

Activity 

Bacterial Vaccines 

Research Contracts 
Interagency Agreements 
Research Grants 
Career Awards 
Fellowships 

Enteric Diseases 

Research Contracts 
Interagency Agreements 
Research Grants 
Career Awards 
Fellowships 
Program Projects 
Training 

Viral Vaccines 



Research Grants 
Conference Support 



Number 



Amount 



Totals 



13 


$ 816,197 


1 


10,821 


18 


1,076,870 


2 


50,551 


2 


32,400 


7 


762,565 


1 


33,750 


35 


2,105,048 


1 


16,200 


1 


14,200 


1 


355,100 


1 


36,504 


7 


548,327 


1 


29,750 


209 


$19,368,453 



Program Summary 

The initial portion of this fiscal year (through March) was heavily involved 
in completing studies directed at the control of influenza A/USSR/77 virus, 
the most recent strain shift. Clinical studies to determine vaccine safety, 
reactogenicity, and antigenicity, as well as its sensitivity to amantadine, 
were performed. Two other areas of concentrated effort in the influenza 
program were further development and study of attenuated vaccine candidates 
and determination of the role of amantadine in influenza disease control. 
A meeting was held in February at the Influenza Research Center to compare 
results with Soviet scientists on respective experiences with amantadine 
and rimantadine. Based on results from investigations supported by this pro- 
gram, there is a revived interest in amantadine. A consensus development 
exercise on the role of amantadine in the prevention and treatment of influ- 
enza is currently being planned. 

Following the advice of a Workshop on the Development of Antiviral s, the 
Antiviral Substances Program (ASP) developed a request for proposals for 
the development of targeted antivirals, and a contract has been awarded. 
Having determined that adenine arabinoside monophosphate (ara-AMP) was 
not effective as a topical drug against herpes labial is, clinical studies 
were initiated using acyclovir which appears to hold greater promise 



9-2 



based on animal studies. Protocols have also been developed to evaluate 
systemic ara-AMP against herpes infections, and phase 1 studies are proceeding 
with acyclovir. If these latter studies prove satisfactory, this drug will 
be considered for clinical efficacy studies. Final determination on the 
establishment of interferon standards and their distribution has been made 
with WHO. This is particularly important based on the renewed efforts in 
clinical application of interferon against infectious diseases and cancer. 
Through the efforts of this program, the first complete summary of all 
clinical studies with interferon was published ( Journal of Infectious Diseases , 
139:109-123, 1979). One of the most exciting findings of the ASP during the 
past year was the apparent efficacy of adenine arabinoside in neonatal 
herpes. Although the data are still being analyzed, the results appear 
similar to the efficacy of this drug against herpes encephalitis. 

A new initiative for the viral vaccine program was the release of a request 
for proposals to evaluate candidate varicella vaccines. Herpes varicellas 
produces a highly contagious but mild disease (chicken pox) in healthy 
children; however, it causes severe disease with increased morbidity and 
even death in immunosuppressed children. At a Workshop on Experimental 
Herpesvirus Vaccines, it became clear that varicella vaccines with clinical 
potential were available and definitive clinical studies should be performed. 

Two other new areas in the Branch's vaccine program are based in the Bacterial 
Vaccine Program. The current Rocky Mountain Spotted Fever vaccine was with- 
drawn from the market based on lack of efficacy, this created a need for 
accelerated work with a new vaccine candidate developed by USAMRIID. Studies 
are underway on epidemiology and animal studies in anticipation of future 
clinical studies. An International Symposium on Pertussis, principally co- 
sponsored by this program and the BoB, raised many questions concerning the 
pertussis vaccine and considerations on possible improvement of this vaccine 
are underway. Another major effort of this program was the release of a 
request for applications for studies on infant (less than two years of age) 
infections. This is an offshoot of the studies on meningitis vaccines. In 
these studies, it was shown that bacterial polysaccharide antigens were not 
as effective in this target population as desired. 

The Hepatitis Program is completing phase 1 and 2 studies prior to efficacy 
studies of the hepatitis B (HBV) vaccine. It has been determined that the 
HBV vaccine containing alum adjuvant is preferable to the aqueous preparation. 
The alum vaccine will be used in the proposed hemodialysis center study. 
Clinical studies to evaluate the possible effect of hepatitis B immune globu- 
lin to prevent transmission from hepatitis antigen positive mothers to newborns 
are underway. The woodchuek appears to be a useful animal model for hepatitis 
pathogenesis studies; attempts to develop this model are underway. 

Epidemiologic studies on diarrheal diseases initiated in FY77 are beginning 
to yield interesting results. Trends related to age, crowding, and etiological 
agents are emerging. Studies with cholera vaccine in endemic areas continue. 
Currently two vaccines (provided by the Wellcome Foundation), an alum precipi- , 
tated whole cholera cell and a purified toxoid complexed with aluminum hydrox- 
ide, are being tested individually and in combination. A major breakthrough 



9-3 



in the treatment of cholera has been the use of chlorpromazine in reducing 
fluid loss in these patients. Of considerable interest was the finding of 
cholera vibrios in U.S. estuaries. This is being followed with great care. 

In FY79 the DAB was involved in the organization and conduct of six major 
meetings: International Symposium on Pertussis, Workshop on Collaborative 
Pneumococcal Vaccine Studies, Workshop on Hepatitis B Vaccine, Experimental 
Herpesvirus Vaccine Workshop, Conference on Cholera, and Workshop on Influ- 
enza B Viruses. Summaries on each of these meetings, with the exception of 
the Pneumoccal Vaccine meeting, will be published. 

Research Highlights 

Influenza 

The Influenza Program has experienced another very active year. As with the 
previous year, the major development in influenza has been the reappearance 
of viruses which are antigenically very closely related to those which pro- 
duced epidemic disease during the 1947-1957 period. The prototype of this 
new era was the A/USSR/77 (HINT) virus which has been shown to be, during 
the last year, antigenically and biochemically quite similar to the A/FLW/50 
(HINT) strain which circulated in 1950. Recent data suggest that the differ- 
ence between A/USSR/77 (HINT) and A/FLW/50 (H1N1), a 1950 strain, may be only 
eight base pairs in the nucleic acid. Although it was widely anticipated 
that a change in the predominant type could be expected later in the 1970s, 
the reemergence of the HINT strains was totally unexpected. These new 
variants have been very restricted in their ability to produce disease and 
virtually all cases have been seen in individuals born after 1955 or age 
to 24 years. The new variants also appear to have supplanted the earlier 
H3N2 strains as the predominant virus. Only scattered isolates of H3N2 
viruses have been reported during the last winter, none from the United 
States. 

During the last year, the Influenza Program completed the evaluation of the 
A/USSR/78 inactivated vaccines. Over 2100 subjects participated, including 
approximately 1300 under the age of 24 years and 300 subjects considered at 
high risk to influenza. Final reports of these studies are being completed 
and will be submitted for publication during this fiscal year. Field trials 
of three other vaccines under development were also conducted. The two 
attenuated vaccines were subjected to extensive tests. These include the 
preliminary clinical evaluation of these vaccines in approximately 200 
subjects under closed conditions, 200 subjects to determine transmissibility, 
and over 2100 subjects during an open-field trial at Texas A & M University. 
However, the test population did not experience a large enough natural 
challenge, and it will not be possible to determine vaccine efficacy. The 
vaccines did not produce any significant reactivity. The subjects will be 
followed to determine their antibody status and their clinical experience 
after natural challenge with related H1N1 viruses. In addition, some of 
these vaccinees will be challenged by related and homologous viruses to 
obtain information on the duration of immunity induced by these vaccines. 
The third vaccine, a purified subunit vaccine, was also tested and a formula- 
tion was developed which induced excellent antibody response in subjects 
with HAI titers less than 1:8. 

9-4 



The utility of amantadine as a prophylactic and therapeutic agent in influ- 
enza was clearly demonstrated last year. Studies will continue to explore 
the use of amantadine in high-risk patients and in the therapy for pneumonia. 
Rimantadine will also be studied. In collaboration with the Antiviral Sub- 
stances Program, pilot studies on the possible synergistic effect of riman- 
tadine and ribavirin have been started. Soviet investigators had earlier 
reported a synergistic effect with these drugs. Studies using a plaque- 
reduction assay have shown that these drugs are remarkably effective when 
used in combination. 

The results of the studies presented above should contribute directly to the 
control of influenza. The basic scientists supported by the NIAID and 
working with influenza viruses have continued to make significant contri- 
butions to our understanding of myxo and paramyxoviruses. Recent data 
suggest the presence of a ninth viral protein. This same investigator has 
also shown that antibody to the F protein of Sendai virus inhibits syncytia 
formation. This may have important implications with regard to infections 
produced by related viruses, such as respiratory syncytial virus infection, 
and to the possible design of vaccines for these viruses. Other investigators 
have shown that certain genes in influenza are not randomly reassorted 
during recombination. In fact, two pairs appear to be "linked." Since these 
pairs involve the RNA polymerase genes, this observation may be quite important 
in attenuated vaccine development. This group has also found that resistance 
to amantadine is linked to the M protein. Persistent viral infections of 
influenza viruses have also been obtained independently by two investigators. 
The significance of these observations is not yet clear, but these in vitro 
systems should enable a variety of studies to examine the biochemical aspects 
of viral interference and viral persistence. 

The supplemental funds provided to the Influenza Program during the swine 
flu and Russian flu era have been expended, and there is a resulting reduc- 
tion in directed program activities. There are currently 12 contracts 
compared with 15 last year. This includes one new contract awarded this 
year to identify alternate cell substrates for diagnosis and cultivation of 
influenza virus. Among the projects which were terminated was the influenza 
surveillance network which had effectively served as a sentinel system since 
1976. In contrast, the number of investigator-initiated projects has dramat- 
ically increased with the addition of four grants to a total of 24, two 
young investigator awards and two fellowships. 

Respiratory Diseases 

The magnitude of infection and illness from parainfluenza viruses types 1, 
2, and 3, and respiratory syncytial virus (RSV) is difficult to document, 
but estimates from several epidemiologic studies indicate that these viruses 
cause severe morbidity in pediatric populations. Of all severe lower re- 
spiratory diseases in infants and young children, perhaps 40 percent could 
be prevented if effective vaccines against these agents were available and 
used. 



9-5 



Efforts to develop an attenuated respiratory syncytial virus vaccine have 
produced a temperature sensitive mutant that has been satisfactorily tested 
in animal models and human adults. Studies in children are underway. The 
testing of the live vaccine developed by Merck Sharp and Dohme has been 
completed in animal models. These studies were not promising, and results 
from an efficacy trial (R. Bel she, personal communication) indicate no 
Benefit. The NIAID does not plan any further tests with this product. 

Studies on the epidemiology and pathogenesis of RSV and parainfluenza infec- 
tions continue at Baylor University and the University of Rochester. Longi- 
tudinal family studies have shown a positive correlation between serious RSV 
disease and age at time of first infection. These studies have also shown 
that maternal antibodies are protective in the first months of life. Several 
factors such as crowding of living quarters, hygienic practices and number 
of persons having regular contact with the infants, may be related to the 
likelihood of developing disease. Month and year of birth are other obvious 
variables. 

Viral Hepatitis 

The goal of the Hepatitis Program is the control of hepatitis A (HAV), B 
(HBV), and non A-non B (NANB) infections. Precise data on morbidity, mor- 
tality, and economic burden to the country are difficult to obtain; however, 
as investigations continue on the etiology, epidemiology, and long term 
sequelae of viral hepatitis, it has become apparent that many cases have 
been misdiagnosed or underreported. In 1977, approximately 56,000 cases of 
viral hepatitis were reported to the CDC. Allowing for misdiagnosis and 
underreporting, it can be estimated that 560,000 cases of viral hepatitis 
occurred in the U.S. in 1977, with a distribution of approximately 50 per- 
cent HBV, 25 percent HAV, and 25 percent NANB. Estimates of death from HBV 
range from 1,500-3,000 (approximately 1 percent fatality rate) per year. 
Costs incurred from hospitalization, professional care, laboratory work, and 
days lost from employment are estimated to exceed $600 million annually. 

Type B hepatitis has an incubation period of 60-180 days, and may exhibit 
an acute and chronic phase. Type A hepatitis has a shorter incubation 
period of 20-40 days and does not exhibit a chronic infection. NANB hepa- 
titis, with an incubation period similar to HBV, is responsible for approxi- 
mately 90 percent of the post-transfusion hepatitis remaining in the U.S. 
Although the virus(es) responsible for this type of hepatitis has not been 
identified, the disease has been successfully transmitted to chimpanzees. 

Because of its more serious clinical manifestations, type B is a disease of 
major public health importance in the U.S. However, the global impact of 
the disease is more significant when 176,000,000 chronic carriers remain as 
a reservoir for infection. In addition to serving as reservoirs of virus, a 
close association of chronic HBV and primary hepatocellular carcinoma (PHC) 
has been demonstrated. The role of the HBV in PHC remains to be defined, 
but cause-and-effect will be difficult to ascertain. 



9-6 



During the past year, efforts have continued in evaluating experimental 
hepatitis B vaccines in humans. As noted last year, aqueous formalin- 
inactivated vaccines prepared by the NIAID were shown to be non-reactogenic 
and non-infectious in volunteers. To date, 16 volunteers have been immunized 
with the adw vaccine and, although most have developed humoral antibody, the 
long delay between boosters and appearance of antibodies indicates that 
these vaccines would not be practical for clinical use. 

Further testing of the ayw vaccine has not proceeded since one of the five 
volunteers participating in the initial safety tests developed NANB hepatitis. 
At the present time, evidence suggests that the vaccine was not infectious, 
but further definitive analyses must await the development of serologic 
markers for NANB virus. 

Independently, Merck Sharp and Dohme has been exploring the development of a 
type B hepatitis vaccine. Their procedure, similar to the NIAID procedure, 
employs isopycnic banding and rate zonal centrifugation of the 22 nm surface 
antigen with subsequent inactivation with formalin. They have evaluated two 
types of vaccines in chimpanzees and humans -- aqueous and adjuvanted (alum- 
inum hydroxide). Their data with aqueous vaccine are comparable to the NIAID 
data, safe, but not adequately immunogenic. However, the alum absorbed vac- 
cine did appear highly immunogenic with approximately 65 percent of the vol- 
unteers developing antibody three and one-half months following a two dose 
regimen and 90 to 95 percent presenting antibody following a booster at six 
months. Merck has offered their vaccine for testing for efficacy by the 
NIAID. The independent evaluation by the NIAID of a potential vaccine that 
is earmarked for licensure will lend additional credence to data generated 
in efficacy trials, and represents the testing of vaccine in a population 
that is at extremely "high risk" for B virus infection and disease, i.e., 
hemodialysis patients and staff. 

In preparation for an HBV efficacy trial, the New York Blood Center has 
been documenting the incidence of viral hepatitis in patients and staff 
within selected hemodialysis centers. The results of this study show that 
the current attack rates appear low compared with rates observed in the 
early 1970s, yet are still 10-20 times higher than in other groups of hos- 
pitalized patients and staff. Based on incidence of HBV infection, avail- 
ability of the test population, and ability to give informed consent, this 
population represents a feasible group for vaccine efficacy. The efficacy 
study will start approximately September, 1979, and will be a randomized, 
double-blind placebo-controlled trial. It is estimated that 1,000 patients 
and 900 staff will be randomized into the trial in a two year period. The 
vaccine to be employed will be a monovalent ayw , alum-absorbed preparation 
provided by Merck Sharp and Dohme. It is expected that efficacy data should 
become available in the Fall-Winter, 1982. 

Another approach to prevention and control of HBV infection and sequelae is 
to interrupt maternal -fetal transmission. Neonatally acquired infection is 
of major magnitude in parts of Africa and the Far East. For example, 85 
percent of newborns born of HBsAg+ HBeAg+ mothers in Taiwan become infected 
within the first six months of life. The NIAID and BoB/FDA are collaboratively 

9-7 



supporting a study to evaluate Hepatitis B Immune Globulin (HBIG) for interrup- 
tion of this transmission route. During the past year, 115 babies have been 
randomized into the double-blind, placebo-controlled trial. Seventy more babies 
will be entered into the study prior to January, 1980. Follow-up of the babies 
for one year will be required before the data can be interpreted. Thus far, 
there have been no adverse reactions to the HBIG, but since the code has 
not been broken, there are no results available. 

The virus of hepatitis B has not been cultivated in vitro . Attempts to 
develop continuous hepatocyte cultures of human and chimpanzee origin have 
been unsuccessful, and contractual support for this activity is terminating 
this year. Hopefully, investigator initiated approaches to this requirement 
will be forthcoming. Successful cultivation of hepatitis A virus was reported 
during the past year by investigators at Merck Sharp and Dohme. 

Exciting observations have been made on a virus isolated from woodchucks 
( Marmota monax ). This virus has many characteristics in common with human 
HBV, including morphology, DNA of similar size and structure, presence of 
endogenous DNA polymerase, and association with hepatitis and PHC. Infection 
of woodchucks with this virus may represent the best and only animal model 
for hepatitis and its relation to PHC, and studies are now underway to 
better characterize the animal-virus system. A continuing problem will be 
the availability of woodchucks, an animal that hibernates from late fall to 
early spring. It is clear that research will be severely hampered until 
young animals of known serologic status are readily available to investigators. 

Recent breakthroughs in DNA recombinant research have the potential for 
rapidly chanqinq approaches to molecular aspects of HBV. At least three 
laboratories have reported the successful cloning of the HBV genome in 
Escherichia col i , with one laboratory reporting expression of HBcAg. The 
ease of which this technology has been applied to HBV bodes well for develop- 
ing nucleic acid probes for studies of viral persistence and hepatoma and, 
perhaps, the elaboration of HBsAg or its component polypeptides as a source 
of future vaccine antigen. An NIAID grantee, Dr. William Robinson, Stanford 
University, is a pioneer in this area of investigation. 

The use of antiviral drugs in eradication of the HBV carrier state is also 
being assessed and is described in the Antiviral Substances Program section. 

Antiviral Substances 



Virus diseases are a leading cause of morbidity and mortality throughout the 
world, and the use of antivirals is an important means of treating and 
preventing these illnesses. Although development of host resistance remains 
the first line of defense against viral diseases, there are many viral 
diseases for which vaccines are not available or cannot feasibly be given to 
all people at risk. Thus, chemotherapeutic agents with low toxicity and 
high efficacy are needed. 



9-8 



Herpes infections are a major cause of disease with the symptoms varying 
from mild labial is infection to fatal encephalitis. Carefully controlled 
clinical trials were supported to determine the efficacy of adenine arabinoside 
(ara-A, Vidarabine) for treatment of herpes encephalitis, and the use of 
this drug reduced the mortality from 70 percent to 28 percent. The results 
from these trials led to the licensure of adenine arabinoside for herpes 
encephalitis in October, 1978. The use of adenine arabinoside for other 
types of herpes infections is still under study, including studies with 
herpes zoster and neonatal herpes. Preliminary results indicate that adenine 
arabinoside will also have some beneficial effect in these diseases. The 
code for the neonatal herpes study was recently broken, and preliminary 
analysis of the data indicates a reduction in mortality of approximately 40 
percent. 

Systemic herpes infections can cause devastating illnesses and a wide range 
of antivirals is needed to treat these diseases. Adenine arabinoside is a 
difficult drug to administer because of its low solubility in water. Better 
antivirals should be developed. Animal model studies supported by the 
Antiviral Substances Program have demonstrated that adenine arabinoside 
monophosphate (ara-AMP) and 9-(2-hydroxyethoxymethyl )guanine (acyclovir, 
ACV) also hold promise for treatment of systemic herpes disease. These 
drugs are being considered as candidates for evaluation in future clinical 
studies against serious herpes infections in protocols similar to those 
utilizing ara-A. 

In addition to systemic herpes infections, topical herpes infections are a 
major cause of disease in the United States, with type two herpes being the 
second most frequent genital infection in the United States, and type one 
herpes (herpes labialis) causing recurrent infections in over 30 percent of 
the adult population. Work to define the natural history of herpes labialis 
has been supported by the Program. This work led to a subsequent clinical 
trial which demonstrated that ara-AMP was not effective as a topical treatment. 
During 1979, a double-blind clinical trial to evaluate the effectiveness of 
topical application of ACV for herpes labialis was started. These studies 
are also designed to evaluate the general use of ACV in humans, including 
analysis of any potential antiviral resistance to the drug and drug absorption 
through the skin. 

Interferon, a broad-spectrum antiviral, is a candidate antiviral for both 
RNA and DNA virus infections. Through grant sponsored research, we are 
learning more about the mechanism of action and the antigenic and biochemical 
nature of interferon. There are three types of interferon: fibroblast and 
leukocyte (type one interferons) and immune interferon (type two interferon). 
Much of our understanding on the nature of interferon has come about through 
the use of NIAID reference interferon preparations, which have allowed the 
comparison of results from laboratories throughout the world. During the 
past year, the World Health Organization has designated the NIAID reference 
reagents for human fibroblast, rabbit, and mouse interferons as international 
standards. The use of NIAID antisera to human leukocyte, human fibroblast, ; 
and mouse interferons has also enabled investigators to identify specific 
biochemical events brought about by this antiviral, and has played an important 

9-9 



part in developing better methodologies for its purification. A number of 
studies on purification have been initiated, and considerable headway has 
been made in the characterization of this elusive moiety. 

Human leukocyte interferon is being evaluated in clinical trials for the 
treatment and prevention of herpes, hepatitis B and respiratory viral infections 
Recent clinical trial data indicate that interferon can reduce the reactivation 
of herpes infection after cranial nerve surgery by 43 percent. The total 
incidence of virus shedding was reduced from 41 percent in placebo patients 
to 8 percent in interferon patients. These studies are continuing to deter- 
mine optimal dose schedules. Interferon has also been shown to be efficacious 
in the treatment of herpes zoster infections in the immunosuppressed patient. 
When administered in combination with antihistamines, it appears to hold 
promise for treatment of respiratory viral infections. 

Currently, there are over 175 million carriers of hepatitis B for which 
there are no satisfactory therapies. The Antiviral Substances Program is 
conducting studies to determine optimal regimens for modifying or eliminating 
the carrier state. Recent results suggest that the best treatment for 
hepatitis B will be combination therapy with interferon and another antiviral, 
such as adenine arabinoside. 

Since 1969, the Antiviral Substances Program has been the primary source of 
funding and support for interferon studies in the United States, and these 
studies have led to an understanding of the potential of interferon in the 
clinic. Based on the results from this Program, the National Cancer Institute 
and American Cancer Society have initiated carefully controlled clinical 
trials with interferon in a variety of cancers. Cooperation among interferon 
researchers continues to be an important objective of the Program. 

A critical part of the effort to develop clinically effective antivirals has 
been the establishment of a series of animal-viral disease models which 
closely mimic some common viral diseases of man. During the past year, the 
effectiveness of acyclovir against herpes infections, including genital and 
systemic infections, has been demonstrated in these animal models. A new 
antiviral, phosphonoformate, is being studied to determine efficacy and 
relative toxicity. These animal studies are important in determining optimal 
dose schedules and routes of administration, information necessary for 
planning successful clinical trials in man. 

A new initiative to develop targeted antivirals was started by the Program 
during 1979. Based on our knowledge of the nucleic acid sequences in virus 
genes, antivirals will be developed which will specifically interact with 
the nucleic acid without interfering with host cell metabolism. Targeted 
antiviral development is a field of the future and will be supported by both 
the grant and contract mechanisms. 

Bacterial Vaccines 

Pneumococcal Diseases : Studies sponsored by the NIAID during the past decade, ; 
along with studies by manufacturers, have led to the development and licensure 
of a 14-valent pneumococcal polysaccharide vaccine (Merck Sharp & Dohme). It 

9-10 



is expected that a second manufacturer (Lederle) will have a licensed product 
by the Fall of 1979. 

It is currently recommended that pneumococcal vaccine be used in those indi- 
viduals over two years of age considered at high risk for pneumococcal 
infections. There are a number of disease conditions which predispose to 
pneumococcal infections; however, there are little data on the immunogenicity 
and efficacy of pneumococcal vaccine relating specifically to these populations. 
The Bacterial Vaccines Program has developed a collaborative effort through 
which pneumococcal vaccines are being studied in patients with the following 
conditions: sickle cell disease, splenectomy, bone marrow transplantation; 
Hodgkin's disease, renal transplantation, chronic dialysis, lymphoma, myeloma, 
diabetes, lupus, lymphoblastic leukemia, alcoholic cirrhosis and chronic 
obstructive pulmonary disease. There are approximately 25 studies underway 
in various stages of completion. Although no direct funding is being given 
to the investigators by the NIAID, these studies are being coordinated by 
staff and all antibody assays are being done through the contract-supported 
serology laboratory at SUNY, Downstate Medical Center. Although these 
studies have not been completed, some important information is already 
evident: (1) Sickle cell and splenectomy patients appear to respond well to 
the vaccine; (2) Hodgkin's patients who have undergone splenectomy and 
chemotherapy respond poorly to vaccine; however, Hodgkin's patients given vaccine 
prior to therapy appear to respond normally; (3) antibody response is diminished 
in renal dialysis patients; and (4) simultaneous administration of pneumococcal 
and influenza vaccines does not have a negative effect on response to the 
individual antigens. When the collaborative studies are completed, they 
should allow for specific recommendations on the use of pneumococcal 
vaccine in various patient populations. 

Pneumococci are estimated to be responsible for 40 to 50 percent of acute 
otitis media in children. This is an important childhood disease which often 
causes hearing disorders and subsequent learning disabilities. Almost every 
child will have at least one bout of otitis media; 20 to 30 percent will have 
six or more occurrences. 

A study of the value of pneumococcal polysaccharide vaccines in preventing 
otitis media was initiated in 1975. Two double-blind efficacy trials of an 
octavalent vaccine for the prevention of disease in children who have had 
previous middle ear infections are now underway. The code has not been broken 
on these studies; therefore, no data on efficacy are available at the present 
time. Data from one of the trials (Huntsville Hospital) will be analyzed in 
the S ummer of 1979 and the other trial (Boston City Hospital) will be completed 
in the Spring of 1980. A third project is designed to establish the optimum 
dose and booster regimen for maximum antibody response of infants and young 
children. Certain polysaccharides (i.e., type 3) are good immunogens in infants 
when given in one dose. Some cause measurable responses when given in two doses 
(i.e., types 7 and 18), while most of the others are poorly immunogenic in infants 
with any dose regimen. 

To better understand the pathogenesis and immunology of pneumococcal otitis 
media and the efficacy of pneumococcal polysaccharide vaccine in preventing • 



9-11 



experimentally- induced middle ear disease, an animal model system has been 
developed. Utilizing this chinchilla model, investigators have been able to 
correlate vaccination with protection from disease, even in cases in which 
there has been no measurable serum antibody response to the polysaccharides. 
These data suggest that pneumococcal vaccines may prevent some otitis media in 
young infants in which serum antibody response is poor. 

Meningitis : It is estimated that approximately 20,000 cases of bacterial 
meningitis occur in the U.S. each year, most in young children and infants. A 
pure polysaccharide vaccine for H_. influenzae type b, developed and tested 
under our program, has been shown ineffective in children under 18 months of 
age. Because most of the H_. influenzae meningitis occurs in this younger age 
group, program efforts are now directed toward developing vaccines effective in 
infants. For the past few years, considerable laboratory and clinical effort 
has been concentrated on a new H. influenzae complex vaccine. This preparation 
is a complex of polyribose phosphate (PRP) and cell wall material, primarily 
polypeptides and 1 ipopolysaccharide. Designated PRPc, this material showed 
promise in animal studies. Adult human studies have shown the new vaccine to 
be potentially a better antigen; however, it is considerably more reactive than 
pure PRP and efforts to significantly reduce the toxicity have met with limited 
success. Plans are being made to continue studies of this in children; however, 
alternate vaccine development approaches are also being followed. The alternate 
approaches include: (1) extraction and characterization of outer membrane pro- 
teins from H_. influenzae to determine their role in disease and their potential 
as immunogens; (2) preparation of a complex of PRP with a pure protein such as 
the diptheria toxoid; (3) preparations of complexes of highly immunogenic 
bacterial polysaccharides with determinant groups of PRP; and (4) preparation 
of very high molecular weight molecules of PRP. 

In addition to efforts to improve the infant's response to bacterial meningitis 
vaccines, protection of the neonate and young infant by hyperimmunization of 
the mother is being attempted. This is being done in humans by giving H_. 
influenzae PRP to women who plan pregnancies and to a control group of women 
who do not plan to become pregnant. Serum samples from mothers and children 
will be studied to determine if the level of infant antibody to H. influenzae 
can be increased during the crucial first few months of life. Hyperimmunization 
of the mother is also being studied in primates; pregnant females will be 
immunized to determine the possible harmful effects of vaccine administration 
during pregnancy as well as to follow antibody levels in the mother and offspring. 

Group A and C meningococcal polysaccharide vaccines have been licensed for use 
in adults and older children. The group A vaccine has been shown to be effective 
in children down to three months of age in an efficacy trial in Finland sponsored 
by the NIAID. Group C meningococcal polysaccharide vaccine has been shown to 
be poorly immunogenic in very young children. A capsular polysaccharide extracted 
from a variant group C strain has been prepared at Rockefeller University. 
This new material has a slightly different chemical structure from the licensed 
group C vaccine, but it is antigenically cross-reactive. Through the NIAID i 
program, this variant C vaccine has been shown superior to the licensed material 



9-12 



in adults and in children over two years of age. Studies in infants will begin 
in the immediate future. 

During the course of the Finnish trial of the efficacy of group A meningococcal 
vaccine in children (mentioned above), it was discovered that persistence of 
antibody and the importance of the booster regimen on persistence of antibody 
appear to be age related. The Finnish study is being continued to further study 
persistence of antibody as a function of the type of dose and booster regimen 
used. 

Group B meningococcal polysaccharide is non-antigenic in humans. A protein 
antigen vaccine candidate, from the outer membrane layer of the organism, has 
been developed by the BoB. Phase I safety and antigenicity tests in adults 
showed reactions to the vaccines to be acceptable, but antibody responses were 
somewhat disappointing. Plans are underway, however, for studies in teenage 
children and children over age 2. A second cell envelope protein antigen pre- 
pared at the Naval Medical Research Institute will be tested in adults in the 
near future. 

Rocky Mountain Spotted Fever : The presently licensed vaccine for RMSF is not 
considered effective and is no longer commercially available. A new inactivated, 
whole-cell vaccine has been developed at the U.S. Army Research Institute for 
Infectious Diseases (USAMRIID). Because of our mutual interest, the NIAID has 
assisted with support of phase I clinical trials of the vaccine in adults at 
the USAMRIID facilities at Fort Detrick. The vaccine appears to be superior to 
the commercial material in these studies and additional clinical studies may be 
sponsored by NIAID. Three new contracts have been awarded this fiscal year for 
additional animal model studies of RMSF and the USAMRIID vaccine and for studies 
of the epidemiology of RMSF. 

Enteric Diseases 

The Enteric Disease Program of the NIAID is concerned with reducing the morbidity 
and mortality of infectious diarrheas in this country and around the world. 
This includes viruses, bacteria and their products, and the unicellular parasites. 
The Program is seeking to define the scope of the diarrheal problem in infants, 
children, and adults, to identify the etiologic agents and elucidate their 
pathogenesis and to gain knowledge for developing practical means of control. 

The Institute funded three enteric disease study centers in FY 1977 (D. C. 
Children's Research Hospital, The University of Michigan, and the University of 
Texas in Houston) for epidemiological studies leading to finding methods of 
controlling diarrheal diseases. Several trends are beginning to emerge: (1) 
Younger children have more diarrhea episodes than older children, i.e., 
57.4 visits to the doctor's office/100 persons/year in the 0-4 year olds; 13.9 
visits to the doctor's office/100 persons/year in the over 20 year olds in a 
small town and rural population and 1.7 episodes of diarrhea in the first 12 
months of life in an urban area and approximately 0.6 episodes in the second 12 
months. (2) Overcrowding of children in day care centers produces a higher , 
attack rate of diarrhea, even in the more affluent populations. There were 
several instances of secondary cases among family members. Three of five 
outbreaks were associated with Shigella infections and two were associated 

9-13 



with both rotavirus and Giardia infections. (3) Rotavirus is a common cause of 
infant diarrheas with a peak attack rate in the winter as high as 71 percent 
during a one month period. (4) Some families (16 percent) with infants did not 
report any episodes of diarrhea in the first 12 months of life; about the same 
number (15 percent) reported two or more episodes of diarrhea. (5) Type 2 
rotavirus was detected about three times more often than type 1. (6) There is 
•a suggestion that uncultivatable adenoviruses play a role in acute diarrheal 
diseases. 

The seventh world pandemic of cholera continues into its 18th year. There were 
40 countries reporting cholera in 1978 as compared to 34 in 1977. This includes 
the United States where the first multicase outbreak of cholera since 1911 
occurred in Louisiana in August. Crabs were implicated as the source, with 
their contamination coming from the environment. The Institute program on 
cholera is part of the U.S. -Japan Cooperative Medical Science Program. Cholera 
studies remain important per se, but they also serve as an excellent model for 
studies on enterotoxin mediated diarrheas, including studies of the local 
immune response. 

The intensive activity in cholera research has produced the following: (1) Two 
laboratory-modified live vaccines for testing in volunteers; (2) Two isolates 
from the environment identified as candidates for a modified live vaccine; 
(3) Evidence that cholera toxin produces long lasting immunity in the mucosal 
IgA system of dogs; (4) A flagellar vaccine which produces better protection in 
rabbits than licensed whole cell vaccine. This protection is enhanced 1,000- 
fold when combined with cholera toxoid; (5) Chlorpromazine is capable of drasti- 
cally reducing fluid loss in patients suffering from cholera; and (6) Non 
Group 1 cholera vibrios are present in U.S. estuaries as well as in the Ganges 
River delta, the perennial seat of cholera infection. These vibrios produce no 
detectable toxin; however, some activate adenylate cyclase in a manner similar 
to cholera toxin. 

The treatment of cholera with fluids has been refined many times in the past 
decade. This past year provided a major new breakthrough in treatment when it 
was determined that chlorpromazine was capable of greatly reducing the fluid 
loss. This is the first example of a safe, practical, pharmaceutical agent 
with this capability. The implications are far-reaching. 

The Wellcome Foundation Ltd., Beckenham, Kent, England, has provided an alum 
precipitated whole cell cholera vaccine, a cholera toxoid complexed with adju- 
vant, and the two products combined for field testing in Dacca, Bangladesh. The 
trial is being designed to determine the protective role of an alum precipitated 
whole cell vaccine and the additive or synergistic effect of the toxoid combined 
with the vaccine. 

Studies on the interaction of cholera vibrios with the intestinal mucosa of 
mice indicate chemotaxis and protease (mucinase) contribute to virulence. This 
suggests there are other antigens which may be useful in new cholera vaccines, 
(e.g., mucinase) an d also characteristics which, when absent, may render live 
mutants avirulent and thus of potential value as living oral vaccines. Vibrio 
ecology studies are of increasing importance with the report of cholera in 



9-14 



Louisiana traced to seafood. V_. cholerae isolates in marine estuaries are 
usually the avirulent non Group 1 vibrios. Selected isolates will be tested 
for virulence in volunteers. Laboratory tests to date indicate no toxin pro- 
duction; however, there is an indication of ability to activate adenylate cy- 
clase as cholera toxin does. The history of cholera in Louisiana indicates 
that cases may recur. Similar situations may exist in other areas in this 
hemisphere and pose an obvious threat to places with poor sanitation. A better 
understanding of the role of vibrios in the environment is needed to suggest 
methods of control . 

Considerable progress has been made in studies of disease caused by toxigenic 
•E_. col i by using techniques developed in cholera studies. Evidence continues 
to show that these strains are a major cause of diarrhea among travelers to 
developing countries and among children and adults in these countries. They 
occasionally cause diarrhea in the U.S., especially in newborns in crowded 
areas with poor sanitation. Both the heat labile (LT) and heat stable (ST) 
enterotoxins of E_. col i have been purified. The LT has a subunit structure 
(A&B) very similar to cholera toxin, and they are immunologically related to 
A&B cholera toxin subunits. There are minor differences in both E_. col i sub- 
units compared to the cholera toxin subunits; however, the biologic activity is 
comparable on a weight-for-weight basis. The structure and mechanism of action 
of ST are being studied. Diarrhea from E_. col i appears to be related to the 
organism's ability to attach to the intestinal mucosa. Two distinct pilus-like 
colonizing factors have been identified on human pathogens (CFS I and CFA II). 

Antibody studies indicate there may be a protective immune response to the CFAs, 
and this is being pursued. It appears there are other factors of E_. coli which 
contribute to diarrheas. One is a S. dysenteriae- 1 ike toxin which is pathogenic 
in rabbits. Also, some E_. coli without known pathogenic entities cause diarrhea 
in human volunteers, indicating other virulence mechanisms. Genetic manipula- 
tion of the LT gene of E_. col i may lead to the production of a strain for 
immunoprophylaxis. 

Viral Vaccine Program 

The viral vaccine program covers those areas where research should be stim- 
ulated toward the goal of an effective vaccine but which is not covered by the 
other specific programs of the Branch. During the past year an Experimental 
Herpesvirus Vaccine Workshop was co-sponsored with BoB, NCI, and the Fogarty 
International Center. It became clear that although attempts at vaccine devel- 
opment against Herpes simplex , cytomegalovirus, and varicella viruses had been 
made, only the varicella vaccine showed promise. Since varicella virus causes 
serious disease in immunosuppressed children or children with malignant diseases . 
an RFP to evaluate the efficacy of this candidate vaccine was released. Work 
with this vaccine has been initiated through a grant with the University of 
Texas at San Antonio for phase 1 studies. The program continues to support 
research to determine the antigenic structure and other characteristics of the 
herpes viruses in the hope of developing other more effective vaccine candidates. 

The Development and Applications Branch also supports five General Vaccine 
Evaluation Centers which form the backbone of the Branch's efforts at control 



9-15 



of important disease problems. Through these groups, different populations, 
including all age groups, normal and "at risk," are available. These groups 
are utilized for the evaluation of all vaccines, viral and bacterial, and 
antiviral s of interest to the Branch. During this past year these Centers have 
been overtaxed and a backlog of work has developed. 

FY 79 has been a successful year with significant progress in vaccine develop- 
ment, licensure of adenine arabinoside for herpes encephalitis, promising 
results in the efficacy of ara-A for neonatal herpes, and interferon for 
trigeminal neuralgia, zoster, and chronic hepatitis B. 



9-16 



EPIDEMIOLOGY AND BIOMETRY BRANCH 

The Epidemiology and Biometry Branch personnel supports the research program 
of the Institute through it's own projects and through collaboration with 
other units in the MIDP and the Institute, Collaborative arrangements included 
consultation in study design, evaluation of the epidemiological approach of 
studies, advice on management of large research data sets, analysis of research 
data and monitoring data from multicenter clinical trails. 

Approximate Level of Support 

Activity Number Amount 

HLA and Infectious Disease Epidemiology 

Research Contracts 2 $ 52,452 

Nosocomial Infectious Epidemiology 

Research Contracts 1 500,000 

HLA and Infectious Disease Epidemiology 

The EBB program which addresses questions about HLA and Infectious Disease 
Epidemiology was supported by two projects in FY79. The study of the associ- 
ation between HLA phenotype and recurrent herpes virus type 1 infection, which 
is sponsored jointly in Framingham, MA, by NIAID, The Bureau of Biologies and 
NHLBI, continued through the collection of clinical data and the collection 
of serum specimens. These data may contribute to our understanding of the 
relationships host factors such as HLA as circulating antibody have on a not 
uncommon expression of a universal infection. 

A second project in this program was started in FY79 when investigators at the 
Immunogenetics Laboratory of the Johns Hopkins University were awarded a con- 
tract in The Influence of HLA on the Immune Response to Viral Vaccines in 
Humans. This study is being conducted among families of the Old Order Amish 
which Dr. Wilma Bias, the Principal Investigator, has characterized previously 
with regard to HLA, An association between any differential in the dynamics 
of the various cellular and humoral components of the immune response to polio, 
mumps, rubella or measles vaccines and HLA will be sought. Should crude 
associations be noted it is anticipated the extensive and reliable geneologic 
information and the access to family members for study may allow the investi- 
gators to identify genes separate from but closely linked to HLA genes which 
modify the immune response to vaccines. 

Influenza 



The entire EBB staff participated actively in influenza research activities in 
FY79. Data from the clinical trials of inactivated A/USSR vaccine was vali- 
dated, organized and provided to the various investigators, 

10-1 



Dr. Blackwelder, Dr. David Ailing of the LCI and Sir Charles Stuart-Harris, a 
Fogarty International Scholar, conducted an investigation of relationships 
among total and cardiovascular mortality and influenza activity as measured 
by acute respiratory mortality and influenza mortality. This is an effort to 
fit U.S. data to a model of these relationships reported by British workers, 
If U.S. data produce a sufficiently good model it is anticipated that quanti- 
tative analysis of patterns of death among the elderly will identify clearly 
an influenza-related portion of mortality which will allow preventive strate- 
gies to be brought to bear more efficiently on truly high-risk persons. Policy 
decisions relating to influenza immunization of all people over 65 require 
reliable estimates of morbidity and mortality, and what proportion may be pre- 
vented by various strategies. Epidemiology is central to thorough considera- 
tion of program options, and the EBB expects to continue to contribute 
significantly to the broadened scope of the influenza program. 

Clinical Trials 

The expanding clinical trials programs within the NIH has stimulated develop- 
ment of new administrative and research technologies in study design, manage- 
ment of data and data monitoring and analysis. This is reflected in MIDP 
as well, and the EBB has been actively supporting several new initiatives, 
particularly two sponsored by CSB. In a multicenter study of treatment 
regimens for cryptococcal meningitis, EBB staff will be monitoring research 
data periodically for adherence to protocol and outcome. In another CSB- 
sponsored clinical trial, EBB staff consulted in the study design and expect 
to be involved with monitoring clinical data in a multicenter trial of 
penicillin prophylaxis of group B streptococcal sepsis in premature newborns. 

EBB staff assisted the DAB bacterial vaccine program officer in two areas con- 
cerning pneumococcal vaccines. In two separate but similar trials of vaccine 
in prophylaxis for pneumococcal otitis media, EBB staff conducted a preliminary 
analysis of efficacy data and will assist in the final analysis. The larger 
commitment in pneumococcal vaccines, however, was Dr. Blackwelder 's work with 
Dr. Gerald Schiffman to devise a sound but simple way to express pneumococcal 
polysaccharide antibody titers in the myriad of clinical trials for which 
Dr. Schiffman is providing the serological data. Dr. Blackwelder also assisted 
intramural scientists in the NIAMDD with the analysis of data from a clinical 
trial of pneumococcal vaccine in patients with systemic lupus erythematosis, 

EBB staff provided consulation to IAIDP in two areas in FY79, including parti- 
cipation in the design of a study of the efficacy of skin tests to the major 
and minor group determinants of penicillin. The material, which was developed 
by Dr. Bernard Levine, can detect persons who are allergic to penicillin, but 
it is not known how many patients in whom penicillin is the drug of first choice 
are denied the drug in the basis of an unconfirmed history of allergic reaction 
to it. This study should help to place the history of penicillin allergy and 
routine use of the skin test in clinical perspective, 

Ms. Jacqueline Smith continued to program for computerized analysis the Kidney 
Transplant Histocompatibility Study (KTHS) data. This multicenter project 
sponsored by IAIDP has collected a variety of clinical data judging the impact 



10-2 



of histocompatibility typing on the survival of kidney transplants, A pre- 
liminary analysis prepared in March showed a surprising differential in 
survival rates among participating centers which persisted when controlling 
for other factors such as the extent of tissue matching, age, and proportion 
of living related donors. Ms. Smith continues to be a consultant in computer 
programming for IAIDP staff and the contractors who are responsible for the 
KTHS. 

Impact of Infections 

In FY79 the EBB staff began with the consultant advice of Dr. Fred J, Payne a 
process of accessing on a routine, ongoing basis the morbidity, mortality and 
cost-of-illness data of allergic, immunologic and infectious diseases. This 
continuing project will for the first time develop these data from the same 
sources annually to allow a demonstration of trends and comparisons between 
years. Earlier results showed, as expected, the infectious diseases to be a 
tremendous burden to society in terms of morbidity but not mortality. This 
project is attempting to dissect out the the contribution of infectious disease 
complications to the high mortality rates of heart disease, cancer and diabetes. 
Previously only the underlying cause of death data were analyzed in studies 
of hospital records, but the extent to which infections effect adversely the 
course of chronic illness has not been established. A clearer understanding 
of the magnitude of the impact of allergic, immunologic and infectious diseases 
throughout the clinical spectrum expressed in terms of episodes of illness, 
direct cost of care, indirect costs such as days out of work or school and 
rates of complications in chronic illness is fundamental to the process of 
allocating research resources and planning research initiatives in these 
diseases. 

Epidemiology of Nosocomial Infections 

EBB negotiated partial funding of the large Study of the Efficacy of Nosocomial 
Infection Control Program (SENIC), a CDC sponsored study which is in the 
final stages of data acquisition. This comprehensive investigation of the 
effectiveness of standard hospital infection control procedures is expected 
to identify more precisely patterns of infections acquired in modern hospitals. 

Trans-NIH 

In the Trans-NIH areas covered by EBB staff, the Epidemiology Committee com- 
pleted the draft for an NIH Epidemiology Associates Program, When implemented 
this program will provide three years of training in epidemiology to fifteen 
physicians per year. The EBB will be a preceptorship for one associate per 
year who will be trained in infectious disease epidemiology. Dr. Curl in 
represented the Institute in the NIH Diabetes Coordinating and NIH Digestive 
Diseases Committees, 



10-3 



MOLECULAR MICROBIOLOGY AND PARASITOLOGY BRANCH 



The Molecular Microbiology and Parasitology Branch plans and conducts research 
grant, program project grant, contract, training grant, fellowship and career 
award programs in molecular microbiology, biochemistry, genetics, DNA recombin- 
ants, physiology, parasitology and medical entomology. It also coordinates 
the activities of the Parasitic Diseases Panel of the U.S. -Japan Cooperative 
Medical Science Program. 

Appropriate Level of Support 

Molecular Microbiology Section 

Activity Number Amount 



Research Grants — ' 






202 


$14,289,517 


Research Contracts 






1 


337,903 


Training Grants 






4 


322,748 


Fellowships 


Sub- 


■ total 


24 
231 


285,635 




$15,235,803 



1/ Includes 13 Career Awards ($440,792) 

Parasitology and Medical Entomology Section 

Research Grants — ' 

Program Project Grant 

Research Contracts 

Training Grants 

Fel lowships 





184 


$11 


,991,405 




1 




349,238 




5 




338,199 




10 




569,576 




11 




150,100 


Sub-total 


211 


$13,398,518 



2/ Includes 6 Career Awards ($197,921 



11-' 



Branch 



Number 



Amount 



3/ 



Research Grants — 
Program Project Grant 
Research Contracts 
Training Grants 
Fellowships 



Sub-total 



386 


$26,280,922 


1 


349,238 


6 


676,102 


14 


892,324 


35 


435,735 


442 


$28,634,321 



3/ Includes 19 Career Awards ($638,713) 

Molecular Microbiology 

Subsequent to the submission of the FY 1978 Annual Report, Dr. Delappe was a 
student in a course at Aspen, Colorado where he learned the Maxam-Gilbert 
method of DMA sequencing, including isolation of restriction fragments, 
labeling with polynucleotide kinase, specific chemical cleavages, and gel 
display of the sequence. 

During the year a number of scientific meetings, both foreign and domestic, 
relevant to the research supported by this Section were attended. A meeting 
on Recombinant DNA and the Eukaryotic Genome, sponsored by the French National 
Institute of Health and Medical Research (INSERM), was held in the village of 
Blois, France in November, 1978. A great many aspects of recombinant DNA 
research and their impact on eukaryotic genome structure and function were 
discussed in the presence of an international audience. The meeting was a 
productive and profitable one to attend. 

In June, 1979, Dr. Delappe gave an opening lecture at the Fourth International 
Symposium on Antibiotic Resistance at the Castle Smolenice near the city of 
Bratislava, Czechoslovakia. The proceedings will be co-published by AVICENUM 
(Czechoslovak Medical Press, Prague) and Springer-Verlag, (Heidelberg, Germany' 

In May, Dr. Delappe was asked to become a member of the Project Advisory Group 
of the Bureau of Drugs, FDA. This is the immediate advisory group for BOD 
research contracts. He was also asked to become a member of an FDA study 
section, but elected not to accept the appointment. 

In October, an announcement was made of the forthcoming award of a Nobel Prize 
to Dr. Hamilton Smith of The Johns Hopkins University. Dr. Smith shared the 
prize with two other scientists. The award recognized the development of 
restriction endonucleases, enzymes that can be used to study genetic organi- 
zation and to manipulate DNA. This was one of the origins of the recombinant 
DNA technology. At the time he made his contribution (in 1969), Dr. Smith 
was supported by a Research Career Development Award and a research grant, 
both of which were funded by the National Institute of Allergy and Infectious 



11-2 



Diseases. He was the tenth grantee of the branch to receive the Nobel Prize. 

Program Summary 

In addition to the basic free-ranging research of relevance to the Institute 
there are two structured programs supported by this section of the Branch, 
one of which involves mechanisms of resistance to antimicrobial agents and 
the other, recombinant DNA. 

(1 ) Mechanisms of Resistance to Antimicrobial Agents 

During the past ten years, the problem of microbial resistance to therapeutic 
agents has become increasingly apparent. The relevance of this phenomenon to 
other interests of this Institute such as hospital-associated infections due 
to staphylococci, gram negative bacteria, and other organisms, together with 
investigations of gonococci and pneumococci, is obvious. 

At this point, there is substantial clinical and epidemiological evidence that 
development of antibiotic resistance, and especially plasmid (extrachromosomal ) 
mediated drug resistance, represents a major problem in medical care. Most of 
the projects are concerned with defining the molecular and biochemical basis 
for resistance, the principal goal being to elucidate the fundamental biolog- 
ical mechanisms involved in the development of drug resistance by microorgan- 
isms. Specific goals involve investigation of the origin, development, 
evolution, expression and mechanism of drug resistance in a variety of specific 
microorganisms. Examples of microorganisms of particular interest are (but 
not limited to): Enterobacteriaceae , Pseudomonas , Neisseria , staphylococci, 
streptococci, mycobacteria, mycoplasmas, and pathogenic fungi . 

Research Highlights 

AI 14938-02 C. Cech (University of Colorado ): This investigator has studied 
the mechanism of rifampicin inhibition of Escherichia coli RMA polymerase with 
a newly developed steady state assay for RNA chain inititation and by analysis 
of the products formed with several 5 '-terminal nucleotides. The major effect 
of rifampicin was found to be a total block of the translocation step that 
would ordinarily follow formation of the first phosphodiester bond. These 
effects were incorporated into a steric model for rifampicin inhibition. 
Additional minor effects of the enzyme bound inhibitor were to increase 
slightly the lifetime of RNA polymerase on the \Pr promoter and to increase 
by two the apparent Michaelis constants of the initiating triphosphates. The 
products formed by RNA polymerase in the presence of rifampicin belong nearly 
exclusively to the class triphosphate purine phosphodiester nucleotide. No 
evidence for the accumulation of such molecules was obtained in vivo . 

AI 10971-08 M. Hiqqins (Temple University School of Medicine ): Dr. Higgins 
has examined the morphological effect of the antibiotic cerulenin on cell 
shape of Streptococcus faecal is . This drug, which inhibits fatty acid 
synthesis, causes an elongation of growth zones and a blockage of cell division 
similar to that observed when DNA synthesis is inhibited in these cells. This 
is an interesting observation in that he had predicted that a signal at the 



11-3 



end of a round of DNA synthesis is necessary for an inhibition of autolytic 
activity and a concomitant stimulation of cell division. Recently, it has 
been shown that a lipid intermediate is involved in a reduction of autolytic 
activity is this organism, and it is supposedly at this level that cerulenin 
works to block cell division. 

AI 12835-05 R. Martinez (University of California, Los Angeles ): Studies 
involving the bactericidal capabilities of normal rabbit serum (NRS) have 
revealed the presence of two principal components capable of reducing the 
viability of Bacillus subtil is , in vitro . These components, rabbit lysozyme 
and a non-lysozyme factor, comprise the classical 8-lysin system in rabbits, 
long noted for its heat-stability and principal activity aqainst gram positive 
bacteria. Both of these factors have been Durified to homogenietv. examined 
for bactericidal activity (singly, and in combination), and have been (or are 
in the process of being) characterized at the molecular level. The results 
indicate that the primary bactericidal component of NRS active against a 
variety of organisms is a single, low molecular weight, proteinaceous factor, 
presumably released from platelets during the coagulation process. Although 
the secondary factor, lysozyme, has also been shown to exhibit bactericidal 
activity, it has been demonstrated that when examined at concentrations found 
in dilute NRS, the presence of this enzyme can account for only a minor portion 
of the antibacterial action observed for NRS. These results have been confirmed 
in whole serum studies to which specific inhibitors of lysozyme enzymatic and 
bactericidal activity have been added. 

AI 12500-05 J. Murphy (Harvard Medical School ): During the past year he has 
published a series of experiments that have demonstrated for the first time 
that iron has a direct effect on inhibition of diphtheria toxin production by 
cells that were "derepressed" for toxin. In addition, it has been demonstrated 
that the decay kinetics of toxin release were identical following either iron 
or rifamoicin. By preventing reinitiation of RNA polymerase, the decay 
kinetics imposed by rifampicin block mimic transcriptional control. Thus, the 
similarity of inhibition kinetics of toxin release imposed by iron and rif- 
ampicin suggested that the control of toxin production was at the transcrip- 
tional level. Further evidence to support this hypothesis was provided by 
RNA:DNA hybridization experiments. He has shown that there is a marked 
stimulation of hybridization of total ^H-RNA extracted from iron starved 
corynebacteria to e-phage DNA. Total labeled RNA extracted from cells that 
were in iron excess or from the non-lysogenic, non-toxigenic C_. diphtheriae 
C7 (-) hybridized to the same extent. 

AI 11756-12 C. Gilvarg (Princeton University ): This investigator has isolated 
the major component of the cell wall of Bacillus megaterium M46, and his studies 
indicate that it is a complex polysaccharide over twenty times the size of 
previously reported teichuronic acids. The molecular size and shape of this 
unusual teichuronic acid were elucidated by a combination of hydrodynamics, 
chemical and electron microscopic studies. This should contribute to a greater 
understanding of the synthesis and assembly of the bacterial cell wall which 
plays an important role in resistance to antimicrobial agents. 



11-4 



AI 10311-10 D. Dubnau (Public Health Research Institute of the City of New 
York ) : This investigator has reported the construction of several plasmid 
chimeras by molecular cloning, thus demonstrating the utility of the B_. subtil is 
system for recombinant DNA experiments and providing a collection of new 
plasmids for studies on replication, incompatibility, and transformation, as 
well as for the engineering of better cloning vectors. A plasmid pEl 94 , 
obtained from Staphylococcus aureus confers resistance to macrolide, 
lincosamide, and streptogramin type B ("MLS") antibiotics. For full express- 
ion, the resistance phenotype requires a period of induction by subinhibitory 
concentrations of erythromycin. A copy number in the range of 10 to 25 copies 
per cell is maintained during cultivation at 32°C. It is possible to transfer 
pE194 to Bacillus subtil is by transformation. In B_. subtil is , the plasmid is 
maintained at a copy number of approximately 10 per cell at 37°C, and resistance 
is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin 
structurally and to which erythromycin induces resistance, lacks inducing 
activity. Two types of plasmid mutants were obtained and characterized after 
selection on medium containing 10 yg of tylosin per ml. One mutant class 
appeared to express resistance constitutively and maintained a copy number 
indistinguishable from that of the parent plasmid. The other mutant type had 
a 5- to 10-f old-elevated plasmid copy number (i.e., 50 to 100 copies per cell) 
and expressed resistance inducibly. Both classes of tylosin-resistant mutants 
were shown to be the result of alterations in the plasmid and not to modifi- 
cations of the host genome. 

(2) Recombinant DNA Molecular Research 

During the past few years, the development of certain techniques in the area 
of molecular biology has made it possible to construct functional DNA 
molecules in vitro which contain segments derived from diverse biological 
sources. The scientific innovations which led to this technological break- 
through were mostly derivative from basic studies on the mechanism of 
restriction, which normally acts as a barrier to gene flow among microorganisms, 
and on the molecular biology and genetics of bacterial plasmids, especially 
those specifying drug resistance. Grantees of this Institute played a pre- 
ponderant role in both of these areas. Whereas DNA recombination in nature has 
depended on random processes, the experimental techniques now available enable 
the in vitro construction and subsequent replication of DNA molecules needed 
for specific experimental goals. This basic advance, coupled with refinements 
of the existing technology, should provide greater knowledge of the mechanisms 
of pathogenicity (at the molecular level) of viral, bacterial, mycotic and 
parasitic agents. This information, in turn, may lead to improved prevention, 
diagnosis and treatment of infectious diseases. Notwithstanding the potential 
benefits of recombinant DNA molecule research, there may be associated poten- 
tial biohazards which must be avoided by the design, construction and testing 
of safer host-vector systems for use in these studies. 

The primary goal of this research is the development and utilization of recom- 
binant DNA molecule technology to increase fundamental knowledge and ultimately 
enhance control of the etiological agents of infectious diseases. Another 
goal is the synthesis of a variety of biologically useful substances through 
the construction of bacterial cells containing functional DNA of either plant 



11-5 



or animal origin. An equally important goal is the identification, assessment 
and elimination of any and all potential biohazards encountered in the 
exploitation of this technology. 

Research Highlights 

AI 10311-10 P. Dubnau (Public Health Research Institute of New York ) : The 
ability to carry out molecular cloning in Bacillus subtil is would be useful 
for a variety of studies on sporulation, transformation, and gene expression. 
In addition, such a capability might be industrially significant, because 
Bacillus species are of considerable commercial importance. Antibiotic 
resistance chimeric plasmids have been constructed by in vitro enzymatic 
manipulation and introduced into Bacillus subtil is by transformation. The 
parental plasmids used had been introduced into B_. subtil is from Staphylococcus 
aureus by transformation. Seven recombinant plasmids have been constructed 
using restriction endonucleases. Although all of the recombinant plasmids 
replicate and express their antibiotic resistance characters, three of them 
have suffered a loss of DNA, either in vitro or, more likely, in vivo . The 
deletion event in all cases involved one of the two termini used to join the 
parental plasmids. The plasmid chimeras reported should prove useful for the 
study of plasmid replication, incompatibility, and recombination. In addition, 
the utility of the B_. subtil is system for molecular cloning has been clearly 
illustrated. 

AI 8619-12 S. Cohen (Stanford University Medical School ): DNA fragments 
generated by the EcoRl or Hindi II endonucleases from the low copy number 
antibiotic resistance plasmids R6 and R6-5 were separately cloned using the 
high copy number ColEl or pML21 plasmid vectors. The hybrid plasmids that 
were obtained were used to determine the location of the EcoRl and Hindlll 
cleavage sites on the parent plasmid genomes by means of electron microscope 
heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation 
of the cloned fragments in cesium chloride gradients localized the high 
buoyant density regions of R6-5 to fragments that carry the genes for resis- 
tance to streptomycin-spectinomycin, sulfonamide, and mercury and a low 
buoyant density region to fragments that carry the tetracycline resistance 
determinant. Functional analysis of hybrid plasmids localized a number of 
plasmid properties such as resistance to antibiotics and mercury and several 
replication functions to specific regions of the R6-5 genome. Precise 
localization of the genes for resistance to chloramphenicol, kanamycin, 
fusidic acid and tetracycline was possible due to the presence of identified 
restriction endonuclease cleavage sites within these determinants. 

AI 8619-12 S. Cohen (Stanford University Medical School ) : Messenger RNA 
that encodes the common peptide precursor for the hormones corticotropin 
and e-lipotropin was purified from the neurointermediate lobe of bovine 
pituitaries, and double-stranded complementary DNA species synthesized from 
this template were cloned in Escherichi a, col j x1776 by inserting them into 
the Pst I endonuclease cleavage site of the pBR322 plasmid. Certain of the 
cloned cDNA inserts contain nucleotides corresponding to the complete amino 
acid sequence of bovine corticotropin and a coding sequence that corresponds 
to at least the first portion of bovine 8-1 ipotropin. The nucleotide sequences 



11-6 



coding for corticotropin and s-lipotropin are separated on the cDNA by a 
6-base-pair sequence encoding lysine and arginine, indicating that the carboxyl 
terminus of corticotropin is connected on the precursor peptide with the 
amino terminus of 8-1 ipotropin by these two amino acids. In addition, the 
cloned cDMA insert is characterized by an usually high C+G (cytosine and 
guanine) nucleotide base content as well as by a number of DMA sequence 
duplications. 

K04 AI 119-03 P. Lovett (University of Maryland ): The technique of molecular 
cloning involves the in vitro insertion of fragments of DNA into small repli- 
cons, plasmids, or phage genomes, followed by selection of chosen recombinant 
molecules by transformation of appropriate recipient cells. Direct application 
of recombinant DNA technology to the study of Bacillus subtil is wil 1 ultimately 
provide a general method for constructing partial diploid strains which will, 
in turn, permit genetic complementation analyses of specific mutations and 
provide a source of easily obtainable DNA highly enriched for genes of chromo- 
somal origin whose in vitro expression may be of special interest such as 
sporulation genes. Plasmid pUBllO, originally detected in Staphylococcus 
aureus , specifies resistance to neomycin and has been transformed into Bacillus 
subtil is 168. In B_. subtil is , pUBllO is stably maintained at about 50 copies 
per chromosome and renders the host resistant to neomycin sulfate at 5 pg/ml . 
pUBllO was transferred by transduction from B_. subtil is to strains of B_. 
pumilus and B_. licheniformis . 

Parasitology 

On September 18-20, 1978 the Branch was represented at a "Symposium on Water- 
borne Transmission of Giardiasis" sponsored by the U.S. Environmental 
Protection Agency. The goal of the symposium was to discuss the state of the 
art regarding Giardia species and the disease as they relate to water supplies, 
and to identify areas where further research is needed. The topics discussed 
included the organism, the disease, epidemiology, detection methodology and 
water treatment technology. It was concluded that more research on al 1 
aspects of giardiasis is needed. 

The 13th Joint Conference of the U.S. -Japan Parasitic Diseases Panel was held 
in Okayama, Japan on October 4-6, 1978. Forty-seven Japanese and ten American 
scientists attended this conference at which 42 papers were presented on a 
broad range of studies of both schistosomiasis and filariaris. In addition 
to the formal scientific sessions, many of the U.S. participants took advantage 
of opportunities to develop potential collaborative research relationships 
with their Japanese counterparts. 

The Branch was also represented at a conference on "Pharmaceuticals for 
Developing Countries" sponsored by the National Academy of Sciences on 
January 29-31, 1979. Particularly relevant to the Parasitology program were 
the presentations on cellular regulatory processes in parasitic helminths and 
those on the pharmacological exploitation of biochemical differences between 
parasites and hosts. 



11-7 



Late in 1978, a Request for Application for Tropical Disease Research Units 
program project grants was issued. The basic objective of this new initiative 
is to bring together relevant biomedical knowledge and technology in a multi- 
disciplinary attack on the world's tropical and parasitic diseases. Nine 
applications were received and reviewed in June, 1979 and, following the 
October, 1979 Council, it is hoped that funds will be available to permit 
the funding of 2 or 3 of these program projects. 

On June 15, 1979, an RFA was issued for New Investigator Research Awards in 
Tropical Diseases. To help bridge the transition from training status to that 
of a productive investigator, this special grant program will provide support 
for young scientists and physicians with meritorious ideas for research on 
the world's tropical diseases. Emphasis will be placed on research on malaria, 
schistosomiasis, filariasis, trypanosomiasis, leprosy, leishmaniasis and the 
immunology of these diseases. The first of these awards will be made in 
July, 1980. 

Program Summary 

In addition to the basic free-ranging research of relevance to the Institute, 
there are two structured programs supported by this section of the Branch-- 
biological regulation of vectors and immunology of parasitic infections. 

(1 ) Biological Regulation of Vectors 

This program has as its goal the advancement of fundamental studies which 
might lead to effective methods of biological regulation of vectors. For the 
past 30 years, control of pests and disease vectors has been based primarily 
on the use of synthetic organic compounds which had the "advantages" of long 
residual action and toxicity to a broad spectrum of target organisms. It 
has now been shown that because of these very characteristics, many of these 
pesticides are more deleterious than beneficial, when all effects on man and 
his environment are considered. Furthermore, resistance to broad spectrum 
chemical pesticides has reduced their effectiveness in many vector control 
programs. For these reasons the search for alternative methods of pest control 
has become imperative, and it is generally agreed that the best approaches will 
consist of integrated pest management programs which combine biological 
control, in the broadest sense, with the judicious use of more specific 
chemicals and management of the physical environment. This approach to vector 
control must be based on adequate information about the ecology of the target 
organism, the environment in which the control program is to be conducted, 
effects of control measures on non-target organisms in the environment,, and 
the biology of the disease organisms being transmitted. 

Research Highlights 

AI 10986-06 P. Sonenshine (Old Dominion University ) : The identity of the 
female sex pheromone (2,6-dichlorophenol ) of the ticks Dermacentor andersoni 
and D_. variabilis was confirmed. Dr. Sonenshine also demonstrated the role 
of fovea! glands as the source of this compound. Pheromone-impregnated 
materials, especially dusts, caused delay of mating and confusion of mate- 



1-8 



seeking males. The use of sex pheromones in an integrated control program 
would provide a negative pressure effect on the reproductive capacity of the 
tick population surviving the direct effects of insecticides. 

AI 11847-11 R. Barr (University of California, Los Angeles ): Dr. Barr has 
found that Holbachia pipientis , a rickettsia-1 ike symbiont of Culex pipiens , 
affects the sperm of male mosquitoes in such a way that the sperm are not able 
to fertilize eggs unless the eggs are also infected with Wolbachia . If the 
eggs are infected with Wolbachia of different geographic origin, the cross may 
be incompatible. Research is continuing to determine whether Wolbachia might 
be cultured and used as a means of controlling mosquito reproduction. 

AI 11123-07 N. Alger (University of Illinois ): Dr. Alger has been studying 
the use of the microsporidian Nosema algerae as a biocontrol agent against 
Anopheles mosquitoes. Her most recent studies in Pakistan have shown that 
it is possible to infect field populations of mosquito larvae, and that the 
resultant adult mosquitoes will produce fewer eggs, some of which will be 
infected and will not live long enough to transmit malaria. Non-target aquatic 
invertebrates are not infected; thus, the use of N_. algerae in mosquito 
biocontrol appears to be safe. 

AI 13295-03 C. Bayne (Oregon State University ): In a study of the suscep- 
tibility of the vector snail Biomphalaria glabrata to Schistosoma mansoni , 
Dr. Bayne has discovered that sporocyst-kill ing cells isolated from the snails 
are actually amebae of the genus Nuclearia which attach to the surface and 
penetrate the schistosome sporocysts. These amebae-sporocyst interactions 
may be important determinants of snail susceptibility in nature. 

(2) Immunology of Parasitic Infections 

The complexity of structure and function of parasites has made the study of 
the immunology of these infectious agents exceptionally challenging and 
rewarding. Exciting opportunities for the elucidation of mechanisms and 
manifestations of immunological responses to parasites now exist as the 
result of the impressive developments in immunology in recent years. Major 
ultimate goals of studies on the immunology of parasitic infections are the 
development of effective vaccines for the prevention of parasitic diseases 
(such as malaria, schistosomiasis and filariasis), the intervention in the 
host response to prevent or ameliorate disease processes which are immuno- 
logically mediated, and the development or improvement of immunodiagnostic 
procedures for parasitic infections, especially as they relate to the immune 
status of the host. 

A related goal of these studies is to contribute to an understanding and 
solution of basic and clinical problems associated with other disease entities, 
especially immunological disorders and hypersensitivity states. A number of 
parasitic infections are excellent models for such studies as: (a) the 
mechanisms of intracellular immunity, (b) the enhancement or suppression of 
concurrent infections or tumor development, (c) immunopathological mechanisms, 
(d) development of disease processes in immunosuppressed or immunostimulated 
hosts, (e) the biochemical and genetic mechanisms for the development of 



11-9 



pathogen variants with different immunological characteristics, (f) the 
genetic basis for variations in host response and (g) the role of IgE and 
other cytotropic antibodies in hypersensitivity. 

Research Highlights 

AI 15235-02 H. Shear (New York University ) : In a study of macrophage acti- 
vation in experimental malaria (P. berghei ) , Dr. Shear showed that early in 
an infection macrophages appear to be activated and parasitized erythyocytes 
are ingested; later, the macrophages appear to be "blocked." Indirect 
evidence points to the possibility of inhibitory immune complexes in the 
serum of infected animals. An analysis of this blocking activity should help 
clarify the animals' inability to cope with the disease ultimately. 

AI 12770-04 M. Wittner (Albert Einstein College of Medicine ) : In a study 
of mechanisms of immune resistance to Trypanosoma cruzi , Dr. Wittner has 
investigated the role of macrophages in the in vivo resistance to T. cruzi 
infection. Mice genetically resistant to T. cruzi were depleted of peritoneal 
macrophages prior to infection with the parasite. Increased parasitemia and 
mortality in this group are consistent with the hypothesis that the macrophage 
constitutes an early line of defense in T. cruzi infection. 

AI 12663-05 J. Farrell (University of Pennsylvania ): Using the golden hamster 
as a model for the study of Kala-azar, Dr. Farrell has shown that hamsters 
inoculated intracardially with L_. donovani develop fatal visceral infections 
whereas animals injected intradermal ly usually exhibit self-limiting, non- 
ulcerating cutaneous infections and subsequent resistance to reinfection. He 
is currently investigating the immunological parameters of these different 
manifestations of the disease. 

AI 08989-09 W. Trager (Rockefeller University ): In a series of preliminary 
experiments, Dr. Trager and his colleagues have developed in vitro techniques 
for the collection of relatively pure merozoites of Plasmodium falciparum to 
be used in antigen-antibody studies. Aotus monkeys immunized with this in 
vitro culture-derived antigenic material are protected from challenge infec- 
tion. 

AI 12913-03 D. Boros (Wayne State University ): Dr. Boros has shown that the 
granulomatous inflammatory response around schistosome eggs is mediated by 
inflammatory T lymphocytes. During the chronic phase of the infection certain 
lymphocytes assume a suppressive/regulatory role and exert their influence on 
the inflammatory lymphocytes to diminish their activity which results in 
smaller granulomas and hence diminished host pathology. 

(3) General Parasitology Research Highlights 

AI 14294-03 J. Bennett (Michigan State University ): In a study of the effects 
of chemotherapeutic agents on schistosome physiology, Dr. Bennett has demon- 
strated that the effective schistosomacide, praziquantel, causes a massive 
contraction of the worm and a large influx of calcium. Apparently the drug 
acts by stimulating the influx of calcium which causes the muscle contractions. 



11-10 



AI 15919-01 J. Dubey (Montana State University ): In an investigation of a 
human toxoplasmosis outbreak in a stable, Dr. Dubey found that of the 37 cases 
reported, none had had common meals; thus raw meat could not be implicated in 
the transmission. Apparently the infections occurred either via the hand-to- 
mouth or dust inhalation route, and the source of infection was a group of 
Toxoplasma positive cats which lived in the stable area. 

AI 10250-08 L. Ash (University of California, Los Angeles ) : Further progress 
has been made in the development of a primate host for the human filarial 
parasite, Wuchereria bancrof ti . In addition to establishing patent infections 
in nine monkeys, one of these infections represented the first serial passage 
of the parasite from monkey to monkey. 

AI 14718-02 R. Cypess (Cornell University ): There has long been a need for 
a specific and reliable diagnostic test for toxocaral visceral larval migrans 
(VLM) since this syndrome presumably may be caused by a variety of helminths. 
Dr. Cypess has demonstrated that the enzyme-linked immunosorbent assay (ELISA) 
is superior to indirect hemagglutination, bentonite flocculation and double 
diffusion in agar tests. Based on specificity, sensitivity and positive and 
negative predictive values, the ELISA appears to be the serodiagnostic method 
of choice for toxocaral VLM. 

AI 11942-05 M. Mu'ller (Rockefeller University ): Dr. Miiller, in a study of 
the biochemical cytology of Trichomonas vaginalis , has shown that drug- 
resistant strains of the parasite do not take up metronidazole in the presence 
of oxygen. These studies suggest that the mechanism of resistance is connected 
with alteration in the effect of oxygen on the intracellular reduction- 
oxidation system. 

Contract Activity 

The schistosomiasis supply contract service, located at the University of 
Lowell, has continued to be utilized extensively by most schistosomiasis 
researchers in this country. This invaluable service can provide all the 
human schistosome parasites and their vector snails. 

The filariasis supply contract, located at the University of Georgia, has 
continued to provide filariasis research workers with various filarial para- 
sites and their vectors. The complexity of filarial life cycles and the 
special facilities needed for the housing and rearing of arthropod vectors 
makes this service contract especially valuable to researchers who otherwise 
might not be able to pursue their research interests. Acceleration of research 
in filariasis is in part directly attributable to this service. 

Two research contracts involving the development of techniques for the cryo- 
preservation of various stages of human and animal filariae are currently in 
progress. Short- and long-term cryopreservation of microfilariae and infective- 
stage larvae of both sheathed and unsheathed microfilaria! species has been 
accomplished, and this now provides unique opportunities to transport rare 
and scarce materials to laboratories capable of working with various aspects 



11-11 



of these parasites. In particular, work on Wuchereria bancrofti and Brugia 
malayi infections has been enhanced considerably. 

As a result of a recommendation made at an NI AID workshop on the "Radiobiology 
of Schistosomes and Filariae" held at NIH on April 17-18, 1978, an RFP was 
issued for a contract for the development of techniques for the radio-labelling 
of schistosome and filarial larvae. Six proposals were received and reviewed, 
and a contract was awarded to Cornell University on June 29, 1979 for two 
years. 



11-12 



EXTRAMURAL ACTIVITIES PROGRAM 
NIAID 
TABLE OF CONTENTS 

REPORT OF THE DIRECTOR 12-1 

A. Introduction 12-1 

B. The Review of Research Proposals 12-1 

C. Implementation of the Multi-Axis Coding System (MACS) 12-1 

D. Personnel 12-1 

E. Manpower Development 12-3 

F. Decline of Physicians in Research Training 12-4 

G. Survey of American Society for Microbiology Membership 12-5 

H. Committee Management 12-5 

I. Conference Proposal Review 12-5 



I. CONTRACT MANAGEMENT BRANCH 



II. GRANTS MANAGEMENT BRANCH 



III. PROGRAM ANALYSIS AND EVALUATION BRANCH- 



,13-1 



.14-1 



,15-1 



A. Fiscal Management and Analysis Section 15-1 

B. Program Analysis Section 15-4 

C. Data Control Section 15 ~ 5 

IV. PROGRAM AND PROJECT REVIEW BRANCH 16-1 



A. Introduction 



,16-1 



B. Allergy and Clinical Immunology Research Committee 16-2 

C. Transplantation Biology and Immunology Committee 16-2 

D. Microbiology and Infectious Diseases Advisory Committee 16-2 

E. Review Services Unit xo J 



F. Clinical Trials. 



V. RESEARCH RESOURCES BRANCH 



.16-3 



,17-1 



A. Special Activities *-' 

B. Asthma and Allergic Diseases Program - 

17 1 
Allergen Reagents ±/ -± 

C. Immunological Reagents and Resources 17-2 

1 7 — ? 

D. Microbiology 

E. Research Resources - Research Reagents 17-3 

F. Molecular Anatomy Program 17-4 

G. Processing and Distribution 17-6 

H. Individual Contract and Agreement Tabulation 17-10 

I. Contract Publications 17-16 



REPORT OF THE DIRECTOR, EAP 

A. Introduction 

The Extramural Activities Program (EAP) of the NIAID ended the decade 
of the 70' s by awarding the largest number of regular research 
projects of any year during the decade, although the number of trainees 
supported was smaller during the early 1970 's. The EAP workload, 
associated with the increased number of applications and proposals 
received, was increased by about 25%. This overload was handled by 
part-time employees, overtime, and the increased productivity of all 
the employees. Although there were stressful times when it seemed 
we would not meet our assigned schedules, it was an exciting time to be 
in the EAP. 

B. The Review of Research Proposals 

The Program and Project Review Branch was fully staffed for the first 
time since the reorganization of the Institute. To assist in the 
tremendous workloads of the winter and spring rounds, reinforcements 
were received from the Office of the Director, EAP and from the Office 
of the Director, NIAID. This workload is described later in the 
report from that Branch. All of these reviews have resulted in 
funding of those applications and contract proposals which were approved 
with good priority scores. The staff in this Branch has been 
congratulated for their efforts . 

C. Implementation of the Multi-Axis Coding System (MACS ) 

In Fiscal Year 1979, we introduced and began accumulating data by the 
new multi-axis coding system. This new and more complex system of 
record keeping was brought about at the recommendations of the Data 
System Planning Committee to meet Institute requirements for broader 
scientific data and to meet NIAID, NIH and other requirements for 
administrative data. This activity also required considerable extra 
effort on the part of staff because it was a new system which was 
implemented during the year in which NIAID received and reviewed the 
largest number of application and contract proposals to date. 

This system recommended by the Data Systems Planning Committee, as 
reported in last year's annual report now includes four axes of 
biological data; administrative subjects such as basic, applied and 
development (BAD); scientific base, applied technology transfer, and 
training (SATT) ; and coding for human subjects, clinical trials 
and prevention. This is a management data system and will eventually 
be evaluated on that basis. 

D. Personnel 

There were fourteen departures from the EAP and fourteen new appointments 
during FY 1979. Some of these new appointments were temporary appoint- 
ments which terminated in 1979. The lag time for the process of 

12-1 



appointing new employees remains about the same as it was last 
year, meaning that there were approximately forty man months during 
which there were employee vacancies in the EAP. This represents over 
three man years and considerably hampers the accomplishment of EAP 
objectives. 

Retirements 



Mildred Applestein 
Gwen Northcutt 
Eleanor Wyatt 

Employees who have left EAP 

Virginia Alary 
Mary Baldwin 
Kathy Cherry 
Sue Connors 
Maria Decker 
Melvin Joppy 
Cynthia Jose 
Carol Kimmel 
Harry Levine 
Janice Pusey 
Earle Vance 

New Appointments 

Todd Ball 

Betty Bucher 

Bonnie Dunning 

Diane Edwards 

Melo Ellis (Stay-in-School) 

Debbie Frazier (Temp) 

Carla Goldblum 

John Hamill 

Fran Hisaoka 

Annette Hobbs (Expert Consultant) 

Anne Rabon 

Sara Spencer 

Gary Thompson 

Daryl Thornton (Stay-in-School) 

Delores Walton (Temp) 

Awards (Quality Increase) 

Louis Bourgeois 
Gertrude Cohen 
Grace Ellis 
Fran Schlademan 
Hubert Sumner 
Rick Wiener 



12-2 



Training 

Fifty-five training courses at a cost of just over $5,000 were taken 
by staff. 

Figure I shows the Organizational Chart. 

E. Manpower Development 

The traditional training program increased insofar as postdoctoral 
awards were concerned. The trend in predoctoral awards continued at 
a slight decline. There was a continued decline in the number of 
physicians who were interested in research training and this loss of 
potential clinical investigators remains the major problem in the 
NIAID manpower development program as well as in other similar 
programs at the NIH. 

The amendment to the National Research Service Act (NRSA) which shifts 
more support to institutional grants and less to individual fellowships 
has not made a significant impact this fiscal year. There are 81 
T32 training grants now being administered by NIAID. Of these, 34 
are in the IAID program and 47 in the MID program. 

Table I estimates the total number of trainees being supported by 
NIAID on the authority of NRSA and on the old T01 programs of which 
there are 18 still operating on unexpended funds and support 70 
fellows. 

TABLE I 

All Trainees - Estimated 

PROGRAM 

IAIDP MIDP TOTAL 



Predoctoral 


40 


65 


105 


Postdoctoral 


119 


202 


321 


M.D. 


57 


48 


105 



TOTAL 216 315 531* 

*Includes T01 

Table II estimates the number of individual fellows being supported 
in each of the major programs. The number of M.D.'s in comparison 
to the number of Ph.D.'s is also shown. 



12-3 



TABLE II 
Individual Fellowships (F32) 
PROGRAM 



Degree IAIDP MIDP TOTAL 

Ph.D. 55 94 149 

M.D. 9 8 17 

TOTAL 64 102 166 

Table III estimates the number of trainees both predoctoral and 
postdoctoral in the LAID and MID programs. 

TABLE III 

Institutional Training (T32) 

PROGRAM 

IAIDP MIDP TOTAL 



Predoctoral 37 68 105 

Postdoctoral 97 145 262 

TOTAL 134 213 367 

F. Decline of Physicians in Research Training 

The decline in M.D.'s selecting research as a career still remains of 
concern to those involved with biomedical training. One aspect which 
appears not to have been examined is the long-range effect of 
legislation. Legislation focusing on the production of more physicians 
for medical service began in 1963. It evolved into a federal initiative 
that became increasingly focused on training. The history of this 
legislation was as follows: 

1963, Health Professional Educational Assistance Act provided 
for construction grants and loans for undergraduate students, 

1965, amendments to the Act provided aid for scholarship and 
medical schools operating costs, 

1968, the Health Manpower Act emphasized absolute increase in 
medical school enrollments, 

1971, Comprehensive Health Manpower Training Act designed to 
increase enrollment through capitation, and to increase 
practitioners in rural and shortage areas and family medicine, 



12-4 




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1976, Health Professions Educational Assistance Act, P.L. 94-484, 
established a target of 50% of graduating M.D.'s as primary care 
specialists, and to encourage medical care institutions to 
establish residencies in family practice, and primary care 
specialties. 

These federal actions, plus the leveling of research budgets, 
the low level of stipends, and a pay back requirement for research 
training have made research much less attractive than service 
for the graduating M.D. In the 1980' s, the NIH must reverse 
this trend. 

G. Manpower Survey being Conducted by the American Society for 
Microbiology 

To date, the Society has received about 17,000 responses to the 25,000 
questionnaires sent out. This was 7,000 more than was anticipated 
by the designers of the survey. These have been coded and they are 
being keypunched and computerized. Dr. William Epstein, the technical 
writer, and Mr. John Dirkse the statistician, are working to examine 
the data and prepare a draft of the report. The work on the survey 
is ahead of schedule. A recent supplement has been awarded to assist 
in anslysis of the data acquired from the additional responses received. 

H. Committee Management 

In Fiscal Year 1979, the Committee Management Officer retired and her 
assistant left for another position. The Committee Management Officer's 
position was filled just prior to the May Council by a very capable 
employee and the position of Assistant to Committee Management Officer 
could not be filled in the fourth quarter because of a freeze on 
personnel. This meant that this office also had to operate with a 
series of temporary measures for accomplishing typing, filing, review 
of vouchers and travel claims and travel activities. It is a tribute 
to the new committee management — council secretary that no major 
problems resulted from operating an office as important as that of 
committee management and council services in this manner. 

I. Conference Proposal Review 

The EAP review of all "unsolicited" conference proposals, i.e., those 
arising outside the program areas of NIAID, which was successfully 
initiated and implemented in FY '78, has continued at about the same 
level in FY '79. Prospecti were received from the Fogarty International 
Center, as well as directly from the proposers. Those which were 
accepted for review, primarily on the basis of Institute relevance, 
were subjected to a primary review by an ad_ hoc scientific group 
and a secondary review by the Research Contract Review Group, the 
latter being scheduled once each quarter. A maximum amount of funds 
was set aside for the support of these conferences. Eight R-13 
conference grant applications were also received and reviewed in FY '79. 



12-5 



They received a primary No Study Section (NSS) assignment but were 
peer reviewed by the EAP; these were presented at the appropriate 
Council meetings for final review and approval. A total of 69 
conference prospecti and R-13 grant applications were received by 
EAP in FY '79. Of this number, 39 were reviewed and 35 of these were 
approved for funding and/or sponsorship for a maximum of $276,796. 
Eleven of the 69 will be reviewed for possible FY '80 funding. 
(See Table IV). 



12-6 



TABLE IV 

CONFERENCE/WORKSHOP PROSPECTI REVIEWED BY NIAID 
FY '79 



Received. 



.69 



From Fogarty International Center (FIC) 59 

Sponsorship & Funds 58 

Sponsorship Only 1 

Directly by Institute 2 

From DRG (R13's) 8 

Not Reviewed 19 



Returned to FIC due to lack of Institute relevance. . 15 

Returned to FIC due to Institute policy 3 

Returned to FIC due to lack of justification 

for meeting site 1 



Reviewed. 



,39 



Approved 35 

Sponsorship & Funds 30* 

Sponsorship Only 1 

Approved (No Funds) 4 

Disapproved (NIAID and/or FIC) 4 

Sponsorship & Funds 4 

Sponsorship Only 

To Be Reviewed 11 



FUNDED OR SCHEDULED FOR FY '79 FUNDING (Thru May '79) ** 

Other 
IAIDP MIDP NIAID 



TOTAL 





# 


$ 


// 


$ 


// 


$ 


// 


$ 


R-13 Grant 
Other Extramural 


1 
8 


$18,125 
$75,050 


6 

9 


$80,983 
$76,638 



5 



$26,000 


7 
22 


$ 99,108 
$177,688 


TOTAL 


9 


$93,175 


15 


$157,621 


5 


$26,000 


29 


$276,796 







^Includes 8 R-13's some of which will not be paid 
'^Includes some which were reviewed in FY '78 but funded in FY '79 



12-7 



I. CONTRACT MANAGEMENT BRANCH 

The Contract Management Branch provides management services to the 
Institute's Research Program including solicitation, negotiation, award, 
and administration of all Institute research contracts. The CMB will 
continue to implement contract policies and procedures promulgated by 
higher procurement authority. 

During FY 1979, Mr. Harry LeVine, Contract Specialist, resigned in 
order to further his education. His replacement was Ms. Sara Spencer 
formally of NINDS. Mrs. Mildred Applestein, Contracting Officer, 
retired after 30 years of Government service. Her replacement was 
Mr. John Hamill formally of NCI. 

In accordance with Secretary Califano's initiative to correct major 
deficiencies in the contracting process, CMB is required to prepare 
the following reports: 

1. Justification for Non-Competitive Procurement (JNCP) approved 
monthly. 

2. Non-competitive Procurement and Scheduling of R&D contracts - 
monthly. 

3. Degree of competition (quarterly). 

4. Even distribution of contract awards within the fiscal year 
plus variances (quarterly) . 

5. Major contracts over $500,000 (semi-annual). 

6. Close-out of contracts (quarterly). 

One of the principal objectives CMB achieved during FY '79, was to 
spread the workload more evenly through the fiscal year by shifting 
contract renewal dates and new contracts as equally as possible in 
each quarter. 

It is estimated that there will be 190 active contracts with a FY '79 
allocation of $16,000,000. 



13-1 



II. GRANTS MANAGEMENT BRANCH 

Fiscal Year 1979 was a year of considerable hardship for the GMB as 
the Branch lost the equivalent of one full-time professional and 
one half-time administrative/award clerk, resulting from vacancies 
left unfilled for appreciable periods of time. Nonetheless the 
Branch was responsible for approximately 1,700 awards with a dollar 
value in excess of $115,000,000 during this reporting period. The 
GMB is responsible for the fiscal and administrative management of 
all grants and awards issued by the NIAID. The GMB works very closely 
with the programmatic divisions and provides the fiscal and administrative 
expertise necessary to more effective program management. The GMB also 
serves as an interpreter of grant policy and procedure issued by the 
several echelons within the DHEW. 

There were many policy and procedural changes that impacted 
significantly on the operations of the Branch. Perhaps two of the more 
significant changes were, 1) the loss of the categorical Report of 
Expenditures, which will be replaced by the non-categorical Financial 
Status Report; and 2) in a more favorable change, the initiation of the 
new project-period concept which, simply stated, enables the awarding 
component to carryover unexpended direct cost funds from one project- 
period to another, and/or, to use the total unexpended direct cost 
balance from one project-period as a funding offset to another. 

The staff of the GMB has made significant contributions as members and 
chairpersons of policy and procedure work groups, committees and 
subcommittees both at the NIAID and at the NIH. Examples of such 
activity include: NIH grants management self -appraisal, NIH change of 
institution policy, PHS grants policy statement, refinement of procedures 
for development of recommended grant budgets on those grants assigned to 
PPRB (NIAID) for competitive review, and various other grant policy 
and procedure drafting committees. Additionally, Branch personnel 
have participated as faculty members or attendees at seminars where 
grantee personnel were in attendance, such as the NIH-AAMC Workshop 
on Business Affairs, the AAMC-group on Business Affairs-Continuing 
Education Program, and the several Grants Management Workshops conducted 
by the GMAC for grantee institutions and NIH personnel alike. 

Generally speaking the Branch performed very well in FY '79, even 
under the most adverse circumstances. As a service-oriented Branch 
whose "cradle-to-grave" involvement in the NIAID encompasses all grant 
programs, it is the goal of the GMB to more fully develop the partnership 
role that exists between the management specialist and their health- 
scientist counterpart, enhance the interaction between the management 
specialists and the grantee community, and to encourage and foster 
greater participation of the entire GMB staff in the "total" NIH 
grants management arena. 



14-1 



III. PROGRAM ANALYSIS AND EVALUATION BRANCH 

The title "Program Analysis and Evaluation Branch" replacing "Review 
and Evaluation Branch" indicates a branch emphasis on development of an 
analytical base to develop reports and support the Office of the Director, 
NIAID Program Directors, and the NIAID staff data and information 
requests. A new management data base is being developed by branch 
personnel augmented by three one-time non-tenured personnel. All 
competing projects (996) and non-competing applications (1,812) will 
be entered into the new multi axis coding system, MACS. 

The Branch serves as the focal point for management and budgetary 
data for Extramural programs; referral liaison with the Division of 
Research Grants, ADP Liaison with other Institutes and NIH, and 
reporting of non-program specific reports such as Prevention and 
Toxicology. The Branch was able to add age parameters to the revised 
PHS #398 (the grant application form) for inclusion in the MACS system 
and thus eliminate a search for this data. 

A. Fiscal Management and Analysis Section 

In FY 1979, the Fiscal Management and Analysis Section developed 
detailed operating plans for the use of all research and training funds 
and implemented the final plans. Awards were paid through both "Central" 
and "Program" accounts to assure payment of the highest quality research 
projects and programs of special interest to the aims of the Institute. 
Tables V and VI show final operating plans of EAP, LAID, and MID for 
contracts and grants in FY '79. 

Due to the increase in the overall appropriation for research grants in 
FY '79, NIAID was able to pay about 43% of all research grant approvals 
and 61% of the approved career/academic award applications. In the 
absence of an appropriation for training and fellowships, this budget was 
subject to the conditions of a continuing resolution. Under this 
resolution, NIAID was able to fund 47% of approved individual fellowship 
requests and 24% of approved training applications. 

Requests for fiscal reports increased significantly during the year 
to over three reports per week. Several studies were also undertaken 
by this Section to assess the impact of proposed changes in NIH policy. 
One such study, "Thinner Grant Applications on Longer Awards", was 
cited by NIH as an important factor in making their final decision. 



15-1 



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15-3 



B. Program Analysis Section 

Since October 1978, the efforts of the Program Analysis Section have 
been almost exclusively directed toward an orderly and timely transition 
from the old scientific classification system (three-digit code) to the 
new multi-axis coding system (MACS). MACS is a complex four-digit 
hierarchial coding system consisting of four main axes; 

Axis I - Organisms/Diseases/Study Area 

Axis II - Approaches/Methodologies 

Axis III - Anatomical Systems/Organs 

Axis IV - Hosts 

The scientific information in every active and pending grant application 
received in the NIAID, including all research and training mechanisms, 
are presently being reviewed and coded into the above system. Other 
areas of special interest which are also identified include: 

1. Trans-NIH health problems (arthritis, blood-related 
diseases, bronchopulmonary disorders, digestive 
diseases, etc.); 

2. Human subjects (clinical or non-clinical environment); 

3. Special groups of human subjects (minors, pregnant women, 
etc. ) ; 

4. Age of subject; 

5. Objectives of research/training (diagnosis, prevention, 
treatment, etc.); 

6. R&D conducted at NIH (NIAID): Basic, Applied, and 
Development; 

7. Entire spectrum of R & D activities at the NIH (NIAID): 
^cience base, Applications, Transfer, and Training, Clinical 
trials, one part of Applications in SATT, is- also included 
under Development. This information forms the basis of data 
which is developed into the Annual Inventory of Clinical Trials. 

Analysis of the information captured by this newly developed intricate, 
multi-faceted coding system, coordinated with our assignment of all 
grants/grant applications to one of twenty-four program areas within 
the MIDP/IAIDP will be used to answer, more efficiently and accurately, 
repetitive and non-recurring types of queries from within NIAID 
(scientific management information for program planning and evaluation) , 
NIH (e.g., Trans-NIH, BAD/ SATT) , members of Congress (problems of 
special legislative concern, e.g., Cystic Fibrosis, Sexually-Transmitted 
Diseases), other government agencies, and the public. 

15-4 



C. Data Control Section 

The Data Control Section, using the NIH and Parklawn computer facilities, 
reports management, budgetary, and programmatic activities for the 
Extramural Activities Program. In addition, special and repetitive 
reports are provided for the Immunology, Allergic and Immunologic 
Diseases Program and the Microbiology and Infectious Diseases Program. 

During fiscal year 1979, a new scientific information data base has 
been captured into NIAID's computer records and the system is nearing 
completion. With this implementation, computer generated reports are 
particularly explicit and will minimize selective interpretation in 
responding to requests for scientific data. The system allows for 
determining percentages of support in given areas, for retrieving 
TRANS-NIH and Programs of Institute Emphasis as identified by the 
Program staff involved. The new system has the capability of reporting 
areas of Basic, Applied, Developmental Research, and Scientific, 
Application, Training, and Technology Transfer in response to NIH 
concern. 

The ongoing Contract system of data capture and retrieval of NIAID 
contract information services the contract office, budget office, 
O.D.'s office and continues to be well received. 



15-5 



IV. PROGRAM AND PROJECT REVIEW BRANCH 

A. Introduction 

In FY 1979, there was an enormous increase in the workload of the Branch, 
primarily involving scientific review by the Microbiology and Infectious 
Diseases Advisory Committee (MID). It was estimated that the MID review 
workload alone was at least 2.2 times the optimal workload of an average 
DRG Study Section. The heavy workload was a direct consequence of the 
release of numerous RFAs and RFPs by the MIDP in anticipation of a good 
budget in 1979 and a lean budget in 1980. In the March meeting alone a 
total of 68 program project, exploratory-developmental, and training 
grant applications were reviewed, as were 55 new competing contract 
proposals in response to 6 RFPs and one PL 480 proposal. It would have 
been humanly impossible for one executive secretary to have handled the 
review load. The Committee was therefore divided into subcommittees, 
each to review a bloc of grants or proposals, and then to meet on the 
fourth day as a full Committee for en bloc actions. This worked out 
reasonably well, with commendable teamwork and cooperation among review, 
grants management and contracts management, and other EAP staff. The 
Committee Chairman performed an outstanding job in chairing the two 
separate sessions of the full MID as well as two subcommittees. Still 
it is to be hoped that in the future more prudence be exercised in 
programming efforts that result in nearly unmanageable workloads for 
both EAP review staff and the Committee. 

The Allergy and Clinical Immunology Research Committee and the 
Transplantation Biology and Immunology Committee both had reasonable 
review workloads and gave advice to the IAIDP on a variety of program 
issues. The AIRC advised on increased flexibility in acceptance of 
Allergic Diseases Academic Award (K07) applications. It also indicated 
that site visits should be conducted in a timely manner. 

The increased workload of the MID impacted on the Review Services Unit, 
which serves the dual purpose of serving both the Council and the Review 
Committees, preparing the appropriate books of summary statements or 
applications and proposals for extramural staff and Council or Committee 
members . 

Through all these, the Branch has been operating with a skeleton staff. 
A detailed manpower analysis of the Branch indicates that there is a 
very real need for an additional full-time (assistant) executive secretary 
as well as a clerk-typist to handle the workload in MID. The assistant 
executive secretary need not occupy a slot but may be a consultant. 
There is a need for another part-time worker in the Review Services Unit, 
or for one of the current part-time employees to be converted to full-time. 
Unless these needs are met in the immediate future, the quality of 
scientific review and the service offered by the Review Services Unit 
will suffer. 



16-1 



B. Allergy and Clinical Immunology Research Committee 

The AIRC met three times during the period of November 1978 through 
September 1979, reviewing two program projects, four allergic disease 
center applications, seven training grant applications, and two Allergic 
Diseases Academic Award applications for a total first year requested 
amount of $1,914,788. In addition, an ad hoc contract proposal review 
was conducted on an amount of $2,265,794 for a two-year period. The 
Committee gave advice to the NIAID on policies regarding the Allergic 
Diseases Academic Award and on the program project mechanism. On the 
AADC program, the Committee advised on a much more flexible policy, 
which would allow multiple applications from multi-campus institutions 
and their clinical affiliates to be awarded. It is hoped that this 
liberal policy would move the program ahead. If the policy is accepted 
by Building 1, this would mean an escalation of the review activities of 
AIRC in the future. 

Recommendations were made on program activities including food allergy, 
drug allergy, allergens, and fungal antigens. Concept clearance was 
given on the production and evaluation of horseradish-peroxidase-labelled 
antihuman globulin as an ELISA standard. 

C. Transplantation Biology and Immunology Committee 

The TIC met three times from November 1978 to July 1979, reviewing three 
training grants, one unsolicited contract proposal and four contract 
proposals submitted in response to an RFP for a total first year requested 
amount of $651,253. In addition, an ad hoc contract proposal review was 
conducted on a first year amount of $964,804. The Committee gave 
advice to the NIAID on the analysis and publication of the kidney 
transplant histocompatibility study data, organization and policies of the 
serum bank, and the formation of a hybridoma banking and distribution 
system. It gave recommendations on the development of RFPs for 1) design 
and evaluation of clinical tray configuration, 2) evaluation of alternative 
tissue typing techniques, and 3) absorption of anti-DR antisera, and 
RFAs for 1) post-transplant immunologic monitoring and 2) the role of 
non-MHC antigens in transplantation. 

Dr. Harley Sheffield joined the Branch in FY '79 and has served as 
Executive Secretary of both the AIRC and the TIC. He, along with other 
professionals in EAP, has acted as executive secretary on ad hoc reviews 
of contract proposals within the IAIDP as well as the MIDP. The review 
workload of the AIRC/TIC is summarized in Table VII. 

D. Microbiology and Infectious Diseases Advisory Committee 

The Microbiology and Infectious Diseases Advisory Committee met four 
times from November 1978 through September 1979, reviewing 101 grant 
applications (39 program projects, 39 exploratory developmental grants, 
23 training grants) for a first year requested amount of $24,964,364 
direct costs; and 109 new competing, renewal, unsolicited, and sole 
source contract proposals for a total requested amount of $55,924,888 



16-2 



(Table I). Of the 39 program projects, two (STD program) were in fact 
reviewed by special review committees and site visited; the others 
(ICIDRs, mycology fungal disease research, arthropod-borne viral 
infections and tropical disease research units, and an unsolicited program 
project) were not site visited, but an ample number of consultants expert 
in the specific fields participated in the review and attended the 
meetings. Of the 109 contracts, 71 were reviewed by the full MID and 38 
by ad hoc review groups with two committee members participating in 
each review. Of the 38 reviewed ad hoc, 12 were non-competing renewals 
and 26 were new competing proposals in response to one RFP of which one 
was from an MID Committee member. 

Advice and concept clearance was sought from the full Committee on the 
following initiatives: Rocky Mountain Spotted Fever (epidemiologic 
studies, animal model), the role of Streptococcus viridans in bacterial 
endocarditis, penicillin prophylaxis by group B streptococcal sepsis in 
the newborn, and potentiation of immune responses to vaccines. Except 
for the last item, these have been implemented by RFPs and the proposals 
reviewed by the Committee. 

E. Review Services Unit 

For Council-related work, the Unit processed 2,045 applications and 
their respective summary statements for FY 1979. This compares with 
2,049 applications and respective summary statements for FY 1978 
(Table II). Although this difference is insignificant, Committee-related 
work has increased the volume of work for the RSU during this fiscal 
year, the applications processed increasing from 173 in 1978 to 222 in 
1979, an increase of 28% (Table VIII). All grant applications for program 
projects, Centers, training and Allergic Diseases Academic Awards as 
well as all new contract proposals reviewed by the three NIAID Committees 
are processed by the Unit, with books made, reviewed for conflict of 
interest, and mailed to consultants as well as distributed to concerned 
staff. As was stated previously, the heavy workload of the MID impacted 
on the work of the Unit. 

F. Clinical Trials 

The reporting to DRG of clinical trials active in 1977 and 1978 was 
completed on June 5, 1979, far beyond the initial deadline set by DRG. Of 
the 128 trials reported, 51 were from the IAIDP, 65 from MIDP, and 12 from 
the intramural programs . The MIDP reported 14 new trials supported by 
contracts and 4 supported by grants. The IAIDP reported 4 new trials 
supported by grants and none supported by contracts. According to DRG, 
a formal publication of 1978 trials will be made (but not of the 1976 
and 1977 trials, although printouts of data are and can be made available 
respectively). It is noted that the number of trials reported in 1976 
was 142. The reasons for the seeming drop in number, from 142 to 128, 
could be the transitional quarter in 1976; or because of possible uneven 
reporting by program staff of grant-supported trials. Whereas in the past, 
a member of the Program Analysis Section screened the grants and identified 
clinical trials, in consultation with the NIAID Clinical Trials Coordinator, 
this year program staff have been asked to undertake this activity. 

16-3 



TABLE VII 
REVIEW WORKLOAD, PPRB, 1978-1979 



Category 



1978 
Number Amount 



1979 
Number Amount 



AIRC/TIC/AD HOCS 

Grant Applications 59 

Contract Proposals 45 
Subtotal 
MID/AD HOCS 

Grant Applications 

Contract Proposals 
Subtotal 106 

CONTRACTS FOR OSD, OTHER 5_ 

TOTAL 215 



$13,079,077 
13,550,639 



18 
24 



104 

49 
57 



12,594,364 

16,342,567 



42 



101 
109 



6,111,861 



210 

5 



257 



Grant amounts are first year direct costs only. 
Contract amounts are total costs. 



TABLE VIII 
REVIEW SERVICES UNIT WORKLOAD 



$2,409,163 
5,835,232 



24,964,364 
55,924,888 



4,861,363 



Categorv 



1978 



1979 



For Council: 

Applications Received 
"Pink Sheets" Received 

For Committees: 

Applications Processed 
Proposals Processed 
Total Processed 



2,049 
2,049 



108 
65 

173 



2,045 
2,045 



119 

103 
222 



16-4 



V. RESEARCH RESOURCES BRANCH 

A. Special Activities 

ASM Exhibit - The Research Resources Branch handled arrangements for an 
NIAID exhibit at the American Society for Microbiology Meeting held on 
May 5-8, 1979, in Los Angeles, California. The RRB made the arrangements 
to take the Institute exhibit to Los Angeles while manpower used during 
the exhibit hours was supplied by the Office of Research Reporting and 
Public Response. Additional manpower was volunteered from the NIAID 
Extramural Activities Program, Microbiology and Infectious Diseases 
Program and from Intramural Laboratories. The exhibit was modified 
from the exhibit previously shown at ASM. An NIAID exhibit of different 
origin was repainted and new pictures and video tape installed to 
present the activities currently being carried out by NIAID. The 
exhibit was well received and gave many visitors knowledge of the 
various activities being supported by the Institute. 

Dr. Robert Byrne has continued to serve as Branch Chief with Sylvia 
Cunningham responsible for management of operations including the 
distribution and cataloging of the microbial, allergen and some of the 
immunological reagents. 

The need for well characterized reference reagents as an adjunct to 
research is well recognized, and in support of the concept, the Research 
Resources Branch (RRB) conducts a program which distributes a wide 
range of reagents to research scientists. Reagents for which a critical 
need exist are identified and arrangements for their production are 
made by the pertinent Institute Program area. The RRB then arranges 
for the packaging of the bulk products, catalogs the item and distributes 
the reagent with the information and technical advice on reagent 
characteristics and use. The Branch is also sponsoring a contract for 
the development of a procedure for the processing and packaging of 
recombinant DNA Vector-Host Systems. In addition, the Branch assists 
with the contract for the establishment of a Plasmid Reference Center. 

B. Asthma and Allergic Diseases Program-Allergen Reagents 

A procurement contract has been awarded to the American Type Culture 
Collection to process the minor determinant penicillin products. The 
RRB has made all the arrangements to have these products packaged and 
lyophilized. The actual processing is expected to begin the week of 
July 30; it is expected that approximately 4 months will be required 
to complete all lots of material. After an IND is submitted, these 
products will be supplied to approximately six contractors who will be 
responsible for conducting clinical trials to ascertain the penicillin 
sensitivity of patients. 

The contract with Johns Hopkins University for the preparation of 
polyvalent ryegrass antiserum was completed this year. This material 



17-1 



has been processed by the American Type Culture, undergone certification 
testing by Mayo Clinic and is now available for distribution. 

Although the contract located at University of Pennsylvania for the 
establishment of an allergic dog colony is sponsored by IAIDP, the 
Chief, RRB has continued to serve as a project officer on this contract. 
The contract has been highly productive in the breeding of dogs and 
is ahead of schedule in producing progeny of atopic breeding stocks. 

In addition to the activities listed above, the Branch has continued 
to distribute allergenic products such as Ragweed Antigen E, K, and 
RA3, Ryegrass I, II, and III and venoms of honey bee, yellow hornets, 
white-faced hornets, yellow jackets, paper wasps and hymenoptera venom 
diluent; these venom products are available for distribution under an 
IND as well as for in vitro use. 

C. Immunological Reagents and Resources 

The new contract awarded to Flow Laboratories during FY 1978 for the 
Maintenance and Breeding of Rabbits of Known Genotype for Use in 
Immunological Studies has continued to serve a useful purpose in 
maintaining a colony of 500 specially bred rabbits for use in NIAID 
immunological studies. In addition, these facilities provide reference 
quantities of reagents and limited numbers of rabbits to other 
responsible investigators doing immunological studies. During this 
period, seven investigators have been supplied with a total of 18 
rabbits; twenty-six shipments of rabbit antisera have also been made 
to other investigators. 

During this period, the rabbits had to be moved from their building in 
Rockville to make room for the new Metro station. The rabbits are 
currently being housed in Fairfax, Virginia, in a building being leased 
by Flow from Litton-Bionetics . Flow expects their new animal building 
which is being built in McLean, Virginia, to be completed in September; 
the animals will be moved again when the new building is ready for 
occupancy. 

D. Microbiology 

During the latter part of FY 1977, two contracts were awarded and have 
continued during FY 1979. The University of Alabama is performing 
research and developmental work to determine optimal conditions for 
the preservation, retrieval, storage and distribution of fragile 
bacterial host strains. Three basic methods of storing have been 
studied, i.e., preservation in 1) paraffin-sealed agar stabs, 2) by 
freezing in peptone-glycerol broth and 3) by lyophilization. Results 
to date indicate that lyophilized material stores better at -30°C or 
-70°C than at 4°C or room temperature. The peptone-glycerol broth 
freezing method appears to cause some reversion frequencies of some 
products. This problem is now being carefully analyzed; similar 



17-2 



studies are being conducted on the lyophilized materials to determine 
if the reversion frequencies are also occurring in these samples. 
The MIDP is sponsoring the Stanford University contract for the 
establishment of a plasmid reference center. During the year a 
modification was made to the contract work scope to allow the contractor 
to continue storing and shipping the bulk cultures. RRB will therefore, 
not be responsible for the packaging, storing and distribution of the 
strains collected under this project. However, backup set of these 
strains are being maintained by RRB (to date 90 sets have been 
received for storage). In addition, RRB will assist with the 
publication and printing of the Catalog of Plasmid Strains once a 
suitable format has been developed. To date, the format for the strains 
data sheet has been revised three times. 

E. Research Resources - Research Reagents 

While responsibility for the actual production of reagents is with the 
various Institute program areas, RRB has continued to sponsor certain 
service type contracts which serve the needs of the various program 
elements . 

The Ohio State University has continued to serve as the viral reagent 
testing laboratory. In addition to verifying feedback data from 
reagent users concerning product viability, the contractor has verified 
certain results obtained by the American Type Culture Collection on 
the reagent transfer contract. During this year, the contractor has 
conducted tests on 15 rhinovirus seeds and antisera, 4 enterovirus 
seeds and has confirmed the contamination of reovirus type 1 with SV40. 
The contractor is now initiating cross-neutralization testing on 
adenovirus types 10 and 13 to see if he can verify a report from a 
reagent user that our adenovirus type 13 rabbit serum neutralizes 
adenovirus type 10. 

After a category of reagents has become established, and the area 
of research that it supports is well defined, it is of questionable 
value for RRB to continue storing and distributing that category of 
reagents for a prolonged period of time. During FY 1975, it was 
determined that four viral reagent groups fall into this category. 
RRB therefore, entered into a five year contractual arrangement with 
the American Type Culture Collection (ATCC) to transfer the enterovirus, 
adenovirus, rhinoviruses and arboviruses reagent collections to the 
ATCC whose prime function is to store and distribute these types of 
reagents. 

Under the contractual arrangement with ATCC, samples of each reagent 
type will be preserved as a museum function. The viability or 
homologous antibody activity of a parallel collection of reagents 
will also be assayed. After each viral group has been assayed and the 
results reported and reviewed, RRB will discontinue that reagent group 
from its catalog with a notation that they are available from ATCC 
and all remaining stock of that group of reagents will be transferred 



17-3 



to ATCC for its custodianship. NIH scientists and NIAID contractors 
will be provided reagents without cost until RRB supply is depleted. 

During the first four years of this project, 50 samples of each reagent 
type for the four viral reagents groups have been transferred to ATCC 
for the museum function. The assay of the enterovirus, adenovirus and 
rhinovirus groups has been completed; these three groups have now been 
transferred to the ATCC. During FY 1979, ATCC has primarily been 
involved in the testing of the arbovirus reagent collection which 
is the last of the four collections to be re-assayed before being 
transferred to ATCC. The schedule for assaying and transferring 
reagents originally prepared in 1975 is still as projected; it is 
planned that the assay of arbovirus reagents will be completed during 
FY 1980 with the inventory of arbovirus reagents being transferred to 
ATCC upon completion of review of the data. 

The Branch has also been involved in assisting with plans to have the 
rotavirus reagents labeled and packaged. These materials are being 
prepared under an Interagency Agreement with Plum Island, USDA which 
is being supported by MIDP. 

The Branch has also given assistance with the preparation of other 
contract documentation, such as work-scope, reporting requirements, 
evaluating criteria, etc. , to have additional arbovirus reagents 
prepared. During the latter part of FY 1979, an interagency agreement 
will be made with the USDA for the preparation of an arbovirus grouping 
fluid comprised of African swine fever, bovine ephemeral fever, 
African horse sickness and Rift Valley fever. During FY 1980, requests 
for proposals will be issued to have other arbovirus reagents prepared. 
The reagents resulting from these projects will ultimately be received 
by RRB for processing (if necessary), cataloging and distribution. 

Prior years of intensive work have resulted in completed work on the 
enterovirus, adenovirus, rhinovirus, myxovirus and the agents and 
antigens of hepatitis A & B. In most cases, seed virus preparations 
and corresponding antisera are now available for most of the viruses 
of public health interest. The reagents which have resulted from 
the various projects have been most useful, particularly the 
hemagglutinins, neuraminidases, ribonucleoproteins and the matrix 
proteins of the influenza viruses of man and animals. Since completion 
of this production effort in FY 1978 and listing in the 1978 - 1980 
Catalog of Research Reagents, these materials have become a very 
popular distribution item. Information received thus far from the 
reagent users indicate that these materials are very valuable. 

F. Molecular Anatomy Program 

The Molecular Anatomy Program (MAP) during FY 1979 has been evaluating 
for safety and efficacy in human vaccine trials, subviral forms of 
hepatitis B surface antigen (HBsAg) , purified from the plasma of chronic 
carriers and inactivated. Thus far, limited studies in volunteers have 
shown the vaccines to be free of infectious hepatitis B virus and local 



17-4 



and systemic side reactions. MAP is currently evaluating different 
vaccine formulas and inoculation schedules in volunteers to optimize 
the humoral anti-HBs response. Four vaccine lots have been prepared 
from the original inactivated HBsAg/ adw preparation; (1) untreated, 
aqueous, (2) untreated, alum-adsorbed, (3) ether-tween 80 treated, 
aqueous, and (4) ether-tween 80 treated, alum-adsorbed. The four 
lots passed safety tests and are currently being evaluated in volunteers 
at the NIH. A large lot (1000 doses) of the untreated, alum-adsorbed 
vaccine and an alum placebo were also prepared for more extensive 
testing by Dr. Hollinger at Baylor Medical School under an NIAID 
contract; the vaccine and placebo lots are currently on safety tests. 

The hybridoma technology offers great promise for analyzing the antigenic 
determinants of HBsAg. Hybridomas from spleen cells of Balb/c mice 
immunized with HBsAg/ adw and mouse myeloma cells, P3 X 63Ag8, have been 
developed. 

Many laboratories are now engaged in efforts to detect antigen-antibody 
systems related to the non-A, non-B hepatitis agents. MAP has involved 
in defining the delta antigen and its antibody (6/anti- 5). This 
specificity is found in carriers of HBsAg and appeared to be related to 
the HBV system. A sensitive RIA for anti-6 has been developed and 
found predominately in chronic HBsAg carriers, although a few 
asymptomatic carriers were also positive for anti-6; low and transient 
titers of anti-6 were found in acute HBV hepatitis patients. The 
evidence to date indicates that 6 represents a marker of an agent, 
not HBV, which requires HBV infection to provide certain helper 
functions for its replication. 

The eastern woodchuck ( Marmota monax ) has an HBV-like virus and this 
animal may represent an inexpensive model of virus-induced liver 
disease including hepatocellular carcinoma. MAP has examined approximately 
50 wild-caught animals and based on endogenous DNA polymerase activity 
and virus-like particles, approximately 30% are chronically infected 
with this virus. We showed that the woodchuck virus cross-reacts with 
HBV in both HBsAg and HBcAg components. Two of the carrier woodchucks 
have recently been diagnosed with hepatocellular carcinoma. Further 
evaluation of the woodchuck as a model of HBV infection and disease 
will continue. 

Efforts in respiratory syncytial virus research have concentrated on the 
isolation and identification of surface antigens of the virus. The 
known instability of the virus has been a major problem. However, 1 M 
MgS04 has been very effective in stabilizing the infectivity of the 
virus throughout the purification procedures. Large-scale isolation 
of virus under stabilizing conditions are now in progress. Several 
lines of mouse Balb/c cells have been established that are persistently 
infected with RSV. These cells produce low levels of infectious 
virus, contain large amounts of ribonucleoprotein and express RSV- 
specific antigens on the cell membrane. These cells will be useful in 
the development of murine hybridomas for the production of monoclonal 
antibodies to surface antigens of the virus. 



17-5 



G. Processing and Distribution 

In addition to the various program elements detailed above, the Research 
Resources Branch also distributes coronaviruses , herpes viruses, 
interferons, mycoplasmas and reoviruses. 

Due to the limited number of reagents to be processed during FY 1979, 
there was not a formal research and development contract to handle 
this activity. However, the American Type Culture Collection did 
process four (4) mycoplasma seeds and one antiserum lot by the 
purchase order mechanism. 

The Research Resources Branch reagent collection (exclusive of the 
viral reagents transferred to ATCC) now consists of over 650 individual 
reagents. The repository and distribution contract remains at Flow 
Laboratories. During this period, the repository activity was moved 
from its Rockville, Maryland, to a new building in McLean, Virginia. 
A tabular record of distribution by this facility since FY 1972 follows: 

DISTRIBUTION OF VIRAL, MYCOPLASMAL, 
AND ALLERGEN REAGENTS 



Fiscal Year 

1972 

1973 

1974 

1975 

1976 

1976 (TQ) 

1977 

1978 

1979 (3/4 year) 





Total amps. & vials 


Total Transactions* 




Distributed 


572 




21,801 


575 




19,181 


500 




9,932 


592 




6,751 


762 




10,188 


192 




3,126 


613 




7,633 


605 




6,851 


561 




6,907 



*Does not include the shipments made for testing and packaging purposes 
and reagents transferred to the American Type Culture Collection. 

In addition to the shipments tabulated above, Flow has also made 141 
shipments for DAB, 28 shipments consisting of whole rabbits or rabbit 
serum and 30 interferon shipments for the National Cancer Institute. 
All of this was conducted under the NIAID contract with the NCI 
contributing funds to NIAID for their activities. In addition, 
American Type Culture Collection has made the following shipments under 
the reagent transfer contract: 

(U.S. Investigators and WHO Laboratories) 

# of Shipments # of Ampoules 

FY 1978 96 1,622 

FY 1979 (3/4 year) 87 1,068 

17-6 



During this year, the printing of the new 1978 - 1980 Catalog of 
Research Reagents was completed by the Government Printing Office. 
Over 1900 of the 2200 copies printed have been mailed out to reagent 
users. 

The Branch has continued its efforts to publicize the availability of 
these valuable reagents to the scientific community. The availability 
of reagents was incorporated into the NIAID exhibit which was shown 
at the American Society for Microbiology meeting held in Los Angeles, 
California. 

The following two (2) tables describe the distribution of reagents by 
groups and also by institutional affiliation during the first three 
quarters of FY '79. 



17-7 



RESEARCH RESOURCES BRANCH 

DISTRIBUTION OF SPECIFIC VIRUS GROUPS 

FY 1979 (3/4 year) 

OCTOBER 1, 1978 - JUNE 30, 1979 



// of Items 
Avail. (% of 
Inventory) 


Virus Group 


SHIPMENTS 
If of % of 


it of 


AMPOULES 
% of // 
Distribution 


9***(1.78) 


Adenovirus 


3 


.5 


18 




.3 


17 


(3.36) 


Allergen 


81 


14.4 


1345 




19.5 


224 


(44.27) 


*Arbovirus 


43 


7.7 


647 




9.4 


4 


(.79) 


Coronavirus 


3 


.5 


9 




.1 


20 


(3.95) 


Recombinant DNA 


27 


4.8 


244 




3.5 


20 


(3.95) 


**Enterovirus 
(Typing Pools) 


80 


14.4 


2294 




33.2 


18 


(3.56) 


Hepatitis 


40 


7.1 


240 




3.5 


18 


(3.56) 


Herpes 


2 


.4 


3 




.1 


5 


(.99) 


Interferon 


133 


23.7 


237 




3.4 


86 


(17.00) 


Mycoplasma 


49 


8.7 


837 




12.1 


79 


(15.60) 


Myxovirus 
Paramyxovirus 
and related 


63 


11.2 


575 




8.3 


6 


(1.19) 


Reovirus 


8 


1.4 


37 




.5 






Combinations 


29 


5.2 


421 




6.1 



Figures based on 561 shipments and 6907 ampoules shipped (excludes testing 
packaging and reagents transferred to ATCC). 

* Included in this figure are 11 shipments of Arbovirus Grouping fluids 
totaling 201 ampoules. 

**Included in this figure is 1 shipment to a WHO Laboratory totaling 
1130 ampoules . 

***Seed and antisera for adenovirus types 1 thru 31 have been transferred 
to the ATCC. 



17-8 



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17-9 



H. Individual Contract and Agreement Tabulation 

PROGRAM: IMMUNE SYSTEM AND DISEASE - REAGENTS AND RESOURCES BRANCH: RRB 



Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



Objectives and Findings 



Sullivan, Rudolph R. 
AI 82565 

Flow Laboratories 
$158,700 



To maintain a genetically bred 
rabbit colony of known genotype 
for use in immunologic 
investigations. A maximum of 
500 adult rabbits will be bred 
and maintained by expert 
veterinary care. This is for 
support of high priority research 
on genetic control of the immune 
response and to supply reagents 
and rabbits to other 
investigators . 



17-10 



Individual Contract Agreement Tabulation 



PROGRAM: INFECTIONS - VIRAL REAGENTS 

Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



BRANCH: RRB 



Objectives and Findings 



Hughes , John R. 
AI 62513 

Ohio State University 
$17,313 (FY 1978 Funds) 



This laboratory serves as a 
shelf-life viral reagents 
testing laboratory for members 
of the enteroviruses, rhino- 
virus, adenovirus, myxovirus 
and herpevirus groups. This 
contract will ensure the 
integrity of these reference 
standard reagents by assessing 
the potency and purity of viral 
reagents after long term storage. 
This contract will terminate 
this year. 



17-11 



Individual Contract Agreement Tabulation 
PROGRAM: INFECTIONS - MOLECULAR ANATOMY PROGRAM BRANCH: RRB 



Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



Objectives and Findings 



Gerin, John 

AI 82572 

Georgetown University 

$928,952 



The major objective is to develop and 
apply methods of isolating viruses and 
viral antigens and the application of 
those techniques to research on 
associated diseases. During FY 1979, 
this laboratory has been involved in 
purifying and inactivating subviral 
forms of hepatitis B surface antigen 
(HBsAg) from the plasma of chronic 
carriers. These materials are currently 
being evaluated for safety and efficacy 
in human vaccine trials. Limited 
studies in volunteers have shown the 
vaccines to be free of infectious 
hepatitis B virus and local and 
systemic side reactions. 

The contractor has also developed 

hybridomas from spleen cells of Balb/c 

mice immunized with HBsAg/ adw and 

mouse myeloma cells, P3 X 63Ag8. 

He also has been involved in defining 

the delta antigen and its antibody 

(6/anti-5). This specificity is 

found in carriers of HBsAg and 

appears to be related to the HBV system. 

Evidence to date indicates that 6 

represents a marker of an agent, not HBV, 

which requires HBV infection to provide 

certain helper function for its 

replication. 

The contractor is also now involved in 
the large-scale isolation of RSV under 
stabilizing conditions which have 
been developed. 



17-12 



Individual Contract Agreement Tabulation 
PROGRAM: INFECTIONS - PROCESSING AND DISTRIBUTION BRANCH: RRB 



Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



Objectives and Findings 



Stanley, Thomas 

AI 82542 



Flow Laboratories 
$184,506 (MID) 
$276,758 (IAIDP) 



When the reagents program was given 
the responsibility for production 
of reference reagents, other responsi- 
bilities were also included, i.e., 
the development and maintenance of 
appropriate means to receive, process, 
store and distribute these research 
materials. The contractor fulfills 
these responsibilities. During the 
past year, the contractor has been 
supplying the storage and shipping 
services for the RRB, Developmental 
Applications Branch, the Enteric 
Diseases Branch and the NCI. As 
a result of these combined activities, 
the contractor has made 2548 
shipments totaling 106,614 vials + 
38,670 trays and 570 shipments of 
bulk material. Approximately 95 
incoming shipments have been handled. 



17-13 



Individual Contract Agreement Tabulation 
PROGRAM: INFECTIONS - PROCESSING AND DISTRIBUTION BRANCH: RRB 



Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



Objectives and Findings 



Clark-Curtiss, Josephine 
AI 725333 

University of Alabama 
$171,657 (FY 1977 funds) 
Contract awarded for a 
3-year period 



This contractor is determining the 
optimal processing, packaging 
and storage conditions for vector- 
host systems for recombinant DNA 
molecule research. To date, the 
contractor has investigated 
various methods of preserving 
these materials. The methods 
employed to date include 1) 
preservation in paraffin - sealed 
gelatin slabs 2) preservation by 
freezing in peptone - glycerol, 
preservation by lyophilization. 
In addition, a study is being 
conducted comparing the effects 
of various storage temperatures. 



17-14 



Individual Contract Agreement Tabulation 
PROGRAM: INFECTIONS - PROCESSING AND DISTRIBUTION BRANCH: RRB 



Principal Investigator 
Contract Number 
Contractor 
FY '79 Obligations 



Objectives and Findings 



Donovick, Richard 

AI 75-2526 
American Type 

Culture Collection 
$149,823 



After a category of reagents has 
become established and the area 
of research that it supports is 
well defined, it is of questionable 
value for RRB to continue storing 
and distributing that category 
of reagents. Therefore, in FY '75, 
RRB entered into a cooperative 
enterprise with ATCC to transfer 
the enterovirus, arbovirus, 
rhinovirus and adenovirus reagent 
collections to the ATCC for their 
custodianship and distribution; 
complete transfer of the four 
viral reagent groups will be accom- 
plished over a five-year period. 
During the first three years of 
the contract, 50 samples of each 
reagent type for the four viral 
reagent groups have been transferred 
for museum storage purposes. 
Under the terms of the contract, 
each of the four collection of 
reagents is to be assayed for 
viability or homologous antibody 
before the complete transfer is 
made, therefore, the contractor 
has been assaying the enterovirus, 
adenovirus, rhinovirus groups 
during the initial period. The 
assay of these groups has been 
completed, the test results have 
been reviewed, and the total 
enterovirus, adenovirus, rhinovirus 
collections have been transferred to 
ATCC. The contractor is now 
performing the required testing on 
the arbovirus reagents . 



17-15 



CONTRACT PUBLICATIONS 

Gerin, J. L. and J. W. -K. Shih. Structure of HBsAg and HBcAg. In : 
Viral Hepatitis (Eds., G. N. Vyas, S. N. Cohen and R. Schmid) . pp. 
147-153. Franklin Institute Press, Philadelphia. 1978. 

Feinstone, S. N. , Y. Moritsugu, J. W. -K. Shih, J. L. Gerin and 

R. H. Purcell. Characterization of hepatitis A. virus. In: Viral 
Hepatitis (Eds., G. N. Vyas, S. N. Cohen and R. Schmid). pp. 41-48. 
Franklin Institute Press, Philadelphia. 1978. 

Purcell, R. H. and J. L. Gerin. Hepatitis B. vaccines: A status report. 
In : Viral Hepatitis (Eds., G. N. Vyas, S. N. Cohen and R. Schmid). 
pp. 491-505. Franklin Institute Press, Philadelphia. 1978. 

Dreesman, G. R. and J. L. Gerin. Summary of workshop on structure of 
hepatitis viruses. In: Viral Hepatitis (Eds., G. N. Vyas, S. N. 
Cohen and R. Schmid). pp. 645-648. Franklin Institute Press, Philadelphia. 
1978. 

Shih, J. W. -K. , G. Hess, P. M. Kaplan and J. L. Gerin. The polypeptides 
of HBV (Dane) particles. In: Viral Hepatitis (Eds., G. N. Vyas, S. N. 
Cohen and R. Schmid). pp. 705. Franklin Institute Press, Philadelphia. 
1978. 

Shih, J. W. -K. , M. Rizzeto, C. Langer and J. L. Gerin. 1979. Type B 
hepatitis: Characterization of delta antigen and the development of 
radioimmunoassays for delta and anti-delta. Abstr. , Annl. Mtg. Amer . 
Soc. Microbiol . S(H)80. 

Rizzetto, M. , D. Gocke, J. Shih, G. Verme and J. L. Gerin. 1979. A 

solid-phase radioimmunoassay for antibody to the hepatitis B associated 
delta antigen: Incidence of anti-delta in HBsAg carriers. Abstr. , 
Annl. Mtg., Amer. Soc. Microbiol. S(H)14. 

Rizzetto, M. , J. W. -K. Shih and J. L. Gerin. 1979. Characterization 
of the hepatitis B virus (HBV) associated delta antigen: Incidence 
of anti-delta in HBsAg carriers by a solid-phase radioimmunoassay 
(SP-RIA). Fed. Proc. 38(3). 

Hess, G. , J. W. -K. Shih, J. L. Gerin, R. H. Purcell and K. H. Meyer 
zum Bueschenf elde. Hepatitis B. antigen: Lack of relationship to 
liver-specific protein (LSP) . J. Med. Virol. (In Press). 

Parrott, R. H. , H. W. Kim, C. D. Brandt, M. 0. Beem, L. Richardson, 
J. L. Gerin and R. M. Chanock. Respiratory Syncytial virus. In : 
Diagnostic Virology (Ed., E. Lennette). (In Press). 

Hess, G., J. W. -K. Shih, W. Arnold, J. L. Gerin and K. H. Meyer zum 
Bueschenf elde. Demonstration and partial characterization of 22 nm 
HBsAg and Dane particles of subtype HBsAg/adw. J. Immunol. (In Press). 



17-16 



Shih, J. W. -K. , G. Hess, P. M. Kaplan and J. L. Gerin. Characterization 
of hepatitis B virus (Dane particle). J. Virol, (submitted). 

Rizzetto, M. , J. W. -K. Shih, D. J. Gocke, R. H. Purcell, G. Verme and 
J. L. Gerin. Incidence and significance of antibodies to delta 
antigen in hepatitis B infection. N. Engl. J. Med, (submitted). 

Moritsugu, Y. , J. W. -K. Shih, S. M. Feinstone, J. L. Gerin and R. H. 

Purcell. The hepatitis A virion: Polypeptides of hepatitis A virus 

obtained from stool of a naturally infected patient. J. Virol. 
(submitted) . 

Shih, J. W. -K. , P. J. Cote, Jr. and J. L. Gerin. Production of 

monocolonal antibodies against hepatitis B surface antigen by somatic 
cell hybrids. AABB Annl. Mtg. (submitted). 

Rizzetto, M. , R. H. Purcell and J. L. Gerin. Antibody to the hepatitis B 
virus-associated delta antigen in hemophiliacs: Evidence for parenteral 
transmission of delta through superinfection. AABB Annl. Mtg. (submitted), 

Purcell, R. H. , R. A. Johnson, V. J. McAuliffe, J. L. Gerin, W. T.. London, 
E. Nordenfelt and E. Helgstrand. Effect of antivirals on markers of 
HBV infection in chimpanzees. Lancet (submitted). 



17-17 



OFFICE OF THE SCIENTIFIC DIRECTOR, NIAID 
1979 Annual Report 

Table of Contents 

Summary of Program—Sell I8-I 

Z01 -Al -0001 3-1 6-OSD Studies of Viral Antigens in 18-3 

Virus-induced Tumors--Lewis 

Z01-A1-00018-13-0SD Biological and Biochemical 18-10 

Characterization of Human 
Papovavi ruses--Takemoto 

Z01-AI-00019-05-0SD Studies on the Treatment of 18-14 

Disease with the Interferon 
System — Levy 

Z01-A1-00131-12-0SD Mechanisms of Hypersensitivity 18-19 

in Histocompatible Inbred Guinea 
Pigs--Stone 

Z01-A1-00190-01-0SD The Molecular Genetics of 18-22 

Eukaryotic Cells and Their 
Viruses— Martin 



SUMMARY OF PROGRAM 

Laboratory and Clinical Research, NIAID 

October 1, 1978 through October 1, 1979 

Office of the Scientific Director 

The Annual Report of the Intramural Research Program contains individual sum- 
maries of research projects in the twelve laboratories which constitute the 
research component of the National Institute of Allergy and Infectious 
Diseases. Administrative responsibility for the Intramural Research Program 
resides in the Office of the Scientific Director (OSD). The OSD has been re- 
organized during the period of the past twelve to eighteen months in order to 
provide for more effective management. The reorganization also allowed for 
distribution of responsibility and authority to all working levels. 

Mr. Charles Cri swell has served as the Administration Officer in charge of 
Intramural Operations. Three Administrative Assistants have been appointed, 
each of whom report to his office. These assistants, Mrs. Helen Bednarek 
(Buildings 5 and 8), Mrs. Cathy Sabo (Building 7) and Mrs. Bea McKinley 
(Building 10) have assumed responsibility for all personnel, budgetary and 
other administrative matters in each of their respective areas. Assistance 
has been provided to each of the Administrative Assistants through the assinn- 
ment of one procurement technician and one clerk typist to perform administra- 
tive functions for all laboratories in each of the geographic areas. Also re- 
porting to Mr. Criswell is Mr. Carter Smith who serves as Head of the Animal 
Care Section which currently consists of 19 animal caretakers among which two 
serve as team leaders. The animal care operations at the Bethesda campus have 
been the direct responsibility of this Animal Care Section. Durinn the oast 
year a new office has been added in OSD. The Editorial Office headed by Mrs. 
Betty Sylvester (Editor) is now assisted by two editorial assistants. This 
office utilizes the most modern of word processing equipment to provide support 
primarily for the production of manuscripts. Currently, they have assumed a 
workload sufficient to take care of the needs of four of the in-house Bethesda 
campus laboratories. It is hoped that sufficient resources will be available 
to expand their services to provide support to all intramural laboratories. 

A new office of Special Assistant to the Scientific Director has been establish- 
ed to provide for direct responsibility for Safety and EEO programs in the IRP. 
Dr. Katherine Cook Jaouni has been appointed to this position and has performed 
in a remarkably good manner. Of particular importance was the development of 
a Minority Biomedical Sciences Program which brought forty young undergraduate 
minority scientists to Washington for a meeting in the spring of 1979. From 
amongst this group, ten were selected to participate in research activities of 
the Intramural Program in the summer of 1979. The program was outstandingly 
successful and will be the forerunner of similar programs to be developed in 
future years. 

The Rocky Mountain Laboratory in Hamilton, Montana was reorganized this year 

IS- r 



with the plan finally approved formally on March 16, 1979. This resulted in 
the Division of this large laboratory complex at RML into three scientific 
laboratories, the Laboratory of Persistent Viral Diseases, the Laboratory of 
Microbial Structure and Function and the Epidemioloay Branch. In addition, 
the Administrative activities of RML were assigned to the Operations Branch - 
RML. Mr. Robert Steiner serves as Chief of the Operations Branch. Currently, 
three acting Lab Chiefs are operating the three new laboratory facilities at 
RML and a process of search and selection is underway to identify permanent 
laboratory chiefs for these three new laboratory activities. The reorganiza- 
tion of RML into three scientific areas of research will allow for a focusing 
of research endeavor and allow for the build-up of research expertise at RML. 
It is anticipated that up to 15 new scientists will be added to the Laboratory. 
Because of restriction in total number of personnel allowed within the budnet 
for assignment at RML, this will require a concomitant reduction of support 
personnel. It is anticipated that loss of support personnel will be offset 
through the use of local contracts to provide needed services to the Rocky 
Mountain Laboratory. 

The Laboratory of Viral Diseases was divided during this year in order to 
allow Dr. Wallace Rowe, Chief of LVD, to direct his energies to the areas of 
personal concern and research interests. The Laboratory had been difficult to 
manage because of the division of its laboratory members into two geographic 
areas. The majority of the members of the Staff located in Buildinn 7 will be 
retained in LVD and work directly with Dr. Rowe. The members of the staff 
which had been located in Building 5 have temporarily been assianed to the 
Office of the Scientific Director. 

During the past year, several Intramural Laboratories were reviewed by the 
Board of Scientific Counselors. They reviewed the Laboratory of Parasitic 
Diseases, the Laboratory of Infectious Diseases, the Laboratory of Viral Dis- 
eases, the Laboratory of Streptococcal Diseases and the Laboratory of Biolonv 
of Viruses. In each instance, up to six Ad Hoc Consultants were added to the 
Board of Scientific Counselors in order to provide greater expertise in the 
review of each of the laboratories. Each investigator, including both tenured 
and nontenured investigators in each laboratory submitted a written report of 
their ongoing and future work. At the time of the on-site visit by the Board 
of Scientific Counselors and its Ad Hoc members, there was a neneral presenta- 
tion of Laboratory activities but, in addition, each investigator, both tenured 
and nontenured, was interviewed for a period of an hour by at least two members 
of the Board. This format has allowed an intensive evaluation of overall pro- 
gram and individual contributions to programs within each of the laboratories. 
The Board of Scientific Counselors has been instrumental in providinq useful 
suggestions and recommendations following each of their visits to the labora- 
tories . 

The scientific accomplishments of the Intramural Program have been exciting. 
Many members of the Staff have received significant awards and recognition 
from their peers and from the appropriate scientific organizations. Perhaps 
the most significant award of all was the receipt of the Paul Ehrlich prize 
with its sizeable cash award given to Dr. "all ace Rowe for his work in the 
fields of viral leukemia and immunogenetics . 

13-2 



'SMITHSONIAN SCIENCE INFORMATION EXCHANG E 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl-Al-00013-16 OSD 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Studies of Viral Antigens in Virus-induced Tumors 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI 

Other 



Andrew M. Lewis , Jr. 
Hubert W. Gerry 



OSD, NIAID 
Staff Fellow, 



IIAID 



COOPERATING UNITS (if any) 

Charles Kirkpatrick (NIAID); Arthur Levine, Cephas Patch, Alan Rabson (NCI 
Heiner Westphal, (NICHHD) Robert Martin (NIAAMD) 



lab/branch 

Office of the Scientific Director 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20014 



TOTAL MANYEARS: 

6.5 



PROFESSIONAL: 

2.75 



3.75 



CHECK APPROPRIATE BOX(ES) 
□ (a) HUMAN SUBJECTS 

D (al) MINORS [] (a2) INTERVIEWS 



rjj (b) HUMAN TISSUES 



•J (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) J ne initial Objectives Of thl'S project 

were to use human adenoviruses and adenovirus - SV40(Ad2-240) recombinants as 
tools to study the genetics of DNA tumor viruses, to define the role of viral 
genes and viral antigens in viral oncogenesis and to study the biology of Ad2- 
SV40 recombinants. Due to the lack of proper containment facilities in NIAID 
between January 1973 and May 1978, there has been a 5-year disruption (see past 
reports) in the major thrust of this project. During this interval, a study of 
Ad2-SV40 recombinants associated tumor induction by these agents with the in- 
corporation into the adenovirus - 2 chromosome of a specific segment of SV40 
DNA. To begin to understand the mechanism by which this SV40 DNA segment conveys 
oncogenicity to these recombinants, it was necessary to understand why Ad2 was 
nononcogenic for hamsters. Since Ad2 will transform hamster cells in tissue 
culture, we initiated a study of the oncogenic properties of Ad2 transformed 
""I:*- I? e / e !u! tS thuS far . lead . u ! to suspect that unrecognized host immune 

1- 

m,,. ; .,» m - - ' - :■- -■'■*. segment 

trie SV4U g enome might induce fun ction s in hamc:to r tumor calls ^t interfere 

PHS-6040 
(Rev. 10-76) 



SrlT 1n the c hamster re J' ect Ad2 transformed cells and transformed eel 
induced tumors. Such a concept suggests that the presence of a specific s 
or the SV4 genome might induce fun ction s in h a mct-o r tumor tpIIc tbai in t 

3 HS-6040 ltf-d 



with the rejection process. Future studies will be directed toward more care- 
fully defining these concepts. 

Project Description 

Major Findings : Oncogenicity of SV4Q deletion mutants that induce altered 17K 
t_ proteins 

The nondefective hybrid, Ad2 + ND / ,, which contains the segment of the SV40 genome 
between map position 0.11 and 0.59, induces tumors in hamsters after inactiva- 
tion by UV light (see 1977-1978 Report). Since nonhybrid Ad2 and the Ad2+ND 2 
hybrid which contains the segment of the SV40 genome between map position 
0.11 and 0.43 do not induce tumors after UV inacti vation , these results imply 
that the segment of the SV40 genome between 0.43 and 0.59 is required for 
tumor induction. To more carefully define the region of the SV40 genome asso- 
ciated with tumor induction, we studied the tumor-inducing capacity of a number 
of SV40 mutants containing deletions of the early region between map position 
0.54 and 0.59. These mutants induce a normal 92,000 dalton T protein, but 
induce altered 17,000 t proteins. Twenty to 90% of hamsters inoculated as new- 
borns with SV40 mutants A883, A885, A886 A890, A2001 andA2005 developed 
tumors after a 6 to 12 month latent period. For this study, SV40 wild-type 
virus produced tumors in 90% of inoculated hamsters within 8 months. The dif- 
ferences in the latent period prior to tumor development by SV40 compared with 
the SV40 mutants was statistically significant. Together with the studies on 
tumor induction by the Ad2+ND. hybrid, these studies imply that the DNA sequen- 
ces comprising the initial portion of the early SV40 genome between map posi- 
tions 0.55 and 0.54 are not essential for tumor induction in hamsters. Although 
the 17 k t-protein which is encoded by the SV40 region between map position 
0.65 and 0.54 is not essential for tumor formation, it does appear to reduce 
the latent period prior to tumor development. 

Host response to adenovi rus 2_ transformed hamster embryo eel 1 s 

The finding that a segment of SV40 DNA, by recombining with the nononcogenic 
Ad2 genome, conveyed tumor-inducing properties to the Ad2 ND. raised questions 
about the mechanism by which this change to oncogenicity was effected. Others 
have shown that Ad2 transformed rat cells were incapable of establishing 
tumors in immunocompetent rats. Since there appears to be little or no dif- 
ference between the in vitro transforming efficiency of the Ad2 ND„ and Ad2 ND. 

recombinants and nonhybrid A2, it seemed reasonable to assume that the change 
toward oncogenicity could be a reflection of the manner in which cells trans- 
formed in vivo by hybrid and nonhybrid virions interacted with the host immune 
system. As there was little data about the tumor inducing capacity of Ad2 
transformed hamster cells, it was necessary to examine this problem before the re- 
combinant could be considered. 

Ad2 inactivated by UV light was used to transform LSH hamster embryo cells. 
Evaluation of transformed cell lines produced by Ad2 (strain adenoid 6) or 
isolates of Ad2 obtained from children in Washington, D. C. , or West Bengal, 
India, showed that 13 of 15 lines induced tumors when injected into newborn 
inbred LSH or randomly bred NIH hamsters (10 7 cells/hamster) with a median 
tumor incidence in syngeneic newborns of 55% (range 8 to 100%). Six out of 6 

_ I8-4 



of these cell lines did not induce tumors when 10 cells were injected s.c. 
into weanling animals over 21 days old. In contrast to the Ad2- trans formed 
lines, similarly derived cell lines transformed by Adl 2 or SV40 uniformly 
produced tumors in weanlings following s.c. inoculation of 10? cells. Trans- 
plantation of tumors from animals injected as newborns with Ad2-transformed 
cell lines to other newborns was readily accomplished in 639 of 640, (99.8%) 
inoculated newborns. However, only 53 of 467 (11.3%) weanling hamsters 
challenged with these tumor lines developed neoplasms. Each of these Ad2- 
transformed lines contained Ad2 T antigen detectable by complement fixation 
or immunofluorescence. None of 6 lines contained Ad2 by Sendai fusion with 
human embryonic kidney (HEK) cells or other contaminating agents by a variety 
of assays. The difference in oncogenicity in weanling hamsters of Ad2-trans- 
formed cells compared to Adl 2 - and SV40-trans formed cells was not related 
to the dose of tumor injected or to differences in in vitro growth properties 
of the cell lines (e.g., doubling times, saturation densities, growth in 
spinner, or colony formation in soft agar). The tumor inducing capacity of 
Ad2 newborn tumor lines for newborn and weanling hamsters was not related to 
the dose of tumor injected. With the two tumor lines tested, weanling rejec- 
tion af newborn tumor transplants could not be overcome by inocula contain- 
25 times the usual dose of tumor suspension. These same two newborn tumor 
lines produced progressively enlarging neoplasms in 17-77% of syngeneic 
weanling hamsters which had been thymectomized as newborns. The lack of 
oncogenecity of hamster cells transformed j_n vi tro by Ad2 supported our spec- 
ulation that the SV40 genome in cells transformed i n vi vo by the Ad2 + ND. re- 
combinant could be altering immunological determinants involved in tumor cell 
rejection. 

Viral DNA sequences and gene products in hamster cells transformed by Ad2 . 

Complementary strand-specific adenovirus DNA of full length or from endonu- 
clease Bam HI fragments was used as a probe to estimate the fractional repre- 
sentation and abundance of viral sequences in five hamster cell lines 
AD2HEI-5 transformed with UV-inacti vated Ad2. The fraction of the viral 
genome present in the five transformed cell lines varied from 44% in the 
Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA 
copies per diploid cell equivalent ranged from 1.8 in the Ad2HEl line to 7.1 
in the Ad2HE4 line. 

35 
In vivo labeling with S-methionine followed by immunoprecipitation with an 

antiserum against Ad2 early proteins revealed viral-specific polypeptides 

with molecular weights of 42,000 to 58,000 in extracts from all five hamster 

cell lines. Several other early viral polypeptides were detected in some of 

the Ad2 transformed hamster cell lines. 

Studies are underway to determine the specific regions of the Ad2 genome that 
are contained in the Ad2HEl line of Ad2 transformed hamster embryo cells and 
to determine which of the messenger RNA's produced in these cells are coded 
for by these Ad2 DNA containing regions. Preliminary results indicate that 
RNA coded for by the early region on the left hand end of the right strand 
(the "transforming" region) is present; in addition, RNAs representing 
regions of the Ad2 genome transcribed late during infection (i.e. after viral 
DNA replication begins) are also present but appear in patterns not typical 
of RNA from cells productively infected with Ad2. 

18-5 



Role of the hamster cellular immune response in Ad 2 transformed cell oncogenesis 

The tumor-inducing capacity of two Ad2 transformed hamster cell-induced lines 
(Ad2HTL3 and Ad2HTL6) was tested in immunocompromised hosts. An average of 
35.2% of neonatally thymectomi zed, syngeneic, weanling hamsters developed 
progressively enlarging tumors when injected subcutaneously with tumor suspen- 
sions prepared from these lines, while no tumors were observed in normal, syn- 
geneic, weanling hamsters challenged with the same inocula. The susceptibility 
of neonatally thymectomi zed hamsters to tumor challenge was directly related 
to the degree of immunosuppression observed following thymectomy as indicated 
by the amplitude of the in vitro response of whole blood cultures to concana- 
valin A. Pretreatment of thymectomi zed weanlings with syngeneic adult lymphoid 
cells resultedin a significant reduction in tumor susceptibility (p = 0.03). 
These findings suggest that the maturation of a thymus-dependent cell-mediated 
immune response in hamsters during the first 21 days of life results in the 
rejection of Ad2- transformed cells. 

Developmental hamster immunobioloqy related to tumor rejection in the Ad 2 
transformed hamster cell system 

Transplantation of the Ad2HTL newborn tumor line to suckling hamsters 1-15 days 
old demonstrated that maturation events in the hamsters immune system between 
6 and 7 days were responsible for tumor rejection, \lery little data has been 
published concerning the ontogeny of the hamster immune response; however, 
based on data from other species, it has been predicted that hamster thymus 
and spleen cells should respond to T cell mitogens within a few days of birth 
and that suckling hamsters should be able to reject skin allografts at 8 days 
of age. To begin to evaluate the development of cellular immune responses in 
hamsters, cells from bloods and spleens from different aged animals were test- 
ed for their ability to respond to the mitogen conconavalin A (ConA) and to 
respond in the mixed lymphocyte reaction. The results of these studies indi- 
cate that hamster spleen cells begin to respond to ConA by age 5 days and 
attained 100 percent of adult type responses by age 15 days. Cells in the 
blood did not respond to ConA until age 25 days. 

Hamster spleen cells did not begin to respond in the mixed lymphocyte reaction 
until 18 to 25 days. To determine if the lack of ConA responsiveness of ham- 
ster spleen cells before age 5 days was due to T cell immaturity or immaturity 
of another cell component which was required for this response, X-ray inactiva- 
ted adherent and nonadhereent (to distinguish between lymphocytes and macro- 
phages) adult peritoneal exudate cells were mixed with spleen cells from 3 day 
old hamsters and the mixtures tested for their ability to respond to ConA. 
These results showed that, in the presence of populations of adherent peritoneal 
exudate cells, 3 day old hamster spleen cells readily responded (stimulation 
indices of 13 to 35) compared to spleen cells alone (stimulation index of 1 to 
2) or spleen cells mixed with nonadherent cells (stimulation index of 1 to 3). 
These results imply that immaturity of cell populations which cooperate with 
lymphocytes in the response to ConA are most likely responsible for the apparent 
immaturity of this reaction in hamsters less than 5 days old. . 

As an in vivo corollary to the evaluation of immune cell responses to mitogen 

. 18-6 



and foreign antigens, the inflammatory response to tumor development in new- 
born, weanlings and weanlings thymectomi zed during the first 24 hours of life 
was examined histopathologically in animals injected with suspensions of 
Ad2HTL3 and Ad2HTL6 tumors. In newborn animals multiple tumor nodules devel- 
oped within 5 days. and were quickly enclosed by highly vascular connective 
tissue. New capillaries were seen entering these nodules. During the first 
few days inflammatory cells consisted mainly of polymorphonuclear leukocytes 
with some lymphocytes and histocytes around the forming nodules. In sections 
with cells invading the surrounding connective tissue, there was no inflam- 
matory response. The response to tumor development in thymectomi zed weanlings 
was similar to that observed in newborns. In normal weanlings, tumor nodules 
developed early but were regressing by 5 days. Some new vessel formation was 
present during the first few days of tumor nodule development but this was 
less impressive than the intense vascular response noted in newborns. Inflam- 
matory cells consisted of polymorphonuclear leukocytes during the first day 
followed by the rapid appearance of an intense lymphohistiocytic infiltrate. 
After 7 days, the tumor nodules consisted of mostly granulation tissue with 
many multinucleated giant cells and focal calcification. 

Immunogenicity of Ad 2 transformed hamster cells and transformed cell-induced 
tumor li nes 

To determine the possible role of Ad2 transplantation antigen (TSTA) in the 
rejection of tumors transplanted to weanling hamsters, several types of 
studies are underway. An assay has been developed for Ad2 TSTA using an Ad2 
tumor line (Ad2HTLl ) which had been adapted to grow in adult hamsters. Im- 
munization with Ad2 significantly protected (defined as a resistance index 
*RI) greater than 10) hamsters against a challenge with Ad2HTLl . Immunization 
with SV40 did not convey significant protection (RI between 1 to 10) in these 
experiments. This assay is being used to study the immunogenicity of Ad2 
transformed cell lines and newborn and weanling tumor lines developed from 
these transformed cells. Results thus far indicate that 3 Ad2 transformed 
cell lines (Ad2HEl , Ad2HE3, Ad2HE6) and the tumor lines established from them 
(Ad2HTLl, Ad2HTL3, Ad2HTL6) contain a common Ad2 TSTA which during immuniza- 
tion with x-irradiated cells conveys protection to hamsters challenged with 
Ad2HTLl . Differences in RI did not correlate with the differences in the 
transplantability of the newborn tumor lines to newborn and weanling hamsters. 
Adult hamsters that had rejected tumors induced by viable Ad2HTL3 and Ad2HTL6 
cells were also protected when challenged with Ad2HTLl . These results show 
that whether they are accepted or rejected by weanling hamsters, each of these 
lines contains detectable Ad2 TSTA. Such findings appear to exclude the 
possibility that certain tumor lines can be transplanted to older animals 
because they lose detectable Ad2 TSTA. To determine whether a reducation in 
the concentration of Ad2 TSTA is responsible for the transplantability of 
certain newborn tumor lines to adults, the amount of immunizing antigen in 
suspensions of different tumors is being evaluated. 

Difference i_n th_e transplantability of virus-induced neoplastic cells i_n 
in-bred hamsters 

Tumor cells behave like normal cells in that they are usually rejected when 
transplanted as allografts to histoi ncompatible hosts. Since hamsters are 

- 18-7 



suspected to lack resistance to transplantable tumors of viral origin, we 
have been studying the transplantabi lity of our transformed cells and tumor 
lines to different strains of inbred hamsters. Eleven Ad2, Adl2 and SV40 
transformed LSH cell lines and LSH tumor lines established from them are being 
transplanted to syngenic LSH hamsters, CB hamsters which are histocompatibil- 
ity complex (MHC) disparate, and PD4 hamsters which are MHC identical but dis- 
parate at minor H loci. Initial findings show that Ad2 transformed cells pro- 
duced tumors only in newborn LSH hamsters. Ad2 tumor lines (Ad2HTLl , Ad2HTL3) 
adopted to grow in LSH adults by serial passage in neonatal hamsters produced 
tumors readily (TPD.q 1 2 • 3 to 10 4 - 97 0.2 ml of tumor suspension) when trans- 
planted to LSH adults. These same lines produced tumors inefficiently 
(TPD- l_0°-° to 10^-^/0.2 ml) when transplanted to adult CB hamsters and of 
intermediate efficiency (TPPr- 10 1 - 4 to 10 3 - 7 /0.2 ml) when transplanted to 
PD. hamsters. When 10° to lb 7 Adl2 transformed LSH cells were inoculated 
into adult LSH hamsters 84% developed tumors. When the same cell doses were 
injected into CB hamsters only 33% developed tumors. Tumor lines established 
in LSH hamsters from Adl2 transformed cells produced tumors in a pattern sim- 
ilar to the Ad2 tumor lines when transplanted to adult LSH, CB, and PD. ham- 
sters. When 10 6 and 10 7 SV40 transformed LSH cells were injected into adult 
LSH and CB hamsters, 100% and 97% respectively developed tumors. SV40 tumor 
lines produced tumors with equal efficiency in all 3 hamster strains. Thus, 
our Ad2 induced neoplastic LSH cells produced tumors only in immunoimmature 
syngeneic LSH hamsters; our Adl2 induced neoplastic LSH cells produced tumors 
efficiently in both immunoimmature and immunomature syngeneic LSH hamsters 
but produced tumors less efficiently in allogeneic hamsters; our SV40 induced 
neoplastic LSH cells produced tumors efficiently in both syngeneic and allo- 
geneic hamsters. 

Current ideas about the role of TSTA and allograft rejection of viral induced 
neoplasms do not account for the finding above. In this regard, we are con- 
sidering the possibility that the differences in the transplantabi 1 ity of 
these various cell lines are a reflection of the unrecognized manner in which 
tumor and allograft determinants on the surface of hamster cells are altered 
during transformation events by a particular viral agent. Studies are under- 
way to further clarify these findings. 

Signi fi cance to Biomedi cal Research 

The results of our recent studies impinge on tumor immunology and the possible 
role of antigenic determinants on cell surfaces in determining the outcome of 
viral-induced malignancy. The availability of well-characterized tumor lines, 
which are transplantable to both immunoimmature and immunomature hamsters and 
tumor lines, which are transplantable to immunoimmature but are consistently 
rejected by immunomature animals provides a useful system for identifying and 
studying the maturation of the functions of the hamster cellular immune system 
responsible for the rejection of viral -induced tumors. The central theme of 
tumor virology is the concept that viruses induce the malignant state (i.e., 
the ability to produce tumors in a susceptible host) in cells by a process 
called transformation. A number of studies (including our own work with the 
nondefective Ad2-SV40 hybrids) with different agents have associated trans- 
formation and tumor induction with the functioning of a specific region of the 
viral genome and in some cases, with a specific gene product. Paridoxi cal ly , 

— 18-8 



many virus-transformed cells which possess the properties ascribed to the 
transformed state fail to produce tumors when injected into either syngeneic 
immunomature or immunoimmature hosts. The reasons for this lack of transform- 
ed cell oncogenicity for a host that should be susceptible are poorly under- 
stood. Our studies with viral transformed hamster cells provide data which 
pertain to this question. At this juncture, we interpret our results with 
the Ad2 and SV40 transformed LSH hamster cell system as suggesting that, dur- 
ing the process of transformation, antigenic determinants on cell surfaces 
in addition to TSTA are altered. Some of these determinants are associated 
with graft rejection by allogeneic hosts; others are associated with the re- 
cognition and rejection of tumor cells by syngeneic hosts. An understanding 
of these cell surface modulations and the role of the viral genome in the al- 
teration of their function would be a significant step in understanding the 
mechanism by which viruses convert normal cells to malignant ones. 

Publ i cations 



Patch, C. T. , Levine, A.S., and Lewis, A.M., Jr.: The adenovirus-SV40 hybrid 
viruses. Comprehensive Virology, ]_3: 495-542, 1979. 

Johansson, K. , Persson, H., Lewis, A.M., Jr., Petterson, U., Tibbetts, C. and 
Philipson, L. : Viral DNA sequences and gene products in hamster cells trans- 
formed by adenovirus type 2. J. of Virol., 27: 628-539, 1978. 

Cook, J.L. and Lewis, A. M. , Jr.: Host response to adenovirus 2 - transformed 
hamster embryo cells. Cancer Res., 39: 1455-1461, 1979. 

Lewis, A.M., Jr. and Martin, R. G. : The oncogenicity of simian virus 40 dele- 
tion mutants that induce altered 17k t-proteins. Proc. Nat. Acad, of Sci . , in 
press. 

Patch, C.T., Hauser, J., Lewis, A. M. , Jr., and Levine, A.S. : A method for 
determining the extent and copy number of overlapping and non-overlapping 
segments of integrated viral genomes. J. of Virol., in press. 

Cook, J.L. and Lewis, A.M., Jr.: Age-related and thymus-dependent rejection 
of adenovirus 2 - transformed cell tumors in the Syrian hamster. Cancer Res., 
in press. 

Lewis, A. M., Jr. and Cook, J.L.: The association of tumor induction by ultra- 
violet light inactivated adenovirus 2-SV40 recombinants with a specific seg- 
ment of SV40 DNA. J. of Nat. Cancer Inst., in press. 



18-9 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZCH-AI-00018-13 OSD 



PERIOD COVERED 



TITLE OF PROJECT (SO characters or less) 

Biological and Biochemical Characterization of Human Papovaviruses. 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI: Kenneth K. Takemoto OSD, NIAID 

OTHER: Sheila Bond OSD, NIAID 

Tatsuo Miyamura, Visiting Fellow OSD, NIAID 



COOPERATING UNITE 



Peter Howley, Ming Fan Law, LP, NCI; George C. Fareed, UCLA, Los Angeles, CA: 
Hawley Linke, UCLA, Los Angeles, CA., L.W. Law, NCI. 



LAB/ BRANCH 

Office of the Scientific Director 



SECTION 

Cellular Virology Section 



INSTITUTE AND LOCATION 



IIAID. NIH. Bethesda. Maryland 20205 



TOTAL MANYEARS: 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS 

D (al) MINORS Q (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



SUMMARY OF WORK (200 words or 

The principal goal 
biochemical characteriz 
the past six years, mos 
toward studies with BKV 
of various types. JCV, 
vitro, and therefore ha 



year, however, a major 
different readily avail 
This will now enable us 
this important virus. 



less - underline keywords) 

of this unit continues to be the 
ation of the human papovaviruses, 
t of the research efforts of this 

since this virus could easily be 

on the other hand, has been diffi 
s remained a relatively unknown vi 
advance was made when we were able 
able cells, human amnion and human 

to conduct detailed investigation 



detailed biological and 
BKV and JCV. During 
unit were directed 
grown in cell cultures 
cult to propagate i_n 
rus. During the past 
to grow JCV in two 
embryonic kidney cells, 
s on the biology of 



Studies are continuing on the mechanism of persistent infection of human 
fetal brain cells by BKV. It has been established that viral genomes exist in 
episomal form in these cells, and that rare cells in the culture spontaneously 
undergo lytic cycles to produce infectious progeny. This type of virus-cell 
interaction may explain how thesp ^a^va viruses persist indefinitely in 

' PHS-6040 
(Rev. 10-76) 



the human 



PROJECT DESCRIPTION 

Persistent BK virus infection . We reported last year on the establishment of 
a unique, heretofore unrecognized type of persistent viral infection in cell 
culture. In this system, BK virus was shown to persist indefinitely in trans- 
formed human fetal brain cells. The mechanism whereby virus persists in the 
cells has been determined. It was found that all cloned lines contained viral 
DNA in non-integrated, episomal form. By restriction endonuclease analysis, 
the episomal viral DNA was shown to be identical to parental DNA and was 
infectious. The number of epi somes per cell was between 5 to 10. It is 
postulated that in an occasional cell, the episomal DNA is spontaneously 
"induced" to go into a complete replicative cycle and viral progeny is 
produced. The persistent state is thus perpetuated, even in the presence of 
potent antiserum. We speculate that this type of virus-cell interaction can 
perhaps explain how these viruses persist in their natural hosts. 

Another unusual feature of these cells is that they are transformed and 
have most of the properties of transformed cells. They grow in low serum 
medium, form colonies in soft agar, are immortal, and produce tumors in nude 
mice. However, unlike the usual papovavirus transformed cells, they do not 
contain T-antigens detectable by immunofluorescence. By immunoprecipitation 
and analysis in gels, there appears to be an abberant T-protein produced with 
a molecular size in the range of 40K. Whether this protein is of viral or 
cellular origin is being determined. 

Replication of JC virus in non-fetal human cells . JC virus (JCV) has now been 
shown to be the causative agent in progressive multifocal leukoencephalopathy 
(PML), having been isolated from or identified in over 30 cases of the disease. 
Studies on this important virus have been severely limited due mainly to the 
difficulty in growing the virus; until recently, it has been grown only in 
human fetal glial cells, which are difficult to obtain. In the past year, we 
have successfully adapted JCV to grow in 2 types of commercially available 
cells, human embryonic kidney and human amnion cells. Both kinds of cells 
support JCV replication with full yields of virus. Serological and biochemi- 
cal analysis of the adapted JCV showed that the virus was identical to the 
original strain grown in human fetal glial cells. Detailed biological and 
biochemical investigations on this virus can now be performed and a more 
complete characterization of JCV can be expected within the next few years. 

BKV helper function for adenovirus replication . Previous comparative studies 
on common early viral functions between the simian and human papovavi ruses, 
SV40 and BKV, have established a relatedness between the 2 viruses. Thus, 
they share immunologically related T-antigens and BKV can complement the 
growth of temperature sensitive SV40 mutants which dre defective in early gene 
functions. To understand more fully the functional similarities between BKV 
and SV40, the helper function for adenovirus growth by BKV in non-permissive 
monkey cells was studied. During the course of these experiments, it was 
observed that replication of BKV in CV-1 monkey cells was primarily abortive 
at 37 C. However, when infected cells were incubated at high temperature 
(40 C) the cells became permissive for BKV replication and high virus yields 
were obtained. 

--I8-II 



Based on this finding, experiments to determine helper function for 
adenovirus growth in monkey cells by BKV were performed at abortive (37 C) and 
permissive (40°C) temperatures. Results of these experiments showed that 
while enhancement of adenovirus replication occurred in cells co-infected with 
BKV and incubated at 37 C, there was a 10-fold greater degree of enhancement 
at 40 C. This was probably due to an increased production of T-antigen at 
the higher temperature. These experiments thus provide additional information 
on the high degree of relatedness between the simian and human papovavi ruses 
and is in agreement with the recent findings of extensive homology between the 
genomes of the 2 viruses obtained by DNA sequencing studies. 

Common sequences in the genomes of JC, BK, and SV40 . In studies done in 
collaboration with Drs. Law and Howley (NCI), the DNAs of the primate papova- 
viruses SV40, BK, and JC were analyzed for nucleotide sequence homology. Under 
non-stringent conditions, extensive homology was found throughout the genomes 
of the 3 viruses, which correlate well with the previous observations that the 
structural (viral) as well as non-structural (t-proteins) antigens of the 
primate papovavirus are immunologically related. The region of strongest 
homology among the 3 genomes was localized in the late region between 0.76 to 
0.85 map units coding for VP2. 

Significance to Biomedical Research . The human papovavi ruses are ubiquitous 
viruses which are world-wide in distribution, infecting children at an early 
age and persisting thereafter throughout life. One of them, JCV, has clearly 
been determined to be the causative agent of PML. The papovaviruses represent 
a class of persistent viruses whose possible role in chronic diseases, includ- 
ing cancer, need to be determined. They have been demonstrated to be oncogenic 
in tissue culture and in animals, thereby providing important models for the 
study of viral oncogenesis. 

Proposed course . The ability to propagate JC virus in non-fetal cells will 
now enable us to conduct detailed investigations on the biology and biochem- 
istry of this virus which were heretofore not possible. An immediate 
objective is to analyze the DNA of JCV virions since previous data indicated 
extensive heterogeneity which probably accounted for the poor growth and low 
yields of virus. The system of persistent BKV infection in T-antigen 
negative, transformed human fetal brain cells will continue to be investigated. 

Publications 



Israel, M.A. , Takemoto, K.K., Martin, M.A., Soloman, D. , Howley, P.M., 
Aaronson, S.A. and Khoury, G. Evaluation of normal and neoplastic tissue for 
BK virus. Virology 90, 187-196, 1978. 

Bond, S.B., Howley, P.M. and Takemoto, K.K. Characterization of K virus and 
its comparison with polyoma virus. J. Virology 28_, 337-343, 1978. 

Law, M.F., Takemoto, K.K. and Howley, P.M. Characterization of the genome of 
the murine papovavirus K. J. Virology 30, 90-97, 1979. 



18-12 



Takemoto, K.K., Linke, H., Miyamura, T. and Fareed, G.C. Persistent BK 
papovavirus infection of transformed human fetal brain cells. I. Episomal 
viral DNA in cloned lines deficient in T-antigen expression. J. Virology 30, 
1177-1185, 1979. 

Takemoto, K.K. , Howley, P.M. and Miyamura, T. JC human papovavirus replication 
in human amnion cells. J. Virology 3£, 384-389, 1979. 

Law, M.F., Martin, J.D., Takemoto, K.K. and Howley, P.M. The co-linear 
alignment of the genomes of papovaviruses JC, BK, and SV40. Virology (In 
Press) 

Miyamura, T. and Takemoto, K.K. Helper function for adenovirus replication 
in monkey cells by BK human papovavirus. Virology (In Press). 

Awards and Honors 



Invited lecturer, Virology '79 Lecture Series, The Institute for Medical 
Research, Camden, New Jersey, January 4, 1979. "The Papovavirus Group." 

American Society of Microbiologists Symposium on Viruses and Human Cancer, 
May, 1979, Los Angeles, California. "Human Papovaviruses: Search for 
evidence of possible involvement in human cancer." 



18-13 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

Z01-A1 -0001 9-05 OSD 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Studies on the Treatment of Disease with the Interferon System 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 

OTHER: 



Hilton B. Levy 
Freddie Riley 
Clarence Corey 



Biochemist 

Chemist 

Bio-lab technician 



OSD, NIAID 
OSD, NIAID 
OSD, NIAID 



COOPERATING UNITS: Dr. Arthur Levine, NCI; Dr. King Engel , NIANDS; Dr. John 
Hooks, NIDR; Maj. Edward Stephen, USAMRIID; Dr. Chester Liu, USAMRIID; Maj. Don 
Harrington, USAMRIID; Dr. Martin Lerner, Wayne State Univ.; Dr. Larry Crane; 
Wayne State Univ; Drs. Herbert Oettgen and Susan Krown, Sloan Kettering Inst.; 

cooperating units (if any) Dr. Beatrice Lampkin, Children's Hospital Center, Cincinnati, 
Ohio; Dr. Mosmorine, Connaught Laboratories, Canada; Dr. Goodwin Hilfenhaus, 
Behringeverke, Germany; Dr. Tagir Bektemirov, Gamalaya Institute, USSR; 
Dr. Edward Lvosky, Litton Bionetics. 



lab/branch 

Office of the Scientific Director 



SECTION 

Molecular Virology Section 



INSTITUTE AND LOCATION 

NIAID, Bethesda, Maryland 20014 



TOTAL MANYEARS: 



PROFESSIONAL: 

2 



CHECK APPROPRIATE BOX(ES) 
& (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



(b) HUMAN TISSUES 



[] (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The nuclease-resistant, primate-effective interferon inducer, poly inosinic 
polycytidylic acid, poly lysine, carboxymethyl cellulose (Poly ICLC) has a vari- 
ety of physiological activities. In addition to inducing interferon in primates 
it is an immune adjuvant and a radioprotective agent. It is currently being 
evaluated in 3 collaborative clinical studies, and 3 more are being developed. 
It appears to have had ameliorative effects in 4 patients; one with lymphoblasti 
leukemia, one cancer patient whose widely disseminated Herpes lesions disappeared 
after treatment, and 2 patients with chronic relapsing polyneuropathy. A drug 
without carboxymethycellulose has been prepared and is being evaluated. 



I8-14 



PHS-6040 
(Rev. 10-76) 



Project Description 

This past year has been one of preparing to do larger scale testing of poly 
ICLC in man. This has included actual clinical studies and studies of a more 
basic type. 

Programs are ongoing with the Sloan Kettering Institute, where a phase I study 
of the drug in terminal cancer patients is being done. They are using a dif- 
ferent dosage and administration schedule than that which we used with Dr. 
Levine of the NCI. All that can be said at this writing is that the patients 
are making good levels of interferon. One leukemia patient had widely dissem- 
inated herpes continuously for 1 and 1/4 years. One week after going onto the 
drug, the herpes lesions disappeared and have not reappeared. 

A phase II study has been initiated with the Children's Cancer Study Group, a 
multi-institution collaborative organization. The present protocol is designed 
to treat acute lymphoblastic leukemia at stages earlier than terminal. No data 
are avai Table as yet. 

The Policy Network of NCI accepted poly ICLC as a high priority drug, and is 
undertaking its manufacture, packaging and distribution. They also are begin- 
ning a long-range toxicology study, the results of which will be available for 
NI AID use in treating patients with viral disease. We have developed a proto- 
col together with NCI to treat a variety of solid tumors and leukemias. The 
protocol has passed NCI, Clinical Center and NIAID review. There are extensive 
immunological studies described in this protocol. 

There have been written the following protocols for clinical studies which are 
almost ready to submit for NIAID approval. 

1) With Drs. Alexanian and Gutterman of M.D. Anderson Hospital - to treat 
patients with multiple myeloma in comparison with their study using exogenous 
interferon. 

2) With Dr. Brian Durie of University of Arizona - to treat multiple myeloma 
and to study the effect of the drug on a variety of immunological parameters. 

3) With Drs. Mosely and Rakela of University of Southern California Medical 
School liver unit - to treat patients with hepatocellular carcinoma associated 
with chronic hepatitis B infection. 

Poly ICLC, like the parent poly I Poly C, is pleiotropic. In addition to its 
ability to induce interferon, it has demonstrated the following action: To 
date, it has been shown to be even at low doses, an effective adjuvant with 
the following weak vaccines; Venezuelan Equine encephalitis, Japanese B ence- 
phalitis, Swine Flu, hemophilus influenzae polysaccharide, Rift Valley fever 
and Herpes envelope antigens. With strong antigens, such as albumin and 
pneumococcal polysaccharide type III, if anything, there is an inhibitory action 
on antibody production. Monkeys receiving poly ICLC showed a marked, but 
transient increase in the number of small lymphocytes in the paracortex of the 
nodes draining the site of injection, as well as an increase in migration rate 
of lymphocytes from high endothelial venules. 

_ 18-15 



Poly ICLC, like a number of other interferon inducers is a radio protective 
agent. Mice given Poly ICLC can tolerate a significantly increased amount of 
X-irradiation. While the mechanism of protection is not clear, it is probably 
associated with a strong stimulation of marrow stem cells as shown by an in- 
crease in endogenous colonies in the spleen. 

In a study with Dr. Engel of NIAMDS a year and a half ago, a patient with 
chronic relapsing polyneuropathy was started on poly ICLC. After six weeks of 
treatment the patient went from a state of almost complete paralysis to one 
in which he was able to walk six miles, and lift weights over his head. He 
continues to be well, but requires weekly or biweekly injections. Otherwise, 
weakness begins to set in again. 

Another patient with a similar illness also made a dramatic recovery but doesn't 
appear to require continued treatment. Immunologic studies are being done on 
these patients. 

Connaught Laboratories, the manufacturer of Salk type polio vaccine, has had a 
serious problem in obtaining enough monkey kidney tissue culture to grow the 
polio virus. The monkeys are chronic carriers of foamy virus, and there is 
barely time to get a harvest of poliovirus from the primary tissue culture 
growth, before the foamy virus destroys the tissue culture. They could increase 
the amount of tissue -ul ture by 10 fold if they could make secondary cultures. 
In two collaborative experiments they found that by treating the monkeys with 
poly ICLC before removing the kidneys, they could reduce the virus titer suf- 
ficiently so that secondary cultures could be made. They plan to make the poly 
ICLC themselves and try it again. If successful, they will consider applying 
it to their production schedule. 

Commercial interest in poly ICLC has been expressed by Pasteur Development 
Corporation in France, Ciba-Geigy in Switzerland and Merck, Sharpe and Dohme 
in this country. 

There has been some concern over the presence of carboxymethylcellulose (CMC) 
in poly ICLC. While CMC has been used in parenteral medicines for years, the 
fact is that there is no known enzymatic pathway for its degradation. Monkeys 
and humans who have received poly ICLC over two years ago have shown no adverse 
reactions, nor have those who received CMC as a vehicle for steroid administra- 
tion. However, because it would be easier to formulate and might allow for the 
preparation of a more concentrated solution, we have tried to prepare a poly I 
poly C poly lysine complex without CMC. By taking advantage of the known 
effects of ionic strength and temperature on the components, we have succeeded 
in preparing a series of such complexes that are resistant to hydrolysis by 
RNase, and which induce interferon in monkeys and mice. Much work needs to be 
done before we can give this poly ICLC to people. 

We have studied the effect of changing the size of the poly lysine and that of 
the poly I and poly C on Tm, nuclease resistance and capacity to induce inter- 
feron in monkeys. Poly lysine of molecular weight 2000 forms an ineffective 
complex, poly lysine of molecular weight 27,000 is close to maximally effective. 
9s Poly I and Poly C form a much more effective complex than does smaller poly 
I or poly C. Our standard poly ICLC is made with 9s Poly I, 9s Poly C, 27,000 

18-16 



mol . wt. poly lysine and high viscosity CMC. 
Future Plans : 

1) It is hoped in the next year to expand clinical work with patients with 
malignancies, emphasizing the following questions: a) Are there some tumors 
that are responsive? b) What are the effects on virus diseases in these 
patients? c) What are the effects of the drug on the immune system in man? 

2) With NCI it is planned to do long-term toxicological studies in mice and 
monkeys. 

3) Through the use of the contract mechanism we plan to examine the question 
of stability of the drug, as well as some biochemical and immunological 
matters that bear on therapy. 

4) In a collaborative study with Dr. Liu of USAMRIID we plan to examine the 
effect of poly ICLC and interferon on a large variety of cardiovascular 
functions in monkeys. 

Publications 



Harrington, D. G. , Crabbs, C. L. , Hilmas, D. E., Brown, Jr. R. , Higbee, S.A., 
Cole, . E., Levy, H.B.: Adjuvant effects of low doses of a nuclease-resistant 
derivative of polyinosmic -acid on antibody responses of monkeys to inactivated 
Venezuelan equine encephalomyelitis virus vaccine. Infection and Immunity , 
Apr. 1979, p. 160-166. 

Levine, A. S., Sivulich, M., Wiernik, P. H., Levy, H. B. Initial clinical 
trials in cancer patients of polyribonosinic-polyribo cytidylic acid stabilized 
with poly-L-lysine, Carboxymethylcell ulose [Poly(ICLC)] , a highly effective 
interferon inducer: Cancer Research 39: 1645-1650. May 1979. 

Hilmas, D. E. , Stephens, E. L. , Spertzel , R. 0., Levy, H. B. Use of PICLC for 
the prophylaxis and treatment of Venezuelan equine encephalomyelitis virus 
infection in nonhuman primates. Current Chemotherapy . 1978. 

Levine, A. S., Levy, H. B. Phase I- 1 1 trials of PIC stabilized with poly-L- 
lysine. Cancer Treatment Reports ., Vol. 62, No. 11. Nov., 1978. 

Levy, H. B., Lvovsky, E. Tropical treatment of vaccinia virus infection with 
an interferon inducer in rabbits. Journal of Infectious Diseases , Vol. 137, 
No. 1. January, 1978. 

Levy, H. B., Hilmas, D. E. Evaluation of a nuclease-resistant derivative of 
PLCLC as a radioprotective agent. Radiation Research , 77, 1979. 

Stephens, E. L., Hilmas, E. E. , Levy, H. B., Spertzel, R. D. Protective and 
toxic effects of a nuclease-resistant derivative of PIC on Venezuelan equine 
encephalomyelitis virus in Rhesus monkeys. Journal Infectious Diseases , Vol. 
139 No. 3, 1979. 



18-17 



Lerner, M. E., Levy, H. B. Physiological accompaniments of sustained 
interferonemia induced in man by poly ICLC. lrvf_. Immun . Accepted. 



TB-I8 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



Z01-A1-00131-12 OSD 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Mechanisms of Hypersensitivity in Histocompatible Inbred Guinea Pigs 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI: Dr. Sanford H. Stone 



Chief, Immunology Section QSD, NIAID 



cooperating units (if any) Dr. Cedri c S. Raine, Division of Neuropathology, Albert 
Einstein College of Medicine, New York, N. V. ; Dr. Richard H. Quarles, D.M.N, 
NINCDS, Bethesda, MD. ; Dr. Robert B. Nussenblatt, CB, NEI, Bethesda, MD. 



lab/branch 

Office of the Scientific Director 



Immunology Section 



institute and location 

NIAID, Bethesda, Maryland 20014 



TOTAL MANYEARS: 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (a1 ) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



CX(c) NEITHER 



SUMMARY OF WORK (200 *ords or less - underline keywords) 

For some years, we have been using the unique tool 
guinea pigs for investigation of mechanisms of hypersen 
genesis of and the protection against infectious and au 
vantages lie in the ability to perform viable adoptive 
and in the capacity to study genetic factors in certain 
autoimmune phenomena. We have concentrated recently on 
age-dependent induction of cataracts in immature guinea 
paralysis and atrophy of autoimmune encephalomyelitis an 
treatment of this condition. The cataracts are reminis 
experimental entities shown to be due to nutritional de 
alteration and may be secondary to the autoimmune react 



of inbred histocompatible 
sitiv 
toimm 



n'ty in the patho- 
lune diseases. Ad- 



transfer of lymphoid cells 
hypersensitivity and 
the pathogenesis of an 
pigs undergoing the 
d on the prevention and 
cent of some clinical or 
ficiency or enzymatic 
ion in the host. 



-18-19 



PHS-6040 
(Rev. 10-76) 



Project Descri ption 
Subproject I - Cataract formation in allergic encephalomyelitis of guinea 
pigs 

An unusual pathological event occurred in immature guinea pigs sensitized 
to spinal cord antigens either actively or passively by lymph node-cell trans- 
fer. A significant number of these developed cataracts bilaterally during a 
severe acute attack of allergic encephalomyelitis (EAE). The lens histology 
was significant for posterior migration of the epithelium with some eyes show- 
ing vacuolization under the capsule. The vitreous, choroid and retina did 
not show foci of inflammation and the intra-ocular and intracleral portion 
of the optic nerve showed no evidence of cell drop-out nor round cell infil- 
tration. Histological preparations from the central nervous system (CNS) of 
the guinea pigs showed the classic picture of acute EAE. 

The age-dependency of this eye lesion was quite evident- Both wean- 
lings and newborns, but no adults, developed the opacities; the cataracts 
were not strain-specific, occurring in both strain 13 and Hartley guinea pigs. 
The evidence points to the probability that these cataracts are not the direct 
result of an immunological response, but secondary to the acute paralytic 
syndrome of EAE. 

Subproject II - Protection against chronic EAE by injection of myelin 
basic protein in incomplete Freund's adjuvant 

CNS lesion morphology was examined in detail in inbred strain 13 guinea 
pigs sensitized for chronic EAE in which the disease was either allowed to 
develop or was suppressed by injections of myelin basic protein (MBP). Path- 
ologic changes correlated well with the clinical picture; in chronic animals 
clinical disease was accompanied by inflammation in the CNS including fibrosis 
and remyel i nation. Relapses showed the CNS to contain recent changes super- 
imposed upon old lesions. In animals in which the disease was suppressed, 
clinical signs did not develop, but some early sub-clinical changes were seen 
morphologically. These lesions were remyelinated promptly and there was no 
progression in lesion formation. This contrasted with the progression in un- 
treated chronic animals; long-standing disease was characterized by large, 
burnt-out plaques, with Schwann cell invasion and peripheral nervous system 
myelination, glial bridges between sub-pial astrocytes and the leptomeninges , 
and fenestrated blood vessels. 

Therapy of established chronic disease (as distinguished from suppression 
experiments) is under investigation currently using MBP in paralyzed strain 
13 guinea pigs. This will require an elaborate set of controls and will be 
cautiously pursued in view of its relevance to the treatment of multiple 
sclerosi s. 

Publ i cations 



Raine, C. S. , Traugott, U. and Stone, S. H. : Chronic relapsing experimental 
allergic encephalomyelitis: CNS plaque development in unsuppressed and 
suppressed animals. Acta Neuropathol . 43:43-53, 1978. 



-18-20 



Raine, C. S. Traugott, U. and Stone, S. H. : Glial bridges and Schwann cell 
migration during chronic demyeli nation in the C.N.S. J. Neurocytol . 7: 
541-553, 1978. 

Traugott, U., Stone, S. H. and Raine, C. S.: Chronic relapsing experimental 
allergic encephalomyelitis: correlation of circulating lymphocyte fluctua- 
tions with disease activity in suppressed and unsuppressed animals. J. Neurol 
Sci. 41 : 17-29, 1979. 



18-21 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE! U.S. DEPARTMENT OF PROJECT NUMBER 
PROJECT NUMBER (Do MOT use this space) IHEALTH, EDUCATION, AND WELFARE 

PUBLIC HEALTH SERVICE „„, ., „„,_„ „, „_ 

hot ice cf Z01 -Al -001 90-01 -OSD 

INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 



j TITLE OF PROJECT (80 characters or less; 

The Molecular Genetics of Eukaryotic Cells and Their Viruses 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI : Malcolm A. Martin OSD, NIAID 

OTHER: Hardy Chan, Senior Staff Associate OSD, NIAID 

Dean Hamer, Staff Associate OSD, NIAID 

Mark Israel , Research Associate OSD, NIAID 

Yoshiaki Ito, Visiting Scientist OSD, NIAID 

Roy Repaske, Microbiologist OSD, NIAID 

Kama! Chowdhury, Visiting Fellow OSD, NIAID 



COOPERATING UNITS (if any) 

Wallace P. Rowe, LVD, NIAID; Edward Scolnick, LTVG, NCI 



LAB/BRANCH 

Office of the Scientific Director 



SECTION 

Molecular Biology 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
□ (a) HUMAN SUBJECTS Q (b) HUMAN TISSUES Q (c) NEITHER 

(a1 ) MINORS Q ( a2 ) INTERVIEWS 



SUMMARY OF WORK (200 words or less - underline keywords) 

The principal goal of this unit continues to be the biochemical characteri- 
zation of eukaryotic and animal viral genes. Attention has been focussed on the 
segments of DNA and RNA Tumor virus genomes responsible for the establishment and 
maintenance of the transformed state. In this regard we have shown that the 
large form of polyoma tumor antigen plays no role in tumorigenesis mediated by 
polyoma DNA. SV40 DNA recombinants have been used to elucidate the mechanisms 
regulating the synthesis and processing of eukaryotic RNA. 

The first phase of risk assessment experiments conducted in the P4 facility 
at Ft. Detnck, Maryland, was recently completed. These studies showed that 
polyoma virus DNA is not transferred out of E. coli K-12 following the 
inoculation of susceptible newborn animals with bacteria containinq recombinant 
plasmids or phage. 



18-22 



PWS-6040 
(Rev. 10-76) 



Z01-A1-00190-01-0SD 
Project Description: 

The DNA Recombinant Research Unit, established in the Summer of 1978, has 
continued to utilize recombinant DNA procedures to investigate the regulation 
and structural organization of eukaryotic cells and their viruses. While the 
main thrust of research activities continues to be the study of the transforming 
region of SV40 and polyoma virus, new projects which involve the molecular 
cloning and biochemical characterization of murine retraviruses and the use of 
the SV40 vector system to evaluate transcriptional regulation of eukaryotic 
genes has been initiated. Experiments evaluating potential risks associated 
with recombinant DNA research have also been carried out at the P4 facility 
located at Ft. Detrick and P2 laboratories on the NIH reservation. 

During the year Dr. Daniel Simmons resigned to take a position as Assistant 
Professor of Biology, University of Delaware. Dr. Dean Hamer joined the Unit 
last fall following post doctoral training in Dr. Phil Leder's laboratory and 
will use SV40 recombinants to investigate transcription of mammalian genes. 
Dr. Yoshiaki Ito joined the group in August following a six-year tenure at the 
Imperial Cancer Research Fund Laboratories, London and will continue to 
characterize transforming polypeptides specified by papovavirus genomes. 
Dr. Roy Repaske became an active participant in the risk assessment experiments 
coordinated by Drs . Martin and Rowe and perfected the in vitro packaging 
procedure utilized in "shotgun" cloning. He has recently mastered nucleotide 
sequencing procedures and will use them to characterize specific segments of 
cloned DNA. 

Research Accomplishments 

I. The Oncogenic Potential of Papovaviruses 

During the past two years the polyoma (PY) virus system has been used to 
evaluate the molecular events associated with transformation and tumori genesis. 
The PY virus system has at least two advantages over SV40: 1) tumors are 
readily and rapidly induced in newborn hamsters; 2) the PY viral genome encodes 
three peptides which play a role in the oncogenic process whereas SV40 DNA 
contains the genetic information for only two of these three. We have extended 
our previous studies involving tumorigenesis in newborn hamsters by evaluating 
the biological activity of viral DNA preparations previously digested with 
restriction enzymes. These experiments clearly indicate that tumori geni city 
of PY DNA is enhanced when the distal portion of the early gene region is 
interrupted. This was an unexpected result since in the more extensively 
studied SV40 system, the entire early region must be intact for the initiation 
and maintenance of the transformed state. Using immunoprecipitation techniques, 
we demonstrated that tumor cell lines, originally induced by PY virions or PY 
DNA, contained the small and middle PY tumor (T) antigens (Ags) but not the 
large form of viral T Ag. Fifteen independent cloned tumor cell lines derived 
from hamster tumors induced by PY virions or PY DNA were analyzed by blot- 
hybridization and shown to contain viral DNA sequences specifying the small 
and middle PY T Ags. PY DNA sequences encoding the large T Ag were invariably 
interrupted. These data imply that PY large T Ag is not required for the 
maintenance of the tumor cell phenotype. Studies are currently in progress 

"18-23 



Z01-A1-00190-01-OSD 

to evaluate whether PY large T Ag plays any role in virus mediated tumori genesis. 
An effort is also being made to clone the PY DNA sequences integrated into the 
tumor cell genome. (Israel, Chowdhury, Martin.) 

II . The Molecular Structure of Murine Retraviruses 

Our unit has initiated a major collaborative effort with Dr. Wallace Rowe's 
group (LVD) to clone and characterize a variety of murine retraviruses. This 
research activity grew out of an ealier joint project with Dr. Edward Scolnick's 
group (Laboratory of Tumor Virus Genetics, NCI) in which Friend Leukemia (FLV), 
and Harvey Sarcoma (HaSV) viruses were cloned in derivatives of E_. col i K12 
using a lambdaphage vector. After refining in vitro packaging procedures to 
permit the "shotgun" cloning of unintegrated viral DNA, large quantities of 
FLV and HaSV DNAs were prepared and extensively characterized. We concentrated 
our energies of the restriction mapping of HaSV DNA and showed that one of the 
six recombinant phage we constructed contained a triplication of viral DNA 
sequences that flank both ends of the integrated HaSV DNA. Work is continuing 
on the stability of the reiterated HaSV DNA sequences in both eukaryotic and 
prokaryotic host cells and determining the fine structure of the reiterated 
DNA segment. (Chan, Repaske, Martin.) 

More recently we have begun the molecular cloning of the endogenous 
retraviral DNA sequences present in the AKR mouse. Thus far, we have obtained 
recombinant phage preparations containing integrated: 1) ecotropic AKV; 
2) xenotropic; and 3) two MCF DNA sequences. The cloned AKV and xenotropic 
preparations appear to represent complete viral genomes. These retraviral DNA 
inserts will be biochemically characterized and subgenomic viral DNA segments 
will be purified for use as a probe in hybridization experiments for the 
detection of specific regions of viral DNA. (Chan, Repaske, Martin.) 

Ill Regulation of Eukaryotic Genes 

In order to provide a biological assay for regulatory sequences present in 
eukaryotic DNA as well as mutants derived from them, we have developed SV40 
host vector systems that allow us to introduce new genetic information into 
animal cells. During the past year, we have constructed recombinant molecules 
containing chromosomal mouse globin genes linked to an SV40 vector and infected 
monkey cells. We find that the mouse globin gene signals for transcription, 
processing and translation are recognized in monkey cells resulting in the 
production of substantial quantities of globin. Further, by preparing mutant 
forms of SV40, we have characterized discontinuous eukaryotic genes and the 
splicing of mammalian RNA. Specifically, the minimum size of the recognition 
site, the species, cell type, and distribution of the splicing enzymes, and the 
physiological role of splicing in stable RNA formation has been investigated. 

We are now attempting to extend this system to the human globins and are 
particularly interested in using SV40 recombinants to elucidate the molecular 
lesions in various hemoglobinopathies. We have also initiated a project to 
form SV40 recombinants carrying hepatitis virus type B sequences. Such hybrids 
could be very useful in mapping the hepatitis genome and, eventually, for 
preparing diagnostic and therapeutic reagents. (Hamer) 

- 18-24 



Z01-A1-00190-01-0SD 
IV. Assessment of Risks Associated with Recombinant DNA Research 

In December 1975, Drs. Malcolm Martin and Wallace Rowe were asked by the 
Recombinant DNA Advisory Committee to conduct experiments in NIH containment 
facilities to evaluate potential risks associated with recombinant DNA experi- 
mentation. The first phase of such studies was carried out in P4 containment 
facilities located in Frederick, Maryland or on the NIH reservation and involved 
the inoculation of weanling mice or newborn hamsters with E. coli K12 containing 
polyoma-plasmid or polyoma-lambdaphage recombinants. In both animal systems, 
the polyoma-recombinants were not transferred out of their IE. coli hosts follow- 
ing parenteral inoculation. As controls, polyoma DNA, liberated from the pro- 
karyotic vector DNA, was infectious (mice) or tumorigenic (hamsters). Mice 
inoculated with purified recombinant DNA preparations containing a single copy 
of viral DNA never developed a virus infection whereas, a few animals infected 
with a recombinant phage DNA preparation harboring a head-to-tail dimer of viral 
DNA developed an infection. Intact recombinant DNA preparations as well as 
purified phage particles induced tumors in 5-19% of inoculated animals, a 
value similar to that observed following injection of supercoiled PY DNA (19%). 
It should be emphasized that potentially infectious or tumorigenic recombinant 
DNA molecules are not transferred out of EK2 hosts into mammalian cells in 
either of the animal model systems we used. Even in those cases in which animals 
inoculated with recombinant phage or purified recombinant DNA did develop a 
viral infection or tumors in worst case experiments conducted in a laboratory 
setting, the biological activity of recombinant preparations was at least a 
million-fold less active than polyoma virus particles. 

We have recently received polyoma plasmid and polyoma lambda recombinants 
in EK1 E. coli host cells, which were constructed by investigators in Europe. 
We have agreed to begin animal testing studies in the fall of 1979. In addition, 
we plan to inoculate bacteria containing cloned Harvey Sarcoma and Friend 
Leukemia virus DNAs into appropriate test animals to extend our risk-assessment 
analysis to another virus (retravirus) system. (Drs. Martin and Rowe and 
their friends. ) 

Publications 



Israel, M.A., Chan, H., Rowe, W., and Martin, M.A.: Biologic activity of 
polyoma virus DNA in animals. Journal of Virology , 29:990-996, 1979. 

Israel, M.A. , Chan, H., Rowe, W. and Martin, M.A.: Molecular cloning of 
polyoma virus DNA in E_. coli . I. Plasmid vector system. Science , 203:883- 
887, 1979. 

Chan, H.W., Israel, M.A. , Garon, C.F., Rowe, W.P., and Martin, M.A.: 
Molecular cloning of polyoma virus DNA in E_. coli . II. Lambda phage vector 
system. Science , 203:887-892, 1979. 

Howley, P.M., Israel, M.A. , Law, M.F., and Martin, M.A.: A rapid method for 
detecting and mapping homology between heterologous DNAs. Evaluation of 
polyomavirus genomes. Journal of Biological Chemistry , 254:4884-4891, 1979. 



18-25 



Z01-A1-00190-01-OSD 

Hamer, D. , Smith, K. , Boyer, S., and Leder, P.: "SV40 Recombinants Carrying 
Rabbit B-Globin Coding Sequences. Cell 17:725-735, 1979. 

Hamer, D. and Leder, P.: SV40 Recombinants Carrying,a Functiona Splice 
Junction and Polyadenylation Site from the Mouse B -Globin Gene. Cell 17: 
737-747, 1979. 

Simmons, D.T. and Martin, M.A.: Common methionine tryptic peptides near the 
amino-terminal end of primate papovavirus tumor antigens. Proc. Nat. Acad. 
Sci. U.S. 75:1131-1135, 1978. 

Simmons, D.T., Takemoto, K.K., and Martin, M.A. : Properties of simian virus 
40 and BK virus tumor antigens from productively infected and transformed 
cells. Virology 85:137-145, 1978. 

Simmons, D.T., Chang, C, and Martin, M.A. : Multiple forms of polyoma virus 
tumor antigens from infected and transformed cells. J. Virol. 29:881-887, 1979. 

Honors and Awards 



Dr. Malcolm Martin was awarded the Public Health Service Superior Service 
Award in May for his activities involving recombinant DNA research. Dr. Martin 
continues to serve on the Editorial Boards of Journal of Virology and Journal 
of Biological Chemistry . In June, he completed a four year appointment as a 
member of the Virology Study Section, DRG, NIH. In April, Dr. Martin was 
invited to deliver a lecture at the Cogene-The Royal Society of London Meeting 
on Recombinant DNA held at Wye College, Kent, England. 



^18-26 



LABORATORY OF BIOLOGY OF VIRUSES 

1979 Annual Report 

Table of Contents 



Z01-AI 
Project Number 

Summary 

00123-13 

00124-10 

00125-10 

00126-06 

00127-12 

00128-12 
00156-04 



Messenger RNA: Regulation of Synthesis, 
Modification, and Translation — Moss 

Replication of the Parvovirus, KRV — 
Salzman 

Mechanisms of Viral DNA Replication, 
Transcription, and Integration — Salzman 

Restriction Enzyme Analysis of Vaccinia 
Virus DNA — DeFilippes 

Structure and Function of Genetic and 
Protein Components of Defective 
Parvoviruses — Rose 

Properties of Adenovirus DNA — Garon, 
Rose 

Structural Characterization of DNA Virus 
Genomes — Garon 



Page 
19-1 

19-14 

19-19 

19-23 

19-27 

19-30 
19-33 
19-36 



Annual Report of the Laboratory of Biology of Viruses 

National Institute of Allergy and Infectious Diseases, NIH 

October 1, 1978 to September 30, 1979 

The goal of the Laboratory of Biology of Viruses is to determine 
how viruses replicate. At the molecular level, this involves investigation 
of the structure of the virion and of its purified component parts, of 
mechanisms of replication and transcription and expression of the viral 
genome. Emphasis is also placed on cell-virus interactions including 
the process of cell transformation. We believe that information of this 
kind, although difficult to obtain, is fundamental to understanding 
disease processes and vital to achieving a rational basis for chemotherapy. 

Papovaviruses 

The current studies have tried to define some of the processes 
responsible for the formation of viral messenger RNA molecules. Thus 
far, these studies have provided the precise structures of the viral 
mRNA molecules, have defined the nature of the templates used for the 
synthesis of viral transcripts, have located promoters on the SV40 
genome that are recognized by E. coli RNA polymerase and have compared 
SV40 DNA I and SV40 DNA I nucleoprotein complexes as templates for 
transcription . 

The Structure of Early and Late Viral mRNA 

The mechanism of splicing of the early and late cytoplasmic species 
of SV40 mRNA has been studied using a technique which first involves the 
isolation of specific regions of the viral genome. Restriction enzyme 
cleavage of SV40 DNA generates specific DNA fragments which can be 
fractionated using column chromatography. These fragments are then 
annealed to viral mRNA and the reaction products are fractionated by 
velocity sedimentation. The DNA fragment which is annealed to the viral 
mRNA is used as a primer for the enzyme, reverse transcriptase, and a 
complementary DNA copy of the mRNA is made. The sequence and structure 
of the cDNA has been compared with the known nucleotide sequence of 
SV40. This procedure has been used to characterize the late mRNA which 
codes for the major structural viral coat protein and has provided 
direct physical evidence for two early mRNA species. The regions of the 
DNA that have deleted in both species of early mRNAs have been determined. 
Also, the nucleotide sequences across these spliced regions have been 
determined and correlated with the mRNA coding for the large T and small 
t antigens. These two mRNA species were determined to have the same 5 1 termini 
which would suggest two levels for control of genetic expression. One 
would be the regulation of initiation of transcription at a common 
promoter; the other would involve post- transcriptional splicing. 

To further characterize the mechanism of transcription of early 
SV40 RNA, nascent RNA chains that are attached to template DNA molecules 
are being isolated and characterized by the same methodology employed to 
study the processed cytoplasmic species of early viral RNA. Transcription 



19-1 



complexes have been isolated from infected cells which continue viral 
mRNA synthesized in vitro . The structure of these nascent chains gives 
insight into the initial transcription products and the mechanisms that 
operate to regulate transcription. We are investigating differences 
between RNA's formed in vivo and in vitro . The structure of nascent 
chains will provide precise information about the primary products of 
transcription. We are also studying the use of isolated nuclei for the 
study of the regulation of transcription. Isolated nuclei are able to 
sustain in vitro synthesis of viral RNA. In this case, the SV40 template 
in the cell nucleus is present as a nucleoprotein complex containing the 
five histones. This is in contrast to in vitro synthesis of nascent RNA 
chains described above which uses a less well defined transcriptional 
complex. 

These nascent RNA molecules will be probed with specific SV40 DNA 
fragments to determine their structure. In order to acquire a library 
of these specific fragments, recombinant technology has been employed. 
Cloning of SV40 DNA fragments using the plasmid pBR322 and E. coli 
should provide sufficient amounts of specific DNA fragments which can be 
used to probe nascent RNA molecules and determine the mechanisms operating 
during regulation of transcription. 

Since sufficient amounts of specific SV40 DNA fragments can be 
obtained from cloning, we will be able to study how synthesis of specific 
RNA sequences are affected by mutations within the genome. Further 
characterization of RNA species obtained following Proflavin treatment, 
which blocks RNA processing, will aid our understanding of the mechanism 
that operated to regulate transcription. (Thompson, Bina-Stein, Salzman) 

Localization of RNA Polymerase Promoters on SV4Q DNA 

Transcripts of SV40 DNA synthesized by Escherichia coli RNA polymerase 
have been characterized. This model system has been used for the development 
of new methods applicable to the analysis of the mechanisms involved in 
the synthesis of the viral messenger RNAs in SV40- infected cells. It 
has been previously shown that E. coli RNA polymerase recognizes specific 
initiation sites on SV40 DNA. Except for one of them, determination of 
the location of these sites on the SV40 DNA map is only approximate. No 
specific termination site for transcripts has been identified, and 
consequently RNA polymerase generates a heterogeneous population of 
molecules. The size of some of these RNA molecules is several times the 
length of the viral genome. 

We have shown that, after binding of the E. coli RNA polymerase to 
SV40 DNA, it was possible to cleave such transcriptional complexes with 
"single-cut" restriction endonucleases (Bam H , Eco R_, Hpa II). The 
addition of ribonucleotide triphosphates to these linearized complexes 
leads to the synthesis of defined species of RNA which can be analyzed 
by electrophoresis. The determination of the size of each of these RNAs^ 
together with the assignment of the DNA strand on which they are transcribed 
will allow the precise mapping of the various RNA promoters (Reuveni, 
Lavialle, Salzman) . 

19-2 



Transcriptional Properties of SV40 Nucleoprotein Cores 

Simian Virus 40 (SV40) provides an excellent model system for 
investigating the process of transcriptional regulation in eukaryotic 
cells. Many structural aspects of viral transcriptional complexes and 
mRNA molecules have been determined. In addition, the entire nucleotide 
base sequence of SV40 DNA has been determined. Similar to most eukaryotic 
chromatin, during the SV40 infection cycle, cellular histones ELA, 
H 2 B, , EL, and H. are bound to the viral DNA. Late in the infection 
cycle, nistone H, also becomes associated with the bulk of the viral 
chromatin in a stable nucleoprotein complex. Just prior to encapsidation, 
the SV40 nucleoprotein complex undergoes a redistribution of proteins 
and histone H, is replaced by viral proteins. Since binding of H, on 
chromatin may suppress transcription of DNA sequences, we were interested 
in determining if the viral proteins also acted to modify or regulate 
the process of RNA synthesis. Such regulatory properties could be 
important in a clearer understanding of the process of "early" mRNA 
synthesis during infection of the host cell. In view of the close 
similarity between this SV40 DNA-histone-viral protein nucleoprotein 
complex and cell chromatin structure, such studies would also be of 
interest in a more complete understanding of the general problem of 
transcriptional regulation in eukaryotic cells. 

A nucleoprotein complex can be isolated from purified SV40 virions 
under very mild, physiological conditions. These nucleoprotein cores 
are potentially active transcriptional complexes. These core complexes 
do not contain endogenous RNA polymerase activity; however, 95-100% are 
able to form active transcriptional complexes. The transcriptional 
activity of those complexes is extremely high and is similar to the 
activity obtained with purified SV40 Form 1 DNA. This observation 
contrasts with published data, in which the level of transcription of 
nucleoprotein complexes (SV40 DNA-Histone) is generally less than 20% of 
the activity of deproteinized SV40 DNA. The results suggest that the 
viral proteins may play a role in the "activation" of the nucleohistone 
complex. Analysis of the composition and structure of the SV40 nucleoprotein 
core are being carried out and should allow an understanding of the 
unexpectedly high efficiency of the SV40 core as a template for RNA 
synthesis. (Brady, Lavialle, Salzman) 

Parvoviruses 



Kilham Rat Virus 



The specific biochemical mechanisms involved in the replication 
of the autonomous parvovirus, KRV, has been examined in a rat nephroma 
cell line. The virus, isolated originally from a rat sarcoma, contains 
one molecule of linear, single-stranded DNA and three capsid proteins. 
Little information is available about replication of this single-stranded 
DNA virus in a eukaryotic cell or on the transcription of this DNA to 
make viral proteins. It was found that the virion KRV-DNA can self 
prime in vitro to synthesize the double-stranded replicative intermediate. 



19-3 



To define the structure of this self priming terminus, which is important 
in DNA replication, the nucleotides in the 3 1 DNA terminus have been 
sequenced. The DNA structure which can be deduced from the sequence 
contains a terminal hairpin. This is consistent with the finding that 
DNA replication is a self priming reaction. 

It had previously been found that in an infected cell, two KRV 
specific mRNAs are synthesized. The DNA which is homologous to the 
major 21S viral mRNA has been mapped using both the "Southern" blotting 
technique and measurement of the "R loops" seen in the electron microscope. 
Inhibition studies suggest that these KRV mRNAs are synthesized by 
cellular RNA polymerase II. Preliminary studies also show that there are 
at least three virus induced proteins synthesized during infection. 

Adeno Associated Viruses 

The main objectives in studying defective human parvoviruses (AAV) 
are to define specific biochemical mechanisms used in synthesizing 
their DNA, RNA and proteins, to identify and characterize the helper 
virus ^nediated step(s) required for their replication, to relate biochemical 
findings to normal cellular processes, and to determine conditions for 
selective interference of both AAV and Helper virus (adenoviruses, 
herpesviruses) infection. 

Factors Which Confer Permissivity on Defective AAV 

Human adenovirus (Ad) serotypes provide an early factor (s) that is 
necessary for adenovirus-associated virus (AAV) multiplication in human 
cell lines. However, little if any AAV production occurs in primary 
African green monkey kidney (AGMK) cells co- infected with AAV and a 
helper human Ad (non-permissive infection) unless cells are additionally 
infected with SV.. (permissive infection) . To determine the basis of 
the host restriction of AAV replication in AGMK cells, AAV DNA, RNA and 
protein synthesis were analyzed under various conditions of infection. 
Hybridization reactions revealed no detectable AAV-specific DNA or RNA 
in infections with AAV alone or in combination with SV^q. In co- infections 
with AAV and Ad 5 or Ad 7 , the synthesis of both AAV- and Ad-specific DNA 
and RNA occurred without a significant rise in titre of either virus. 
During non-permissive infection, however, AAV DNA synthesis was abnormal 
in that an expected accumulation of single-stranded progeny molecules 
was not observed. Finally, although intact 20S AAV transcripts were 
present in the cytoplasm of AGMK cells during non-permissive infection 
(in amounts ranging from 50 to 80% of that found during permissive 
infection) , AAV-specific polypeptides were not demonstrable by polyacrylamide 
gel electrophoresis. Taken together, these experiments indicate that 
the host restriction of AAV replication in AGMK cells is exerted at the 
level of translation of the single AAV messenger RNA. In addition, it 
appears that one or more of the AAV polypeptides specified by this 
message is required for the production of single- stranded AAV progeny 
DNA. The fact that coinfection with a simian adenovirus, SV15, permits 
complete replication of both Ad and AAV (analogous to coinfection with 



19-4 



SV40) strongly suggests that an Ad(SV15) gene product also exerts a 
regulatory effect on expression of the AAV mRNA (as well as several late 
human Ad mRNAs) in AQVIK cells. Isolation and characterization of this 
gene product should be relevant to possible approaches for selectively 
interfering with virus infection. (Buller, Sebring, Rose) 

AAV DNA Replication 

Further understanding of mechanisms involved in DNA replication was 
obtained by studying AAV DNA synthesis in cell- free extracts. Extracts 
of nuclei from KB cells doubly infected with adenovirus-associated virus 
type 2 (AAV) and adenovirus type 2 (Ad) were examined for their ability 
to synthesize various molecular forms of AAV DNA. It was found that 
replicating AAV DNA molecules elongate to yield genome length hairpins 
or linear, open-ended duplexes. Both plus and minus DNA strands serve 
as templates for this synthesis which apparently does not reinitiate new 
rounds in vitro . Replicating Ad DNA molecules also could be identified 
in these extracts, but incorporation of [%]TTP into Ad DNA was diminished 
80-90% in coinfections with AAV as compared to nuclei extracts prepared 
from cells infected with Ad alone. These results confirm our previous 
in vivo studies which indicated that a self -priming mechanism is involved 
in the replication of AAV DNA, studies that represented the first reported 
evidence for this mode of initiating DNA synthesis. Investigations with 
extracts will continue with the objective of identifying and characterizing 
enzymatic and regulatory components that participate in viral and cellular 
DNA replication. (Sebring, Buller, Rose) 

Adenoviruses 

The major objective of these studies has been the application of 
physical and biochemical techniques to map and to define the structure 
of specific genetic regions in the genomes of both oncogenic and non- 
oncogenic human adenoviruses, with the ultimate goals of (1) relating 
these sequences to those biochemical activities which permit these 
viruses to influence or gain control of cellular functions (e.g., cell 
lysis and transformation) and (2) of defining basic mechanisms for 
regulation of genetic expression (e.g., initiation of DNA synthesis). A 
number of unique features have been described which may have implications 
in viral replication or carcinogenesis or both. 

DNA Terminal Proteins 

The DNA of adenoviruses is normally isolated as a linear duplex 
molecule of discrete size using standard procedures of extraction with 
protealytic enzymes, phenol and SDS. Recently, it has been possible to 
demonstrate forms of DNA that are either circular or oligomeric by using 
procedures which do not involve proteolytic enzymes. In these latter 
cases, the DNA molecules are always joined at their ends, and it appears 
that either end of a molecule may interact with the other end of the 
same molecule or with either end of another molecule. The circles and 
oligomers are resistant to treatment with 4M guanidinium chloride, 4M 
urea, formimide, sarkosyl or mercaptoethanol. Treatment with proteases, 



19-5 



however, rapidly converts the circles and oligomers to linear double- 
stranded monomers, as does treatment with SDS. It had been previously 
proposed that there is a protein attached to each end of the DNA molecule 
and that this protein is responsible for the formation of the observed 
complexes. Subsequent studies have clearly demonstrated the presence 
of a 55,000 dalton protein very tightly linked to the DNA molecule. 
Although the function of the bound protein is unknown, it has been 
proposed that it may be involved in DNA replication by allowing initia- 
tion or completion of the 5 1 ends of the progeny strands. It has also 
been suggested that the protein may have a structural role by circularizing 
the DNA within the virus particle, may protect the DNA from exonuclease 
digestion or may act as an endonuclease in a hairpin model of DNA replication. 
Furthermore, it has been recently demonstrated that the efficiency of 
transfection with the Ad 5 DNA protein complex is about 100-fold higher 
than that of pronase- treated Ad 5 DNA. 

Brown et al. (Journal of Virology 16, 366) first demonstrated that 
these DNA-protein complexes did not migrate into agarose gels during 
electrophoresis. This observation provided the basis for a useful means 
for detecting not only the presence or absence of terminal protein 
components but the identification and purification of terminal DNA 
sequences as well. We have fractionated the Ad 2 DNA-protein complex on 
hydroxy lapatite columns and analyzed the DNA eluate by agarose gel electro- 
phoresis. Although the initial DNA-protein complexed did not migrate 
into agarose gels, >75% of the initial DNA molecules in the DNA eluate 
were now able to enter the gels in spite of the absence of protease or 
SDS treatment. When the protein in the DNA eluate was labeled in vitro 
with 125i f protein was not seen associated with the DNA that migrated 
into the gels. When the DNA eluate was examined in the electron microscope, 
DNA was found to be essentially linear. The re-addition of a DNA-free 
protein fraction to the DNA eluate produced circularization of about 20% 
of these linear molecules. We conclude that circularization of the 
adenovirus DNA-protein complex as well as its inability to migrate into 
agarose gels is likely due to an aggregate of a virion component (s) in 
addition to the covalently-attached 55K protein previously described. 

Significant quantitative differences were noted when Ad 18, a 
highly oncogenic serotype, was extracted with 4M guanidinium chloride 
and the DNA-protein complexes assayed in similar fashion. Purified 
virions of adenovirus serotype 2 and 18 were applied to gradients 
containing guanidinium, peak fractions were pooled and the DNA-protein 
complexes dialyzed and compared. 35-55% of the molecules from each 
serotype appeared circular in the electron microscope. While the DNA- 
protein complexes from serotype 2 were effectively bound to glass fiber 
filters (99%) under the conditions employed, over 45% of the guanidinium 
purified material isolated from serotype 18 passed unimpeded through the 
filter under identical conditions. Of some interest was the fact that 
nearly all molecules in this fraction were circular. Bound material 
could be effectively released from filters with 1% SDS or pronase 
digestion. DNA molecules from either serotype when extracted with pro- 
teolytic enzymes did not bind to filters. Approximately half of the 
guanidinium purified material from serotype 18 was shown to enter 



19-6 



agarose gels upon electrophoresis. The structural implications of this 
population of molecules which apparently does not bind to glass fiber 
filters and which easily penetrates agarose gels, yet retains the 
ability to circularize, is of considerable biological interest. (Garon, Rose) 



Pox Viruses 



Vaccinia 



Our primary objective is to determine the molecular events that lead to 
the selective transcription of DNA and subsequent processing and translation 
of messenger RNA (mRNA) . Poxviruses provide a unique and important system 
for such studies since the enzymes needed for transcription and mRNA 
modification are packaged within the core of infectious virus particles. 
A single enzyme complex containing three activities - RNA triphosphatase, 
RNA guanylyltransf erase, and RNA (guanine-7-)methyltransf erase - has been 
purified from vaccinia virus. Acting consecutively, the individual activities 
in the complex are capable of modifying the 5 '-end of nascent RNA molecules 
to form a cap structure in vitro . Extracts from uninfected human cells 
were shown to carry out a similar set of reactions, however, three separate 
enzymes were found instead of a single complex. The donor and acceptor 
substrate specificities of the different viral and cellular enzymes 
suggested mechanisms used for processing mRNA in vivo . Evidence for temporal 
control of vaccinia virus gene expression at the transcriptional level 
has been obtained by DNA: RNA hybridization studies and by translation of 
viral mRNA in a cell-free protein synthesizing system. Transcriptional 
and translational maps of the vaccinia virus genome are being prepared 
using DNA fragments, produced by restriction endonuclease digestion, 
that have been cloned in phage lambda using approved DNA recombinant 
technology. 

Major Findings 

1. Post-transcriptional modification of mRNA 

A. Isolation and characterization of viral capping and methylating 
enzymes . 

Members of this research group previously identified the 5 '-terminal 
cap structure m 7 G(5')pppN consisting of a 7-methylguanosine (m 7 G) linked 
by a 5' to 5' triphosphate bridge to one or two consecutive 2 ' -O-methylated 
ribonucleosides (N) present in viral and cellular mRNAs. The mechanism 
of cap formation was determined by purifying the appropriate enzymes from 
vaccinia virus. The latter, included (i) an enzyme complex containing both 
mRNA guanylyltransf erase, which is the capping enzyme, and mRNA(guanine-7-) 
methyltransf erase and (ii) a separate mRNA (nucleoside-2'-) -methyl transferase. 
Further studies of the purified enzyme complex during the past year revealed 
a third activity, RNA triphosphatase. On a molar basis, the RNa tri- 
phosphatase is nearly 100-fold more active than the associated mRNA 
guanylyltransf erase. Consequently, both triphosphate and diphosphate- 
ended RNAs are capped at similar rates and modification of the 5 '-end of 
incomplete or nascent transcripts occurs in the following order: 



19-7 



pppN,-N 2 .. . .-*• ppN-jHSL. . .+ Pi (i) 

GTP + ppN,-N 2 . . .+ G(5' JpppKL-N . .+ PPi (ii) 

AdoMet + G(5 ' )ppFN,-N 2 . . .-> m G(5' )pppN, -N 2 - . .+ MoHcy (iii) 

AdoMet + m 7 G(5')pppN 1 -N : ,...^ m 7 G ( 5 ' ) pppN?-N„ . . . + AdoHcy (iv) 
(Venkatesan, Mass) 

B. Isolation and characterization of cellular capping and methylating 
enzymes . 

Previously, we detected capping activity in crude extracts prepared from 
HeLa cell nuclei. During the past year, this enzyme has been purified 
approximately 1,000-fold and extensively characterized particularly 
with respect to its donor and acceptor substrate specificities. This 
analysis has provided important information regarding the mechanism of 
capping cellular RNA. Of a variety of potential donor molecules, only 
GTP and ITP were utilized by the purified enzyme. These results indicated 
that the mRNA guanylyltransf erase recognizes the oxygen on the purine 
ring of GTP, discriminates between ribose and deoxyribose sugars, and 
requires a triphosphate. The inability to use m GTP as a donor indicates 
that methylation occurs after capping. With regard to acceptor molecules, 
the enzyme is specific for a diphosphate-ended oligo- or polyribonucleotide. 
The inability to efficiently cap triphosphate-ended acceptors is explained 
by the absence of an associated RNA triphosphatase. In that respect, and also 
by the absence of an associated mRNA( guanine- 7-) methyl transferase, the 
HeLa cell capping enzyme differs from the one isolated from vaccinia virus. 
This result is consistent with our previous purification of a specific 
HeLa cell mRNA (guanine-7-)methyltransferase. Although the possibility of a 
pre-transcriptional capping mechanism has been considered by others, our 
finding that the minimum length acceptor is a dinucleotide such as ppGpC 
makes this mechanism unlikely. In fact, the Km for a dinucleotide is 
higher than for a polymer indicating that a longer oligonucleotide or 
short polyribonucleotide is a better acceptor. We suggest that in vivo , an 
RNA triphosphatase removes the third phosphate from the 5 '-end of short 
unfinished primary transcripts and that they are then capped by the 
enzyme that we have described. If this enzyme also caps RNA at sites 
of cleavage, then additional kinases capable of forming diphosphate- 
ends must exist. (Venkatesan, Moss) 

c. Coupled transcription and modification 

One of the major advantages of working with vaccinia virus is that 
all of the enzymes necessary for synthesis and modification are contained 
within the purified virus particle. After irradiating purified vaccinia 
virus particles with ultraviolet light to form pyrimidine dimers in the 
DNA, only short abortive transcription products were made. Analysis of 
these products indicated, however, that they were capped, methylated and 
polyadenylylated. These results suggested to us that neither completion 
of an RNA chain nor processing from a polycistronic precursor was required 
for modification of either end of the RNA. The presence of the poly (A) 



19-8 



tract at the 3 ' -end of the short transcript further suggested that a 
slow-down or cessation of transcription, rather than a specific 3 '-terminal 
sequence, serves as a signal for polyadenylylation. (Gershowitz, Moss) 

2. Organization and regulation of the vaccinia virus genome 

A. In vitro translation of irrmediate early, early and late classes of 
RNA from vaccinia virus infected cells " " " "'" 

Cytoplasmic RNA, isolated at various times after vaccinia virus 
infection, was translated in a message-dependent cell-free system prepared 
from rabbit reticulocytes. When programmed with RNA extracted at 2 hr after 
infection, early viral proteins were made and formation of cellular proteins 
was diminished. Primarily late proteins were synthesized using RNA 
extracted at 4 or more hours after, infection, suggesting that the switch 
in protein synthesis is regulated principally by changes in RNA concentration 
rather than by modification of the translation apparatus of the cell. 
Immediate early RNA was obtained by infecting cells in the presence of 
inhibitors of protein synthesis. Comparisons between immediate early and 
early gene products did not reveal a class of early genes that require 
protein synthesis for expression. On the contrary, seven polypeptides, 
of which a 28,000 dalton species was most prominent, were synthesized in 
relatively greater amounts with immediate early RNA than with early RNA. 
This result suggests the possibility that expression of certain immediate 
early genes is regulated by a feed-back mechanism. (Cooper, Moss) 

B. Analysis of the terminal repetition within the vaccinia virus genome 

Previously we reported that a 3.54 ym duplex was formed by annealing 
the two ends of the vaccinia virus genome. This inverted terminal repetition 
was estimated to be about 10,000 nucleotide base pairs long. To further 
characterize this structural feature, restriction maps were made of the 
ends of the genome using the following enzymes: Eco RI, Hpa II, and Hind II. 
To accomplish this, we developed a novel mapping method, involving use of 
"nick translated" probes prepared from the terminal fragment, to order 
partial digestion products. From this study, we concluded that identical 
restriction sites are present at the 2 ends of the genome for approximately 
10,000 nucleotides confirming our previous electron microscopic analysis 
and work of other investigators. 

To determine whether the inverted terminal repetition is transcribed 
in vivo , 32 P- labeled cytoplasmic RNA was obtained from infected cells. 
This RNA was then annealed to restriction fragments, prepared from the terminal 
repetition and adjacent regions of the DNA that were immobilized to a 
nitrocellulose sheet. We found that early RNA hybridized to the repetition 
except for the terminal 3,000 nucleotide base pairs indicating that it 
is indeed transcribed. (Barbosa, Moss) 

C. Formation of recombinant DNA molecules containing portions of the 
vaccinTa virus genome . 

DNA recombinant technology developed within the past few years 
followed by a relaxation of the guidelines for carrying out such research 

19-9 



has allowed us to clone portions of the vaccinia virus genome in phage 
lambda. Our initial approach has been to clone successive Eco RI 
fragments of DNA starting from one end of the genome. Thus far, this 
has included a 9,500 nucleotide base-pair segment containing most of the 
terminal repetition, a 6,500 nucleotide base-pair segment containing 
unique sequences adjacent to this, and the next 4,000 nucleotides base- 
pair segment. In order to clone the end piece of DNA, it was first 
necessary to remove the terminal cross-link with a single-strand specific 
nuclease and then add synthetic Eco RI linkers. Each of the recombinant 
DNA clones were identified by restriction endonuclease digestion and 
gel electrophoresis. Studies are now in progress using the cloned genome 
fragments to obtain a transcription and translation map. (Wittek, Chan, Moss) 

3. Action of specific antiviral agents 

Isatin-S-thiosemicarbazone is known to specifically inhibit vaccinia 
virus replication at a late stage. At 6 hr after infection, viral protein 
synthesis was inhibited by about 95%. We confirmed that a portion of 
the virus-specific RNA appears to be degraded (B. Woodson and W. K. Joklik, 
1965, Proc. Natl. Acad. Sci. , USA 54: 946-953). Nevertheless, the amount 
of viral RNA that was capped, properly methylated, and polyadenylylated, 
was reduced by only about 50%. Moreover, RNA from IBT-treated cells 
stimulated cell-free protein synthesis to one-half the level obtained with 
RNA from control cells. Polyacrylamide gel electrophoretic analysis 
further demonstrated that RNA from IBT-treated cells was translated into 
late viral proteins in vitro . Thus, it seems possible that the inhibition 
of protein synthesis in IBT-treated cells does not result entirely or 
directly from either an inhibition of mRNA synthesis or from a depletion 
of mRNA caused by accelerated degradation. An alternative possibility, 
that accelerated degradation is secondary to a more immediate effect 
of the drug on protein synthesis, was considered. (Cooper, Moss) 

Structural Studies of the Viral Genome 

Vaccinia DNA has covalently-linked-ccmplementary (CDC) ends which 
define each end of the physical map of the genome. These CLC end segments 
can be identified in each set of segments that are produced by a particular 
restriction enzyme. The set of segments is alkali denatured, neutralized 
and passed through a BND-cellulose column from which only the CLC segments 
elute as duplex chains. The end segments have been identified for 
several restriction enzymes such as Sal I, Hind III, Kpn I and Xho I. 
For Sal I, the end segments are identical and small with a,-mass of 
2.1 X 10 6 daltons. For Xho they are larger (both 3.9 X 10 ) and for 
Hind III they are larger still (17.6 and 13.5 X 10 6 ) . 

The restriction enzymes listed above as well as several others are 
used to digest vaccinia DNA to obtain unique DNA segments which are 
separated by agarose electrophoresis. To obtain a physical map, segments 
produced by one enzyme are isolated from agarose and digested by a 
second enzyme to find overlapping regions. The end segments described 
here serve as reference points from which the maps are continued. The 
Sal I and Hind III map of vaccinia strain WR are now almost completely 
known. Kpn and Xho maps are also near completion. 

19-10 



A particular method which has helped to generate reproducible 
partial DNA segments involves restriction enzyme digestion in the presence 
of actinomycin D. The original observation that the drug blocked 
complete digestion with Hind III has been extended to 3 other restriction 
enzymes, Sal I, Xho and Kpn. None of these digests produce as long a 
partial as the one half length vaccinia DNA molecule generated with Hind 
but they do contain several smaller linked segments which help to establish 
the map. 

Initial experiments designed to locate the origin of replication 
involve pulse labeling HeLa cells with H-thymidine after infection with 
high multiplicities of vaccinia virus. A peak of synthetic activity 
occurs 2.5 hours after infection. From 2.5 to 3.5 hours after infection 
alkaline and neutral sucrose gradients of cytoplasmic (viral) DNA 
reveal a spectrum of molecular sizes with about 10 - 20% of the DNA in 
full length molecules. These DNA species will be examined in a Dintzis 
(Proc. Natl. Acad. Sci. £7: 247, 1961) type experiment. 

Several experimental techniques have evolved during the year. The 
method of Vogelstein and Gillespie (Proc. Natl. Acad. Sci. 76_: 615, 
1979) for extracting DNA segments from agarose with glass powder has 
been modified so that it is now possible to routinely recover intact, 
70% or more of yg amounts of large vaccinia DNA segments. Also, poly 
rC columns have been used to hybridize segments which contain short 
stretches of GC rich regions in duplex DNA. Using this column technique, 
we have found that the largest Sal I segment binds to the column. Since 
GC rich regions may provide a structural basis for a signal element, the 
Sal I segment will be digested with other enzymes to localize the GC 
region. Finally, we have started to use ..malachite green columns to 
fractionate sheared vaccinia DNA (6 X 10 daltons) according to base 
composition. Those fragments with the least affinity for the column 
have the highest average GC content. They will be radiolabeled and 
hybridized to Sal I digests to identify those segments from which they 
originated. (De Filippes) 

Molluscum contagiosum 

Molluscum contagiosum virions were isolated from clinically typical 
skin lesions and the DNA extracted and characterized using techniques of 
electron microscopy and agarose gel electrophoresis. The structural char- 
acteristics determined in these studies tend to place the MCV genome among 
other members of the poxvirus group in terms of genome molecular weight 
and organization. The apparent denaturability of the NCV genome into a 
continuous, single-stranded circle would point to the presence of 
terminal cross-links in the DNA molecule. So far, this structural 
arrangement appears to be unique to members of the poxvirus group. The 
objective of these studies has been to define structural features of 
these molecules and to relate them to the biochemical events involved in 
the replication and growth of this virus in the cells they infect. 

Extracts from molluscum contagiosum lesions when stained with sodium 
phosphatungstate and examined in the electron microscope appears to 



19-11 



contain many structurally mature virus particles of both C and M forms. 
However, the virus has not been successfully propagated in the laboratory. 
Molecular information about this virus has been limited to description 
of size, shape and composition and has been difficult to obtain. The 
structural characteristics determined in our initial studies (Virology 81: 
247) tend to place the MCV genome among other members of the poxvirus 
group in terms of molecular weight and organization. The average molecular 
weight of MCV-DNA was calculated to be 118 X 10 and appeared to be 
extremely sensitive to both mechanical shear and nuclease damage. 
Single-stranded circles measuring twice the length of linear molecules 
were observed following high levels of denaturation. This now appears 
to be a generalized feature of all poxvirus DMAs so far examined. The 
biological function, if any, of the unique terminal-cross- links is 
unknown. Also of special interest was the fact that the most easily 
denaturable regions of the MCV genome (and presumably that of the highest 
AT content) appeared to be the end 19% of the DNA molecule. That the MCV 
genome differs markedly from that of vaccinia is clear from the visual 
denaturation profiles and from the restriction endonuclease cleavage 
patterns obtained in our initial studies. Further differences were noted 
in the extractability of the various poxvirus genomes. Procedures used 
for successfully extracting and purifying the DNA molecules from one 
viral genome proved completely unsuitable for the other. 

Previously, little was known about whether most isolates belonged 
to the same or different MCV strains or whether a given strain was 
always isolated from similar clinical sources. A survey of the restriction 
endonuclease fragment patterns of several independent MCV isolates was 
initiated. Lesions obtained from individual patients were never pooled 
but rather purified, extracted, and assayed as a single isolate. Initially, 
eleven such isolates were processed. In these studies, lesions isolated 
from various sites on an individual patient appeared to contain virus 
whose DNA showed identical gel patterns. Furthermore, virus was isolated 
from the lesions of patients' relatives which, when assayed by the above 
restriction endonuclease procedure, showed gel patterns identical to 
those of the patients themselves. Interestingly, only 3 characteristically 
frequent patterns were observed among eleven independent isolates. 
Restriction endonuclease digestion produced 11 to 20 fragments ranging 
in size from 4 X 10 to 31 X 10 daltons depending on the virus isolate. 
Mixing experiments showed comigration of some fragments in all patterns. 
Mixing experiments showed comigration of some fragments in all patterns. 
Although comigration of DNA fragments does not necessarily mean base 
sequence identity, extensive comigration would imply significant genetic 
or organization similarity among the viral genomes. Direct comparisons 
of these gel purified fragments by hybridization or heteroduplex analysis 
have not been attempted due to limits in the quantity of viral DNA 
obtainable from each MCU isolate. 

Although we have not collected a large enough body of data to make 
any reasonable assessment of the relationship between the clinical 
characteristics of the disease and the restriction endonuclease 
cleavage patterns, it may be possible once the data are accumulated, to 
relate tehse observations and to develop a useful molecular epidemiological 



19-12 



scheme. Such a scheme would make possible rapid identification and 
classification of MCV isolates without the necessity of propagating the 
virus in the laboratory. Furthermore, since the degree of sensitivity 
of such a system is limited to only the number of cleavage sites recognized 
by a given restriction enzyme, other available enzymes may easily be 
substituted. 

We propose to continue to define structural features of these 
poxvirus DNA molecules with the aim of (1) comparing genetic variation 
among members of this group, and of (2) relating these features to those 
biochemical events which are involved in the replication of these agents. 

Honors and Awards 

Dr. NOrman P. Salzman continued to serve on the Editorial Board 
of the Journal of Virology and on the Editorial Advisory Board, Biochemistry, 
and Scientific Board of the Coordinating Council for Career Research. 
He serves as Professorial Lecturer, Georgetown University School of 
Medicine, and was an invited participant and Session Chairman at the 
EMBO/FEBS Workshop on Gene Structure and Formation of ENA of Viruses 
and Cells. 

Dr. Claude Garon received the N.I.H. Merit Award. 

Dr. James Rose was an invited participant at the EMBO/FEBS workshop. 

Dr. Bernard Moss continued to serve as associate editor of Virology 
and on the editorial boards of the Journal of Virology, of Antibiotics 
and Chemotherapy, and of Intervirology. In addition, he received the 
PHS commendation Medal, was an invited principal speaker at the Society 
for General Microbiology (U.K.) Meeting, was a Session Chairman at an 
International Conference on Transmethylation, was invited speaker at 
the EMBO/FEBS workshop on Gene Structure and Formation of RNA of 
Viruses and Animal Cells, and served on a Poxvirus Study Group for the 
World Health Organization. 

Dr. Riccardo Wittek, a guest investigator in the LBV, received the 
Forderungspreis which is awarded annually to the outstanding young 
microbiologist in Switzerland, and also served on a Poxvirus Study Group 
for the World Health Organization. 



19-13 



Smithsonian science information exchange! u.s. department of project number 

PROJECT NUMBER (.Do NOT use this space) [HEALTH, EDUCATION, AND -ELfARE 

PUBLIC HEALTH SERVICE 

NOTICE OF | n;m AT 00123-1^ LP" 

j INTRAORAL RESEARCH PROJECT I * UX ^ UU - L ^ J " LC 

! PERIOD COVERED 

: October 1, 1978 - September 30, 1979 

TITLE OF PROJECT (30 characters or lessy 
I 

Messenger RNA: Regulation of Synthesis, Modification and Translation 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Principal Investigator: Bernard Moss, Medical Director, LBV, NIAID 

Other: Sundararajan Venkatesan Senior Staff Fellow, LBV, NIAID 

Ernest Barbosa Research Associate, LBV, NIAID 

Steven Langberg Staff Fellow, LBV, NIAID 

Jonathan Cooper Visiting Fellow, LBV, NIAID 

Bahige Baroudy Visiting Fellow, LBV, NIAID 

Riccardo Wittek Guest Investigator, LBV, NIAID 



COOPERATING UNITS (if any) 

Ehud Katz, Dept. of Virology, Hebrew University Medical Center, Jerusalem, Israel 



LAB/BRANCH 

Laboratory of Biology of Viruses 



SECTION 

Macromolecular Biology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



'0TAL MANYEARS: 
9 



3SI0NAL: OTHER: 

6.5 4.5 



CHECK APPROPRIATE 80X(E3) 
G (a) HUMAN SUBJECTS £ (b) HUMAN TISSUES ~ (c) NEITHER 

□ (a1 ) MINORS Q (a2) INTERVIEWS 



SUMMARY OF WORK (200 words or less - underline keywords) 

Our primary objective is to determine the molecular events that lead to the 
selective transcription of DNA and subsequent processing and translation of 
messenger RNA (mRNA) . Poxviruses provide a unique and important system for 
such st udi es since the enzymes needed for transcription and mRNA modification 
are packaged within the core of infectious virus particles. A single enzyme 
complex containing three activities - RNA triphosphatase , RNA guanylytransf erase , 
and RNA (guanine-7-)methyltransf erase - has been purified from vaccinia virus . 
Acting consecutively, the individual activities in the complex are capable of 
modifying the 5 '-end of nascent RNA molecules to form a cap structure in vitro. 
Extracts from uninfected human cells were shown to carry out a similar set 
of reactions, however, three separate enzymes were found instead of a single 
complex. The donor and acceptor substrate specificities of the different 



19-14 



PHS-6040 
(Rev. 10-76) 



viral and cellular enzymes suggested mechanisms used for processing mRNA 
in vivo. Evidence for temporal control of vaccinia virus gene expression 
at the transcriptional level has been obtained by CNA;RNA hybridization 
studies and by translation of viral mRNA in a cell-free protein synthe- 
sizing system. Transcriptional and translational maps of the vaccinia 
virus genome are being prepared using DNA fragments, produced by restriction 
endonuclease digestion, that have been cloned in phage lambda using approved 
DNA recombinant technology. 

Major Findings 

1. Post-transcriptional modification of mRNA 

A. Isolation and characterization of viral capping and methylating 
enzymes - Members of this research group previously identified the 5'- 
terminal cap structure m G(5')pppN consisting of a 7-methylguanosine 
(m G) linked by a 5' to 5 ' triphosphate bridge to one or two consecutive 
2 ' -O-methylated ribonucleosides (N ) present in viral and cellular mRNAs. 
The mechanism of cap formation was determined by purifying the appropriate 
enzymes from vaccinia virus. The latter, included (i) an enzyme complex 
containing both mRNA guanylyl transferase, which is the capping enzyme, and 
mRNA (guanine-7-)methyltransf erase and (ii) a separate mRNA (nucleoside-2 ' -) 
methyl transferase. Further studies of the purified enzyme complex during 
the past year revealed a third activity, RNA tr iphosphase . On a molar 
basis, the RNA triphosphatase is nearly 100-fold more active than the 
associated mRNA guanylytransf erase . Consequently, both triphosphate and 
diphosphate-ended RNAs are capped at similar rates and modification of the 
5' -end of incomplete or nascent transcripts occurs in the following order: 

pppN,-N 2 . . . ■> ppN,-N 2 +Pi (i) 

GTP+ppN,-^ ... + G ( 5 ' ) pppN -N 2 +PPi ( ii ) 

AdcMet+G ( 5 ' ) pppN 1 ~N 2 ■> m 7 G ( 5 ' ) pppN.-N 2 . . . +AdoHcy ( iii) 

AdoMet+m 7 G ( 5 ' ) pppN HNL -> m 7 G ( 5 ' ) pppN™-N 2 . . . +AdoHcy ( iv) 

B. Isolation and characterization of cellular capping and methylating 
enzymes - Previously, we detected capping activity in crude extracts prepared 
from Hela cell nuclei. During the past year, this enzyme has been purified 
approximately 1,000-fold and extensively characterized particularly with 
respect to its donor and acceptor substrate specificities. This analysis 
has provided important information regarding the mechanism of capping 
cellular RNA. Of a variety of potential donor molecules, only GTP and ITP 
were utilized by the purified enzyme. These results indicated that the 
mRNA guanylytransf erase recognizes the oxygen on the purine ring of GTP, 
discriminates between ribose and deoxyribose sugars, and requires a tri- 
phosphate. The inability to use m GTP as a donor indicates that methylation 
occurs after capping. With regard to acceptor molecules, the enzyme is 
specific for a diphosphate-ended oligo-or polyribonucleotide. The inability 
to efficiently cap triphosphate-ended acceptors is explained by the absence 
of an associated RNA triphosphatase. In that respect and also by the absence 

19-15 



of an associated mRNA (guanine-7-) methyl transferase, the Hela cell capping 
enzyme differs from the one isolated from vaccinia virus. This result is 
consistent with our previous purification of a specific Hela cell mRNA 
(guanine- 7-)methyltransferase. Although the possibility of a pre-transcrip- 
tional capping mechanism has been considered by others, our finding that 
the minimum length acceptor is a dinucleotide such as ppGpC makes this 
mechanism unlikely. In fact, the Km for a dinucleotide is higher than for 
a polymer indicating that a longer oligonucleotide or short polyribonucleotide 
is a better acceptor. We suggest that in vivo , an RNA triphosphate removes 
the third phosphate from the 5 '-end of short unfinished primary transcripts 
and that they are then capped by the enzyme that we have described. If this 
enzyme also caps RNA at sites of cleavage, then additional kinases capable 
of forming diphosphate-ends must exist. 

C. Coupled transcription and modification - One of the major advantages of 
working with vaccinia virus is that all of the enzymes necessary for 
synthesis and modification are contained within the purified virus particle. 
After irradiating purified vaccinia virus particles with ultraviolet light 
to form pyrimidine dimers in the DNA, only short aborative transcription 
products were made. Analysis of these products indicated, however, that 
they were capped,, methylated and polyadenylylated . These results suggested 
to us that neither completion of an RNA chain nor processing from a poly- 
cistronic precursor was required for modification of either end of the RNA. 
The presence of the poly (A) tract at the 3 '-end of the short transcript 
further suggested that a slow-down or cessation of transcription, rather 
than a specific 3 '-end terminal sequence, serves as a signal for poly- 
adenyly lation . 

2. Organization and regulation of the vaccinia virus genome . 

A. In vitro translation of immediate early, early and late classes 
of RNA from vaccinia virus infected cells - Cytoplasmic RNA, isolated at 
various times after vaccinia virus infection, was translated in a message- 
dependent cell-free system prepared from rabbit reticulocytes. When 
programmed with RNA extracted at 2 hr after infection, early viral proteins 
were made and formation of cellular proteins was diminished. Primarily 
late proteins were synthesized using RNA extracted at 4 or more hours after 
infection, suggesting that the switch in protein synthesis is regulated 
principally by changes in RNA concentration rather than by modification of 
the translation apparatus of the cell. Immediate early RNA was obtained 

by infecting cells in the presence of inhibitors of protein synthesis. Com- 
parison between immediate early and early gene products did not reveal a 
class of early genes that require protein synthesis for expression. On 
the contrary, seven polypeptides, of which a 28,000 dalton species was most 
prominent, were synthesized in relatively greater amounts with imtiediate 
early RNA than with early RNA. This results suggests the possibility that 
expression of certain immediate early genes is regulated by a feed-back 
mechanism. 

B. Analysis of the terminal repetition within the vaccinia virus 
genome - Previously we reported that a 3.54um duplex was formed by annealing 
the two ends of the vaccinia virus genome. This inverted terminal repetition 

19-16 



was estimated to be about 10,000 nucleotide base pairs long. To further 
characterize this structural feature, restriction maps were made of the 
ends of the genome using the following enzymes: EcoRI, Hpall, and Hindlll. 
To accomplish this we developed a novel mapping method, involving use of 
"nick translated" probes prepared from the terminal fragment, to order partial 
digestion products. From this study, we concluded that identical restriction 
sites are present at the 2 ends of the genome for approximately 10,000 
nucleotides confirming our previous electron microscopic analysis and work 
of other investigators. 

To determine whether the inverted terminal repetition is transcribed in vivo , 

P- labeled cytoplasmic RNA was obtained from infected cells. This RNA was 
then annealed to restriction fragments , prepared from the terminal repetition 
and adjacent regions of the DNA that were immobilized to a nitrocellulose 
sheet. We found that early RNA hybridized to the repetition except for the 
terminal 3,000 nucleotide base pairs indicating that it is indeed transcribed. 

C. Formation of recombinant DNA molecules containing portions of the 
vaccinia virus genome - DNA recombinant technology developed within the 
past few years followed by a relaxation of the guidelines for carrying out 
such research has allowed us to clone portions of the vaccinia virus genome 
in phage lambda. Our initial approach has been to clone successive EcoRI 
fragments of DNA starting from one end of the genome. Thus far, this has 
included a 9,500 nucleotide base-pair segment containing most of the 
terminal repetition, a 6,500 nucleotide base-pair segment containing unique 
sequences adjacent to this, and the next 4,000 nucleotides base-pair segment. 
In order to clone the end piece of DNA, it was first necessary to remove 
the terminal cross-link with a single-strand specific nuclease and then 
add synthetic EcoRI linkers. Each of the recombinant DNA clones were 
identified by restriction endonuclease digestion and gel electrophoresis. 
Studies are now in progress using the cloned genome fragments to obtain a 
transcription and translation map. 

3. Action of specific antiviral agents . 

Isatin-6-thiosemicarbazone is known to specifically inhibit vaccinia virus 
replication at a late stage. At 6 hr after infection, viral protein synthesis 
was inhibited by about 95%. We confirmed that a portion of the virus-specific 
RNA appears to be degraded (B. Woodson and W.K. Joklik, 1965, Proc. Nat. 
Acad. Sci., USA 54, 946-953) . Nevertheless, the amount of viral RNA that 
was capped, properly nethylated, and polyadenylylated, was reduced by only 
50%. Moreover, RNA from TBT-treated cells stimulated cell- free protein 
synthesis to one-half the level obtained with RNA from control cells. Poly- 
acrylamide gel electrophoretic analysis further demonstrated that RNA from 
IBT-treated cells was translated into late viral proteins in vitro . Thus, 
it seems possible that the inhibition of protein synthesis in IBT-treated 
cells does not result entirely or directly from either an inhibition of mRNA 
synthesis or from a depletion of mRNA caused by accelerated degradation. An 
alternative possibility, that accelerated degradation is secondary to a more 
immediate effect of the drug on protein synthesis, was considered. 



19-17 



Publications 

Keith, ,J.M. , Ensinger, M.J. and Moss, B.: Hela cell RNA (2 ' -O-methyladeno- 
sine-N -) -methyltransf erase specific for the capped 5 '-end of messenger RNA. 
J. Biol. Chem. , 253, 5033-5041, 1978 

Keith, J.M. , Muthukrishnan, S. and Moss, B. : Effect of methylation of the 
N -position of the penultimate adenosine of capped mRNA on ribosame binding. 
J. Biol. Chem. , 253, 5039-5041, 1978 

Cooper, J. A. and Moss, B. : Transcription of vaccinia virus mRNA coupled 
to translation in vitro . Virology , 88, 149-165, 1978 

Barbosa, E. and Moss, B.: mRNA (nucleoside-2 '-) -methyltransf erase from vaccinia 
virus: purification and ;ysical properties. J^ Biol. Chem. , 253, 7692- 
7697, 1978 

Gershowitz, A., Boone, R.F. and Moss, B.: Multiple roles for ATP in the 
synthesis and processing of mRNA by vaccinia virus: specific inhibitory 
effects of adenosine (6-y-imido) triphosphate. J. Virol. , 27, 399-408, 
1978 

Garon, C.F., Barbosa, E. and Moss, B.: Visualization of the inverted 
terminal repetition in vaccinia virus DNA. Proc. Natl. Acad. Sci. USA 
75, 4863-4867 

Boone, R.F., Parr, R.P. and Moss, B.: Intermolecular duplexes formed 
from polyadenylylated vaccinia virus RNA, J. Virol. 30, 365-374 

Moss, B., Barbosa, E. and Keith, J.M. : Specificity of mRNA methyl- 
transf erase. In Usdin, E., Borchardt, R.T., and Creveling, D. (Ed.): 
Transmethylation, Elsevier/North-Holland, N.Y. 1979, pp. 373-380 

Cooper, J. A. and Moss, B. : In vitro translation of immediate early, early 
and late classes of RNA from vaccinia virus -infected cells. Virology , 96, 
368-380 

Cooper, J. A., Moss, B., and Katz, E. : Inhibition of vaccinia virus late 
protein synthesis by isatin-6-thiosemicarbazone: characterization and 
in vitro translation of viral mRNA. Virology , 96, 381-392 

Gershowitz, A. and Moss, B. : Abortive transcription products of vaccinia 
virus are guanylylated, methylated and polyadenylylated. J^ Virol . , in press 

Cooper, J. A. , and Moss, B. : Translation of specific vaccinia virus RNAs 
purified as RNA-DNA hybrids on potassium iodide gradients. Nucleic Acids 
Res. , in press 



19-18 



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TITLE OF PROJECT (30 characters or less) 

Replication of the Parvovirus, KRV 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Principal Investigator: Dr. Lois A. Salzman Research Chemist LBV NIAID 

Other: Dr. Tonu Wali, Visiting Fellow LBV NTAID 
Ms. Phyllis Fabisch, Microbiologist LBV NTAID 



COOPERATING UNITS (if an 



LAB/BRANCH 

Laboratory of Biology of Viruses 



SECTION 

Biochemical Virology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



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SUMMARY OF WORK (200 *ords or less - underline keywords) 

We have been studying the specific biochemical mechanisms involved in 

the replication of the autonomous parvovirus , KRV in a rat nephroma cell 

line. The virus, isolated originally from a rat sarcoma, contains one 

molecule of linear , single-stranded DNA and three capsid proteins . 

Little information is available about replication of a single-stranded 

DNA virus in a euk^ryotic cell or on the transcription of this DNA to 

make viral proteins. We have demonstrated that the virion KRV-DNA can 

self prime in vitro to synthesize the double-stranded replicative intermediate. 

In an attempt to resolve this self priming terminus, so important in DNA 

replication, we have sequenced the nucleotides in the 3' DNA terminus 

which probably also contains the in vitro origin of DNA replication. 



19-19 



PHS-6040 
(Rev. 10-76) 



We have previously found that in an infected cell two KRV specific mRNAs 
are synthesized. We have mapped the major 21S viral mRNA on the viral 
DNA molecule using both the "Southern" blotting technique and measurement 
of the "R loops" seen in the electron microscope. Inhibition studies 
have shown that these KRV mRNAs are probably synthesized by cellular RNA 
polymerase II. Preliminary studies also show that there are at least 
three virus induced proteins synthesized during infection. 

Project Description 

Parvoviruses are the smallest DNA containing viruses found in vertebrate 
tissues. They infect a wide variety of species including man. The 
parvoviruses are classified as autonomous or defective depending on 
whether or not they require a helper virus for replication. The virions 
replicate best in rapidly dividing cells and produce a wide variety of 
effects on insects, animals and cells in culture. The feline parvovirus 
has been linked to the naturally occuring disease feline panleukopenia . 
Rodent parvoviruses can produce fatal infections and teratogenic effects 
in fetal and newborn hamsters. Parvoviruses are able to persist in a 
latent state and to inhibit homologous and heterlogous virus infection. 
The viruses can also exhibit antimitotic activity and interfere with 
natural or virus-induced tumorigenesis. 

We have continued our studies of the parvovirus, KRV, because of its 
genetic simplicity and the small size of the virus gene. KRV like the 
other parvoviruses possesses a single- stranded DNA genome (1.6 x 106 
daltons or 4500 to 5000 bases) with sufficient information to code for 
only a few proteins equvalent to about 150,000 daltons. The transcription 
process may be very simple and involve only one or two messenger RNAs. 
It is probable that the viral DNA replication and transcriptional components 
are carried out in large part by host functions. Thus, we potentially 
can gain insight into viral functions, host functions and the interaction 
between virus and host. 

In the past year we have concentrated our efforts into studying further 
viral DNA replication, transcription of the viral genome and virus 
induced proteins. We have found that the single-stranded DNA from KRV 
can serve in vitro as a self-primer for the synthesis of a complementary 
linear viral DNA strand. A double stranded viral DNA molecule has been 
proposed as an intermediate in the replication of the viral genome. 
Little is known about the replication of single-stranded linear DNA in a 
eukaryotic cell. Because of the importance of the 3' terminus of the 
viral DNA in self priming viral DNA synthesis and possibly in transcription 
we sequenced the 3' terminal nucleotides. This analysis has lead us to 
propose a 3' terminal hairpin structure with secondary structures and 
the nucleotide sequence of the DNA which at least in vitro serves as the 
origin of replication of the complementary strand. We are currently 
sequencing the 5' terminus of the viral DNA which appears to have a 
unique structure and possibly an associated protein. 

In our studies of the transcription of KRV, we have previously found 

19-20 



that two KRV-specific mRNAs are synthesized in infected RN cells. We 
have also found that only the viral DNA strand is transcribed. Two 
techniques have now been used to map these mRNAs to the viral genome. 
We have mapped the fragments of several restriction enzymes to the KRV 
viral genome. Using the mapped restriction fragments, the major cytoplasmic 
viral RNA (21 S) has been mapped to the viral genome using the "Southern" 
blotting technique. This has been complemented by Electron Microscopy 
of "R loops" or RNA-DNA duplexes. Both techniques indicate that the 21S 
viral mRNA maps from approximately 0.38 to 0.98 on the viral DNA strand. 

Further studies are presently underway to determine if longer transcripts 
are present in the nucleus, and to try and determine if posttranscriptional 
processing occurs by simple cleavage or by "splicing". We are also 
trying to elucidate structural features of the KRV mRNA by assaying for 
RNA cap structures at the 5 1 end and to determine translatability of the 
KRV mRNAs in in vitro assays. 

We have used nuclei isolated from infected rat nephroma cells to study 
the enzymes involved in viral specific transcription. Hybridization of 
RNA synthesized in isolated nuclei indicated that viral specific RNA 
synthesis started at 8 to 9 hours post infection. Viral specific RNA 
was inhibited by 0.1 g of amamitin per ml, suggesting that the viral 
genome is transcribed by cellular RNA polymerase II. The viral RNA 
synthesized in the isolated nuclei was also analyzed on sucrose gradinets. 
The KRV specific RNA varied in length from 26S (full DNA transcript) to 
4S in length. We propose to further study the components involved in 
transcription of the viral genome after gentle disruption of the nuclei. 



19-21 



Publications 

1. Salzman, L.A. and Rabisch, P. Studies on the Replication of KVR 

Single-stranded Linear DNA. J. Gen. Virol. 39, 571-574 (1978). 

2. Salzman, L.A. , Fabisch, P., Parr, R. , Garon, C. and Wali, T. In 

vitro Synthesis of Double-Stranded DNA from the Single-stranded 
KRV DNA Genome. J. Virol. 27: 784-790 (1978). 

3. Salzman, L.A. , McKerlie, L. , Fabisch, P. and Koczot, F. St u dies 

on a Protein Found Associated with Kilham Rat Virus. In D. Ward 

and P. Tattersall (eds.) Parvoviruses. Cold Spring Harbor Laboratory, 

Cold Spring Harbor, L.I., New York (1978) . 

4. Salzman, L.A. , P. Fabisch. Nucleotide sequence of the self -priming 

3 'terminus of the single-stranded DNA extracted from the Parvovirus, 
Kilham Rat Virus. J. Virol. 30: 946-951 (1979). 

5. Wali, T.M. and Salzman, L.A.,. Sedimentation analysis of the RNA 

synthesized in vivo by the autonomous parvovirus, Kilham Rat Virus. 
J. Virol. 1979 (in press) 



19-22 



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PROJECT NUMBER 



ZOl AI 00125-10 LBV 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



i TITLE OF PROJECT (30 characters or less) 

Mechanisms of Viral DNA Replication, Transcription, and Integration 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI: Norman P. Salzman 
Walter Bruszewski 
Minou Bina-Stein 
Nancy Chiu 
Yaf f a Reuveni 
John A. Thompson 
John Brady 
Christian Lavialle 



Chief LBV, 


NIAID 


Staff Fellow LBV, 


NIAID 


Senior Staff Fellow LBV, 


NIAID 


Staff Fellow LBV, 


NIAID 


Visiting Associate LBV, 


NIAID 


Staff Fellow LBV, 


NIAID 


Staff Fellow LBV, 


NIAID 


Visiting Fellow LBV, 


NIAID 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Biology of Viruses 



Biochemical Virology Section 



INSTITUTE AND LOCATION 

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9 .0 



PROFESSIONAL: 



1.0 



4.0 



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G (=) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The structure of SV40 mRNA molecules has been determined by preparing _ 
cDNA copies of the viral mRNAs using reverse transcriptase. Characterization 
oFtranscripts formed when SV40 DNA is transcribed by purified RNA polymerases 
has provided the location of promoters of RNA transcription . The role of 
viral proteins as regulators of transcription has been studied by in vitro 
transcription of nucleoprotein complexes . 



19-23 



PHS-6040 
(Rev. 10-76) 



Papovaviruses 

The current studies have tried to define some of the processes 
responsible for the formation of viral messenger RNA molecules. Thus 
far, these studies have provided the precise structures of the viral 
mRNA molecules, have defined the nature of the templates used for the 
synthesis of viral transcripts, have located promoters on the SV40 
genome that are recognized by E. coli RNA polymerase and have compared 
SV40 DNA I and SV40 DNA I nucleoprotein complexes as templates for 
transcription . 

The Structure of Early and Late Viral mRNA. 

The mechanism of splicing of the early and late cytoplasmic species 
of SV40 mRNA has been studied using a technique which first involves the 
isolation of specific regions of the viral genome. Restriction enzyme 
cleavage of SV40 DNA generates specific DNA fragments which can be 
fractionated using column chromatography. These fragments are then 
annealed to viral mRNA and the reaction products are fractionated by 
velocity sedimentation. The DNA fragment which is annealed to the viral 
mRNA is used as a primer for the enzyme, reverse transcriptase, and a 
complementary DNA copy of the mRNA is made. The sequence and structure 
of the cDNA has been compared with the known nucleotide sequence of 
SV40. This procedure has been used to characterize the late mRNA which 
codes for the major structural viral coat protein and has provided 
direct physical evidence for two early mRNA species. The regions of the 
DNA that have deleted in both species of early mRNAs have been determined. 
Also, the nucleotide sequences across these spliced regions have been 
determined and correlated with the mRNA coding for the large T and small 
t antigens. These two mRNA species were determined to have the same 5 1 
termini which would suggest two levels for control of genetic expression. 
One would be the regulation of initiation of transcription at a common 
promoter; the other would involve post- transcriptional splicing. 

To further characterize the mechanism of transcription of early 
SV40 RNA, nascent RNA chains that are attached to template DNA molecules 
are being isolated and characterized by the same methodology employed to 
study the processed cytoplasmic species of early viral RNA. Transcription 
complexes have been isolated from infected cells which continue viral 
mRNA synthesized in vitro . The structure of these nascent chains gives 
insight into the initial transcription products and the mechanisms that 
operate to regulate transcription. We are investigating differences 
between RNA's formed in vivo and in vitro . The structure of nascent 
chains will provide precise information about the primary products of 
transcription. We are also studying the use of isolated nuclei for the 
study of the regulation of transcription. Isolated nuclei are able to 
sustain in vitro synthesis of viral RNA. In this case, the SV40 template 
in the cell nucleus is present as a nucleoprotein complex containing the 
five histones. This is in contrast to in vitro synthesis of nascent RNA 
chains described above which uses a less well defined transcriptional 
complex. 



19-24 



These nascent SNA molecules will be probed with specific SV40 DNA 
fragments to determine their structure. In order to acquire a library 
of these specific fragments, recombinant technology has been employed. 
Cloning of SV40 DNA fragments using the plasmid pBR322 and E. coli 
should provide sufficient amounts of specific DNA fragments which can be 
used to probe nascent RNA molecules and determine the mechanisms operating 
during regulation of transcription. 

Since sufficient amounts of specific SV40 DNA fragments can be 
obtained from cloning, we will be able to study how synthesis of specific 
RNA sequences are affected by mutations within the genome. Further 
characterization of RNA species obtained following Proflavin treatment, 
which blocks RNA processing, will aid our understanding of the mechanism 
that operated to regulate transcription. (Thompson, Bina-Stein, Salzman) 

Localization of RNA Polymerase Promoters on SV40 DNA 

Transcripts of SV40 DNA synthesized by Escherichia coli RNA polymerase 
have been characterized. This model system has been used for the development 
of new methods applicable to the analysis of the mechanisms involved in 
the synthesis of the viral messenger RNAs in SV40-infected cells. It 
has been previously shown that E. coli RNA polymerase recognizes specific 
initiation sites on SV40 DNA. Except for one of them, determination of 
the location of these sites on the SV40 DNA map is only approximate. No 
specific termination site for transcripts has been identified, and 
consequently RNA polymerase generates a heterogeneous population of 
molecules. The size of some of these RNA molecules is several times the 
length of the viral genome. 

We have shown that, after binding of the E. coli RNA polymerase to 
SV40 DNA, it was possible to cleave such transcriptional complexes with 
"single-cut" restriction endonucleases (Bam Hj, Eco Rj, Hpa II). The 
addition of ribonucleotide triphosphates to these linearized complexes 
leads to the synthesis of defined species of RNA which can be analyzed 
by electrophoresis. The determination of the size of each of these RNAs 
together with the assignment of the DNA strand on which they are transcribed 
will allow the precise mapping of the various RNA promoters (Reuveni, 
Lavialle, Salzman) . 

Transcriptional Properties of SV40 Nucleoprotein Cores 

Simian Virus 40 (SV40) provides an excellent model system for 
investigating the process of transcriptional regulation in eukaryotic 
cells. Many structural aspects of viral transcriptional complexes and 
mRNA molecules have been determined. In addition, the entire nucleotide 
base sequence of SV40 DNA has been determined. Similar to most eukaryotic 
chromatin, during the SV40 infection cycle, cellular histones H2A, 
H2B, , H3 and H. are bound to the viral DNA. Late in the infection 
cycle, histone H]_ also becomes associated with the bulk of the viral 
chromatin in a stable nucleoprotein complex. Just prior to encapsidation, 
the SV40 nucleoprotein complex undergoes a redistribution of proteins 
and histone H, is replaced by viral proteins. Since binding of H, on 
chromatin may suppress transcription of DNA sequences, we were interested 

19-25 



in determining if the viral proteins also acted to modify or regulate 
the process of RNA synthesis. Such regulatory properties could be 
important in a clearer understanding of the process of "early" mRNA 
synthesis during infection of the host cell. In view of the close 
similarity between this SV40 DNA-histone-viral protein nucleoprotein 
complex and cell chromatin structure, such studies would also be of 
interest in a more complete understanding of the general problem of 
transcriptional regulation in eukaryotic cells. 

A nucleoprotein complex can be isolated from purified SV40 virions 
under very mild, physiological conditions. These nucleoprotein cores 
are potentially active transcriptional complexes. These core complexes 
do not contain endogenous RNA polymerase activity; however, 95-100% are 
able to form active transcriptional complexes. The transcriptional 
activity of those complexes is extremely high and is similar to the 
activity obtained with purified SV40 Form 1 DNA. This observation 
contrasts with published data, in which the level of transcription of 
nucleoprotein complexes (SV40 DNA-Histone) is generally less than 20% of 
the activity of deproteinized SV40 DNA. The results suggest that the 
viral proteins may play a role in the "activation" of the nucleohistone 
complex. Analysis of the composition and structure of the SV40 nucleoprotein 
core are being carried out and should allow an understanding of the 
unexpectedly high efficiency of the SV40 core as a template for RNA 
synthesis. (Brady, Lavialle, Salzman) 

Publications 

Chiu, N. , Radonovich, M. , Thoren, M. , and Salzman, N. P., Selective 
degradation of newly synthesized non-^nessenger SV40 transcripts. J. Virol ., 28: 
590-599, 1978. 

Seidman, M. , Garon, C. , and Salzman, N. P. The relationship of SV40 
replicating chromosomes to two forms of the non-replicating SV40 chromosome. 
Nucl. Acids Res . 5_: 2877-2893, 1978. 

Panel V — Virus Task Force. N. P. Salzman, Chairman. NIAID Task Force 
Report, DHEW Publication No. (NTH) 79-1835 

Bina, M. , Thompson, A. , Thoren, M. , and Salzman, N. P. (1979) Rapid 
sequence determination of the late SV40 16S mRNA leader using inhibitors 
of reverse transcriptase. Proc. Natl. Acad. Sci . 76: 731-735. 

Birkenmeier, E. , Chiu, N. , Radonovich, M. , May, E. , and Salzman, N. P. 
Regulation of SV40 early and late gene transcription without viral DNA 
replication. J. Virol . 29: 983-989, 1979. 

Seidman, M. , and Salzman, N. P. Late replica tive intermediates are 
accumulated during SV40 DNA replication in vivo and in vitro. J. Virol . 
30: 600-609, 1979 

Thompson, J. A. , Radonovich, M. , and Salzman, N. Characterization of the 
5 '-terminal structure of SV40 early mRNAs. J. Virol. , in press. 



19-26 



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NOTICE OF i ZOl AI 00126-06 LBV 

INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER (Do NOT use this space 
I 



a5tobe^ 0V F, ED 1978 to September 30, 1979 



TITLE OF PROJECT (30 characters or less, 

Restriction Enzyme Analysis of Vaccinia Virus DNA 



i NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
\ PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Principal Investigator: Frank DeFilippes, Research Physicist LBV NIAID 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Biology of Viruses 


SECTION 

Macromolecular Biology Section 


INSTITUTE AND LOCATION 

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PROFESSIONAL: 
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.2 



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SUMMARY OF WORK (200 words or less - underline keywords) 

The purpose of the project is to study the organization of genes in 
large DNA molecules obtained from animal viruses. The production and 
ordering of unique segments of vaccinia virus DNA provide a physical map 
of the genome. Physical maps have been determined for several different 
restriction enzymes . I am also working to improve the techniques for 
separating and isolating very large pieces of DNA which differ in size 
and base composition by relatively small amounts. In particular, I am 
trying to isolate the DNA segments which contain the origin (s) of 
replication of vaccinia DNA and to test for possible signal elements in 
the DNA without prior knowledge of the nucleotide sequence. 



19-27 



PHS-6040 
(Rev. 10-76] 



Project Description 

Vaccinia DNA has oDvalently-linked-conplementary (CLC) ends which 
define each end of the physical map of the genome. These CLC end segments 
can be identified in each set of segments that are produced by a particular 
restriction enzyme. The set of segments is alkali denatured, neutralized 
and passed through a BND-cellulose column from which only the CLC segments 
elute as duplex chains. The end segments have been identified for 
several restriction enzymes such as Sal I, Hind III, Kpn I and Xho I. 
For Sal I, the end segments are identical and small with admass of 
2.1 X 10 daltons. For Xho they are larger (both 3.9 X 10 ) and for 
Hind III they are larger still (17.6 and 13.5 X 10 ) . 

The restriction enzymes listed above as well as several others are 
used to digest vaccinia DNA. to obtain unique DNA segments which are 
separated by agarose electrophoresis. To obtain a physical map, segments 
produced by one enzyme are isolated from agarose and digested by a 
second enzyme to find overlapping regions. The end segments described 
here serve as reference points from which the maps are continued. The 
Sal I and Hind III map of vaccinia strain WR are now almost completely 
known. Kpn and Xho maps are also near completion. 

A particular method which has helped to generate reproducible 
partial DNA segments involves restriction enzyme digestion in the presence 
of actinomycin D. The original observation that the drug blocked 
complete digestion with Hind III has been extended to 3 other restriction 
enzymes, Sal I, Xho and Kpn. None of these digests produce as long a 
partial as the one half length vaccinia DNA molecule generated with Hind III 
but they do contain several smaller linked segments which help to establish 
the map. 

Initial experiments designed to locate the origin of replication 
involve pulse labeling HeLa cells with H-thymidine after infection with 
high multiplicities of vaccinia virus. A peak of synthetic activity 
occurs 2.5 hours after infection. From 2.5 to 3.5 hours after infection 
alkaline and neutral sucrose gradients of cytoplasmic (viral) DNA 
reveal a spectrum of molecular sizes with about 10 - 20% of the DNA in 
full length molecules. These DNA species will be examined in a Dintzis 
(Proc. Natl. Acad. Sci. 47: 247, 1961) type experiment. 

Several experimental techniques have evolved during the year. The 
method of Vogelstein and Gillespie (Proc. Natl. Acad. Sci. 76: 615, 
1979) for extracting DNA segments from agarose with glass powder has 
been modified so that it is now possible to routinely recover intact, 
70% or more of ug amounts of large vaccinia DNA segments. Also, poly rC 
columns have been used to hybridize segments which contain short stretches 
of GC rich regions in duplex DNA. Using this column technique, we have 
found that the largest Sal I segment binds to the column. Since GC rich 
regions may provide a structural basis for a signal element, the Sal I 
segment will be digested with other enzymes to localize the GC region. 
Finally, we have started to use malachite green columns to fractionate 
sheared vaccinia DNA (6 X 10 daltons) according to base composition. 

19-28 



Those fragments with the least affinity for the column have the highest 
average GC content. They will be radiolabeled and hybridized to Sal I 
digests to identify those segments from which they originated. 



19-29 



{SMITHSONIAN SCIENCE INFORMATION EXCHANGE! U.S. DEPARTMENT OF 

IPROJECT NUMBER (Do MOT use this space; IhEALTH, EDUCATION, AND WELFARE 

PUBLIC HEALTH SERVICE 
NOTICE OF 
i INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



Z01 AI 00127-12 LBV 



PERIOD COVERED 

! October 1, 1978 to September 30, 1979 



j TITLE OF PROJECT (80 characters or lessy 

Structure and Function of Genetic and Protein Components of Defective 
, Parvoviruses 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 


James Rose 


Section Head 


LBV NIAID 


Cther: 


Mark Buller 


Visiting Fellow 


LBV NIAID 




John Janik 


Research Associate 


LBV NIAID 




Edwin Sebring 


Research Chemist 


LBV NIAID 




Frank Koczot 


Research Biologist 


LBV NIAID 




Claude Garon 
James Garrisor 


Research Microbiologist 
Bio Lab Tech (Bicchem) 


LBV NIAID 
LBV NIAID 



COOPERATING UNITS (if an 



LAB/BRANCH 

Laboratory of Biology of Viruses 



SECTION 

Molecular Structure Section 



INSTITUTE ANO LOCATION 

NTATTy NTH. Bethesda, Maryland 20205 



TOTAL MANYEARS: 



PROFESSIONAL: 

4.0 



2.5 



CHECK APPROPRIATE 30x(E3) 
~ (a) HUMAN SUBJECTS 

□ (a1 ) MINORS : ~ (a?) INTERVIEWS 



r^(b) HUMAN TISSUES 



Q (cj NEITHER 



SUMMARY OF *0RK (200 words or less - underline keywords) 

Main objectives in studying these defective human parvoviruses 
(AAV) are to (i) define specific biochemical mechanisms used in synthesizing 
their DNA, RNA and proteins, (ii) identify and characterize the helper 
virus -^nediated step(s) required for their replication and (iii) relate 
biochemical findings to normal cellular processes as well as to potentials 
for selective interference with both AAV and Helper virus (adenoviruses , 
herpesviruses) infection. Among the methods used are nucleic acid 
hybridizations , analytical and preparative sucrose and CsCl sedimentations, 
molecular cleavage with restriction nucleases , gel electrophoresis and 
electron microscopy. 



19-30 



?"S-6040 
(Rev. 10-76] 



Project Description : 

(1) Most, if not all, of the enzymes involved in the replication of AAV 
DNA probably play corresponding roles in cellular DNA synthesis. Among 
these activities, the enzyme (s) responsible for processing AAV concatermeric 
intermediates is of particular interest because of its site-specific 
attack at the origin of DNA replication. We have purified and partially 
characterized two single strand-specific endonucleases from KB cells 

that are possible candidates for such a processing enzyme. Both of 
these KB cell enzymes (KB, and KB„) have relatively high pH optima 
(pH 9.2 and 9.5) and specifically cleave single-stranded DNA. However, 
the KB, enzyme differs from the KB- enzyme with respect to -isoelectric 
point TKB, = 10.3; KB 2 = 6-^) , absolute requirement for Mg (KB 2 
enzyme can also utilize M ) and relative rates of hydrolysis with 
homopolymers (for KB, : dG>dT>dA>dC; for KB-: dA>dT>dC>dG) . Differences 
in rates of hydrolysis among the homopolymers indicate some specificity 
for different nucleotide bonds. Both enzymes hydrolyze poly(dT) 7-8 
times more rapidly than denatured viral or cellular DNA. In addition, we 
have currently identified in KB cells several other distinct endonucleases 
that hydrolyze poly(dT) more rapidly than denatured DNA. It is planned 
to continue the purification and characterization of these endonucleases 
as well as to attempt to identify their roles in the cells and, possibly, 
the viral DNA replication process. 

(2) Human adenovirus (Ad) serotypes provide an early factor (s) that is 
necessary for adenovirus-associated virus (AAV) multiplication in human 
cell lines. However, little if any AAV production occurs in primary 
African green monkey kidney (AGMK) cells co- infected with AAV and a 
helper human Ad (non-permissive infection) unless cells are additionally 
infected with SV- (permissive infection) . To determine the basis of 
the host restriction of AAV replication in AGMK cells, AAV DNA, RNA and 
protein synthesis were analyzed under various conditions of infection. 
Hybridization reactions revealed no detectable AAV-specif ic DNA or RNA 

in infections with AAV alone or in combination with SV._. In co- infections 
with AAV and Ad- or Ad_, the synthesis of both AAV- and Ad-specific DNA 
and RNA occurred without a significant rise in titre of either virus. 
During non-permissive infection, however, AAV DNA synthesis was abnormal 
in that an expected accumulation of single-stranded progeny molecules 
was not observed. Finally, although intact 2 OS AAV transcripts were 
present in the cytoplasm of AGMK cells during non-permissive infection 
(in amounts ranging from 50 to 80% of that found during permissive 
infection) , AAV-specific polypeptides were not demonstrable by polyacrylamide 
gel electrophoresis. Taken together, these experiments indicate that 
the host restriction of AAV replication in AGMK cells is exerted at the 
level of translation of the single AAV messenger RNA. In addition, it 
appears that one or more of the AAV polypeptides specified by this 
message is required for the production of single-stranded AAV progeny 
DNA. The fact that coinfection with a simian adenovirus, SV15, permits 
complete replication of both Ad and AAV (analogous to coinfection with 
SV. n ) strongly suggests that an Ad(SVl5) gene product also exerts a 
regulatory effect on expression of the AAV mRNA (as well as several late 
human Ad mRNAs) in AGMK cells. Isolation and characterization of this 



19-31 



gene product should be relevant to possible approaches for selectively 
interfering with virus infection. 

(3) Further understanding of mechanisms involved in DNA replication was 
obtained by studying AAV DNA synthesis in cell-free extracts. Extracts 
of nuclei from KB cells doubly infected with adenovirus-associated virus 
type 2 (AAV) and adenovirus type 2 (Ad) were examined for their ability 
to synthesize various molecular forms of AAV DNA. It was found that 
replicating AAV DNA molecules elongate to yield genome length hairpins 
or linear, open-ended duplexes. Both plus and minus DNA strands serve 
as templates for this synthesis which apparently does not reinitiate new 
rounds in vitro . Replicating Ad DNA molecules also could be identified 
in these extracts, but incorporation of [ H]TTP into Ad DNA was diminished 
80-90% in coinfections with AAV as compared to nuclei extracts prepared 
from cells infected with Ad alone. These results confirm our previous 
in vivo studies which indicated that a self -priming mechanism is involved 
in the replication of AAV DNA, studies that represented the first reported 
evidence for this mode of initiating DNA synthesis. Investigations with 
extracts will continue with the objective of identifying and characterizing 
enzymatic and regulatory components that participate in viral and cellular 
DNA replication. 



Publications : 

Buller, R. M. L. , Straus, S. E. and Rose, J. A.: Mechanism of host 
restriction of adenovirus-associated virus replication in African green 
monkey kidney cells. J. Gen. Virol. 43: 663-672, 1979. 



19-32 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00128-12 LBV 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Properties of Adenovirus DNA 



NAMES, LABORATORY ANO INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



Principal Investigators: 



Other: 



Claude F. Garon 
James A. Rose 



Research Microbiologist LBV NIAID 
Medical Officer LBV NIAID 



Judy A. Sprague Biologist LBV NIAID 

James W. Garrison Bio Lab Tech (Biochem) LBV NIAID 



COOPERATING UNITS (if any) 

R. Padmanabhan University of Maryland School of Medicine, Baltimore, Maryland 



lab/branch 
Laboratory of Biology of Viruses 



SECTION 

Macromolecular Biology Section 



INSTITUTE AND LOCATION 

NIAID, NTH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 
1.1 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS FJ (a2) INTERVIEWS 



HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The major objective of these studies has been the application of 
physical and biochemical techniques to map and to define the structure 
of specific genetic regions in the genomes of both oncogenic and non- 
oncogenic human adenoviruses , with the ultimate goals of (1) relating 
these sequences to those biochemical activities which permit these 
viruses to influence or gain control of cellular functions (e.g., cell 
lysis and transformation ) and (2) of defining basic mechanisms for 
regulation of genetic expression (e.g. , initiation of DNA synthesis) . A 



number of unique features have been described which may have implications 
in viral replication or carcinogenesis or both. 



19-33 



PHS-6040 
(Rev. 10-76) 



Project Description 

The DNA of adenoviruses is normally isolated as a linear duplex 
molecule of discrete size using standard procedures of extraction with 
protealytic enzymes, phenol and SDS. Recently, it has been possible to 
demonstrate forms of DNA that are either circular or oligomeric by using 
procedures which do not involve protealytic enzymes. In these latter 
cases, the DNA molecules are always joined at their ends, and it appears 
that either end of a molecule may interact with the other end of the 
same molecule or with either end of another molecule. The circles and 
oligomers are resistant to treatment with 4M guanidinium chloride, 4M 
urea, formimide, sarkosyl or mercaptoethanol. Treatment with proteases, 
however, rapidly converts the circles and oligomers to linear double- 
stranded monomers, as does treatment with SDS. It had been previously 
proposed that there is a protein attached to each end of the DNA molecule 
and that this protein is responsible for the formation of the observed 
complexes. Subsequent studies have clearly demonstrated the presence 
of a 55,000 dalton protein very tightly linked to the DNA molecule. 
Although the function of the bound protein is unknown, it has been 
proposed that it may be involved in DNA replication by allowing initia- 
tion or completion of the 5' ends of the progeny strands. It has also 
been suggested that the protein may have a structural role by circularizing 
the DNA within the virus particle, may protect the DNA from exonuclease 
digestion or may act as an endonuclease in a hairpin model of DNA replication. 
Furthermore, it has been recently demonstrated that the efficiency of 
transfection with the Ad 5 DNA protein complex is about 100-fold higher 
than that of pronase- treated Ad 5 DNA. 

Brown et al. (Journal of Virology 16_, 366) first demonstrated that 
these DNA-protein complexes did not migrate into agarose gels during 
electrophoresis. This observation provided the basis for a useful means 
for detecting not only the presence or absence of terminal protein 
components but the identification and purification of terminal DNA 
sequences as well. We have fractionated the Ad 2 DNA-protein complex on 
hydroyapatite columnes and analyzed the DNA eluate by agarose gel electro- 
phoresis. Although the initial DNA-protein complexed did not migrate 
into agarose gels, >75% of the initial DNA molecules in the DNA eluate 
were now able to enter the gels in spite of the absence of protease or 
SDS treatment. When the protein in the DNA eluate was labeled in vitro 
with 125j ; protein was not seen associated with the DNA that migrated 
into the gels. When the DNA eluate was examined in the electron microscope, 
DNA was found to be essentially linear. The re-addition of a DNA- free 
protein fraction to the DNA eluate produced circularization of about 20% 
of these linear molecules. We conclude that circularization of the 
adenovirus DNA-protein complex as well as its inability to migrate into 
agarose gels is likely due to an aggregate of a virion component (s) in 
addition to the covalently-attached 55K protein previously described. 

Significant quantitative differences were noted when Ad 18, a 
highly oncogenic serotype, was extracted with 4M guanidinium chloride 
and the DNA-protein complexes assayed in similar fashion. Purified 
virions of adenovirus serotype 2 and 18 were applied to gradients 



19-34 



containing guanidinium, peak fractions were pooled and the DNA-protein 
complexes dialyzed and compared. 35-55% of the molecules from each 
serotype appeared circular in the electron microscope. While the DNA- 
protein complexes from serotype 2 were effectively bound to glass fiber 
filters (99%) under the conditions employed, over 45% of the guanidinium 
purified material isolated from serotype 18 passed unimpeded through the 
filter under identical conditions. Of some interest was the fact that 
nearly all molecules in this fraction were circular. Bound material 
could be effectively released from filters with 1% SDS or pronase 
digestion. DNA molecules from either serotype when extracted with pro- 
teolytic enzymes did not bind to filters. Approximately half of the 
guanidinium purified material from serotype 18 was shown to enter 
agarose gels upon electrophoresis. The structural implications of this 
population of molecules which apparently does not bind to glass fiber 
filters and which easily penetrates agarose gels, yet retains the 
ability to circularize, is of considerable biological interest. 



19-35 



|SM I THSON I AN SCIENCE INFORMATION EXCHANGE] U.S. DEPARTMENT OF j PROJECT NUMBER 

.PROJECT NUMBEP. [Do MOT use this space; JHEALTH, EDUCATION, AND WELFARE ( 

PUBLIC HEALTH SERVICE 
I NOTICE Cf 

INTRAMURAL RESEARCH PROJECT |Z01 AI 00156-04 LBV 

PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (50 characters or less; 

Structural Characterization of DNA Virus Genomes 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Principal Investigator: Claude F. Garon, Research Microbiologist LBV NIAID 



COOPERATING UNITS (if any) 

Dr. J. W. Burnett, University of Maryland Hospital, Baltimore, Maryland 
Dr. H. B. Bradford, Louisiana Health and Human Resources Administration 
New Orleans, Louisiana 



LAB/ BRANCH 

Laboratory of Biology of Viruses 



SECTION 

Macromolecular Biology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20014 



TOTAL MANYEARS: j PR0FESSI 0NAL: 

0.2 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS £ (b) HUMAN TISSUES [] (c) NEITHER 

L (a1 ) MINORS 2 ( a2 ) INTERVIEWS 



SUMMARY OF WORK (200 »ords or less - underline keywords) 

Molluscum contagiosum virions were isolated from clinically typical skin 
lesions and the DNA extracted and characterized using techniques of 
electron microscopy and agarose gel electrophoresis . The structural 
characteristics determined in these studies tend to place the MCV genome 
among other members of the poxvirus group in terms of genome molecular 
weight and organization. The apparent denaturability of the MCV genome 
into a continuous, single-stranded circle would point to the presence of 
terminal cross-links in the DNA molecule. So far, this structural 
arrangement appears to be unique to members of the poxvirus group. The 
objective of these studies has been to define structural features of 
these molecules and to relate them to the biochemical events involved in 
the replication and growth of this virus in the cells they infect. 



19-36 



PMS-6040 
(Rev. 10-76) 



Project Description 

Molluscum contagiosum virus (MCV) is a member of the mammalian 
poxvirus subgroup which causes a benign tumor of the skin in humans. 
The disease is most common in children. Cases appear to occur world- 
wide and are often associated with poverty, overcrowding and poor hygiene. 
Molluscum contagiosum virions were isolated from clinically typical skin 
lesions and the DNA extracted and characterized using techniques of 
electron microscopy and agarose gel electrophoresis. In some experiments, 
a comparison of the structural features of the MCV genome and other 
members of the poxvirus group was made. 

Extracts from molluscum contagiosum lesions when stained with 
sodium phosphatungstate and examined in the electron microscope appeared 
to contain many structurally mature virus particles of both C and M 
forms. However, the virus has not been successfully propagated in the 
laboratory. Molecular information about this virus has been limited to 
description of size, shape and composition and has been difficult to 
obtain. The structural characteristics determined in our initial studies 
(Virology 81: 247) tend to place the MCV genome among other members of 
the poxvirus group in terms of molecular weight and organization.. The 
average molecular weight of MCV-DNA was calculated to be 118 X 10 and 
appeared to be extremely sensitive to both mechanical shear and nuclease 
damage. Single-stranded circles measuring twice the length of linear 
molecules were observed following high levels of denaturation. This now 
appears to be a generalized feature of all poxvirus DNAs so far examined. 
The biological function, if any, of the unique terminal-cross- links is 
unknown. Also of special interest was the fact that the most easily 
denaturable regions of the MCV genome (and presumably that of the highest 
AT content) appeared to be the end 19% of the DNA molecule. That the 
MCV genome differ markedly from that of vaccinia is clear from the 
visual denaturation profiles and from the restriction endonuclease 
cleavage patterns obtained in our initial studies. Further differences 
were noted in the extractability of the various poxvirus genomes. 
Procedures used for successfully extracting and purifying the DNA molecules 
from one viral genome proved completely unsuitable for the other. 

Previously, little was known about whether most isolates belonged 
to the same or different MCV strains on whether a given strain was 
always isolated from similar clinical sources. A survey of the restriction 
endonuclease fragment patterns of several independent MCV isolates was 
initiated. Lesions obtained from individual patients were never pooled 
but rather purified, extracted, and assayed as a single isolate. Initially, 
eleven such isolates were processed. In these studies, lesions isolated 
from various sites on an individual patient appeared to contain virus 
whose DNA showed identical gel patterns. Furthermore, virus was isolated 
from the lesions of patients' relatives which, when assayed by the above 
restriction endonuclease procedure, showed gel patterns identical to 
those of the patients themselves. Interestingly, only 3 characteristically 
frequent patterns were observed among eleven independent isolates. 
Restriction endonuclease digestion produced 11 to 20 fragments ranging 
in size from 4 X 10 to 31 X 10 daltons depending on the virus isolate. 



19-37 



Mixing experiments shewed comigration of some fragments in all patterns. 
Although comigration of DNA fragments does not necessarily mean base 
sequence identity, extensive comigration would imply significant genetic 
or organization similarity among the viral genomes. Direct comparisons 
of these gel purified fragments by hybridization or heteroduplex analysis 
have not been attempted due to limits in the quantity of viral DNA 
obtainable from each MCU isolate. 

Although we have not collected a large enought body of data to make 
any reasonable assessment of the relationship between the clinical 
characteristics of the disease and the restriction endonuclease cleavage 
patterns, it may be possible one the data are accumulated, to relate 
these observations and to develop a useful molecular epidemiological 
scheme. Such a scheme would make possible rapid identification and 
classification of MCV isolates without the necessity of propagating the 
virus in the laboratory. Furthermore, since the degree of sensitivity 
of such a system is limited only by the number of cleavage sites recognized 
by a given restriction enzyme, other available enzymes may easily be 
substituted. 

We propose to continue to define structural features of these 
poxvirus DNA molecules with the aim of (1) comparing genetic variation 
among members of this group, and of (2) relating these features to those 
biochemical events which are involved in the replication of these agents. 



19-38 



LABORATORY OF CLINICAL INVESTIGATION 
1979 Annual Report 
Table of Contents 



- Z01-AI 
Project Number 

Summary 

00043-14 

00045-11 

00046-11 
00047-10 
00048-09 
00049-09 
00050-09 
00051-0? 

00054-0? 

00055-07 
00056-06 

00057-06 
00058-06 



Immunology and Chemotherapy of Systemic 
Mycoses — Bennett 

Studies on the Interaction of Antibody and 
Complement on the Production of Immune 
Damage — Frank 

Pathogenesis of Delayed Hypersensitivity — 
Kirkpatrick 

Clinical Studies on Patients with Known 
or Suspected Parasitic Diseases — Neva 

The Pathophysiology of Autoimmune Hemolytic 
Anemia — Frank 

Initiation and Regulation of Antigen 
Recognition — Frank 

Clinical Studies of Complement 
Abnormalities of Man — Frank 

Host Defense Mechanism Against Pseudomonas 
Infection in Normal and Immunosuppressed 
Hosts — Aduan 



Page 
20-1 
20-11 

20-14 

20-18 
20-24 
20-29 
20-34 
20-36 
20-40 



The Etiology and Pathogenesis of Viral 20-41 

Gastrointestinal and Respiratory Tract 
Infections in Man — Dolin 

Regulation of the Immune Response in Man 20-44 
and Experimental Animals — Fauci 

Biochemical Pathways of Mediator Release 20-60 
and Mechanism of Tissue Injury in 
Allergic Diseases — Kaplan 

Basic Studies on Pathogenic Fungi — 20-61 

Kwon-Chung 

The Pathogenesis and Chemotherapy of Herpes - 20-66 
virus Infections in Man — Dolin 



Table of Contents (Continued) 



Z01-AI 
Project Number 

00154-04 



00155-04 
00188-01 

00189-01 



Immunologic, Neurophysiologic, Biochemical 
and Cellular Events in Immediate 
Hyp er s ens i t ivi ty — Kal iner 

Phagocyte Cell Function — Gallin 

Rapid Diagnosis of Infections by Enzyme- 
Linked Immunoadsorbent Assays — Straus 

Clinical and Biochemical Studies of Human 
Enteral Adenovirus — Straus 



Page 
20-70 

20-78 
20-86 

20-89 



Summary of Program 
Laboratory of Clinical Investigation 
October 1, 1978 - September 30, 1979 

Michael M. Frank, M. D. , Chief of Laboratory 
and Clinical Director, NIAID 

Introduction 

This has been a highly rewarding year for the Laboratory of Clinical 
Investigation. It has been a year of continuing change and development 
of the new directions of the program. Basically, two years ago a decision 
was made to continue to have represented within the laboratory strong 
programs in infectious diseases, immunology and allergy. Several of the 
senior members of the staff left for other excellent appointments and an 
opportunity was had to change the directions of the laboratory at a time 
when there was no possibility for further growth in positions or space. 
A decision was made to allow for modest increases in the size of the 
commitment toward cellular immunology under Dr. Fauci, toward complement 
research under Dr. Frank, toward allergy research under Dr. Kaliner and 
toward research in the phagocytic cell in host defense under Dr. Gallin. 
Our commitment to mycology continues to be evident and strong and our 
commitment to virology was reaffirmed with the appointment of a new 
leader of the virology program. 

Dr. Fauci had the opportunity to recruit a second senior individual in 
cellular immunology, Dr. Barton Haynes, who joined the senior staff. 
The Clinical Immunology Section recruited one of its former Clinical 
Associates, Dr. Steven Hosea, to join the staff as an independent investi- 
gator developing the area of the pathophysiology of bacterial infection. Dr. 
Kaliner assumed the position of Chief of the Allergy group with the 
departure of Dr. Allen Kaplan and was given a second position to recruit 
a second senior allergist. Dr. Dean Metcalf, one of our former Clinical 
Associates has been recruited and will be joining the laboratory at the 
end of theis fiscal year. Dr. Metcalf served 3 years as a Clinical 
Associate in the Laboratory of Clinical Investigation and then joined 
Dr. Frank Austin's department at Harvard where he continued to develop 
these investigative skills. He will be starting a program in food 
hypersensitivity. Thus, the various groups have continued to be rounded 
out in the face of very limited potential for growth. 

The Clinical Immunology group now has extensive depth in the 
general area of complement protein interactions and contribution of 
complement to medical illness. The group has extended its depth considerably 
in the general area of immune complexes and the pathophysiology of 
immune complexes and has worked very closely with members of the Cancer 
Institute interested in these same problems. The cellular immunology 
program has extended under Dr. Fauci and Dr. Haynes and is deeply involved 
in lymphocyte function in autoimmune diseases. We continue to study the 
pathophysiologic responses of the phagocytic cell and continue to develop 
our capabilities in bacterial infection, various aspects of mycotic 
infection. 

20-1 



During this year Dr. Raphael Dolin left to assume the position of 
Chief of the Infectious Diseases at the University of Vermont. After an 
extensive search Dr. Stephen Straus was added to the program to continue 
and develop our strength in virologic investigation. Dr. Straus comes 
to us from Washington University in St. Louis where he received extensive 
training in infectious diseases. Before that he was trained in basic 
virology in the NIAID Laboratory of the Biology of Viruses. As discussed 
below he will extend and develop our expertise in the the use of antiviral 
agents and in the pathophysiology of viral infection. 

Dr. Charles Kirkpatrick has continued his studies of mucocutaneous 
candidiasis and of delayed hypersensitivity defects in a wide variety of 
clinical diseases. Dr. Kirkpatrick has accepted a position as Professor 
of Medicine in Denver and will be leaving the Institute at the end of 
this fiscal year. Nevertheless, it is hoped that our expertise in 
cellular immunology will be maintained through Dr. Fauci's efforts in 
this area. Dr. Kirkpatrick has made enormous strides in the development 
of rational treatment for patients with mucocutaneous candidiasis and in 
our understanding of the pathophysiologic basis of this disease. It 
can safely be said that his efforts have been responsible for most of 
the available therapies which are now in use in treatment of this disease. 
Moreover, his contributions to the development of our understanding of 
transfer factor have been considerable and it is expected that he will 
be able to continue his efforts to understand the structure of transfer 
factor and its mechanism of action in his new position in Denver. 

Research Accomplishments 

CLINICAL VIROLOGY SECTION 

Dr. Straus is continuing the ongoing effort of the viral disease 
section in exploring the pathogenesis and natural history of herpes 
virus infections in man. Moreover studies of infections mononucleosis 
have been extended during the year and a number of effects of the causative 
agent the EB virus, upon the target B lymphocyte have been examined. 
Moreover effects of EB virus on T cell responses and on helper and 
supressor T cell function have been examined. The group continued and 
confirmed earlier observations on the usefulness of adenine aranoside 
in therapy of serious herpes virus infections. An enzyme linked immuno- 
adsorbant assay was also developed for detecting influenza A hemagglutinin, 
Candida cell wall mannan, human adenoviruses, herpes simplex virus and 
antibodies to pneumococci. These important diagnostic advances will 
make rapid diagnosis and patient evaluation much more practicable as 
they are further developed. Dr. Straus initiated studies of enteral 
adenovirus infection as a cause of gastroenteritis in children. These 
viruses are very difficult to grow and a collaborative study has been 
initiated to try several methods of approach to the culture of these 
viruses and to the study of their pathophysiologic effects. It is hoped 
to add studies of this viral group to the ongoing studies in NIAID of 
the causes of viral gastroenteritis including the addition of viral 
challenges of normal volunteers to define the pathophysiology of these 
infections. 

20-2 



CLINICAL MYCOLOGY SECTION 

Dr. Bennett continued his investigations of host defense reactions 
to Cryptococcus neoformans. The immunodominant groups of the type A 
form of the Cryptococcus were studied in an attempt to determine the 
precise mechanisms of antigenicity. In this Cryptococcal type, defined 
originally by Drs. Kwon-Chung and Bennett, the immunodominant group was 
found to be 1 3 mannan as a backbone with Oacetyl groups and to a 
variable extent the glucuronyl side chain. An ELISA assay was also 
developed for the detection of Aspergillus fumigatus. Under Dr. Kwon- 
Chung efforts were extended to understand the various pathways utilized 
in Cryptococcus neoformans and C. bacillisporus. Also the nuclear states 
of basidiospores in the sexual development of C. neoformans were studied 
as were the mating types among the various groups which have been observed 
in this laboratory. The observation that Cryptococci can be divided into 
distinct species is considered of major importance in understanding the 
biology of this organism and perhaps in explaining some aspects of the 
host defense mechanisms engaged by these organisms. 

CLINICAL PHYSIOLOGY SECTION 

Dr. Fauci's unit has continued to explore the precise mechanisms 
and immunoregulatory events associated with triggering of B lymphocytes 
following nonspecific and antigen induced stimulation. Functionally 
distinct as well as overlapping immunoregulatory lymphocyte subsets have 
been characterized. The precise abnormalities of immunologic reactivity 
have been determined in several autoimmune diseases such as systemic 
lupus erythematosus, Sjogren's Syndrome, infectious mononucleosis, 
sarcoidosis, and tuberculosis. Dr. Fauci's group has succeeded in 
producing an anti-ideotypic antibody which specifically blocked the 
function of human B cells bearing the corresponding antibody ideotype. 
Hybridoma lymphocyte cell lines were established which are secreting 
antibody which binds specifically to functionally distinct subsets of 
human blood lymphocytes. The availability of such antibodies should 
prove of major importance in further exploration of immunologically 
mediated effects in humans. Countercurrent centrifugation techniques 
were adapted from those previously described and which result in 95% 
purity and yield of monocytes from human blood. Again, the availability 
of such highly purified cells should greatly ease further developmental 
work in cellular immunology. Modulation of lymphocyte subsets by a 
number of clinically relavant pharmacologic agents has been further 
defined. Clinical therapeutic studies in this group have yielded highly 
favorable results in the approach to the treatment of systemic necrotizing 
vasculitis. A number of systemic necrotizing vasculities have yielded to 
therapy with cyclophosphamide or cyclophosphamide and glucocorticoids. 

CLINICAL IMMUNOLOGY SECTION 

This Section has continued its studies in three major areas, the 
role of complement in the production of disease, the structure and 
function of immune complexes in disease and the role of the reticuloendo- 
thelial system in the development of autoimmune illness and in host 

20-3 



defense mechanisms. In the first of these areas great strides have been 
made in the purification of large quantities of complement components 
which are available for study in patients. This has required an enormous 
effort in terms of protein purification and new methodologies have been 
developed for the purification and separation of functionally active 
components. Virtually all of the components of the classical pathway 
have now been purified and are now available for study as are certain of 
the regulatory proteins of the complement system. A new method has been 
developed for the evaluation of the opsonic fragment of C3 and it has 
been possible to study the degradation of C3 on a molecular basis in 
precise quantitative detail. This complement protein is of major importance 
in bacterial destruction and further understanding of its mechanisms of 
action is of great importance. Moreover, methods have been developed 
for analyzing these mechanisms of degradation in normal sera. Studies 
of complement receptor function and Fc immunoglobulin receptor function 
have continued to provide great insight into normal pathophysiologic 
mechanisms and disease states. Thus, for the first time a receptor for 
C5 has been identified unequivocally on polymorphonuclear neutrophils 
and the binding of C5 to C3 has been studied extensively. The role of 
phagocytic cells in the degradation of and decay of C3 on opsonized 
particles has also been extensively explored. Tests of in vivo reticulo- 
endothelial function in man have been developed which are now coming 
into wide use around the world. It has been demonstrated for the first 
time that certain kinds of autoimmune diseases associated with the 
presence of circulating immune complexes are also associated with defects 
in the function of the reticuloendothelial system, and other autoimmune 
diseases associated with circulating immune complexes are not. In 
general the autoimmune diseases associated with clearance defects are 
those which have as part of the clinical spectrum tissue deposition of 
immune complexes and tissue damage. Thus in general the most severe 
autoimmune diseases are associated with defects in immune clearance. 
Extensive studies have been performed in lupus erythematosus, mixed 
connective tissue disease, rheumatoid arthritis, mixed cryoglobulinemia, 
Sjogren's Syndrome, primary biliary cirrhosis and chronic active hepatitis, 
etc. Defects in immune clearance which are specific for various types of 
receptors on phagocytic cells have been demonstrated as well. Importantly, 
the standard test for RES function in man, that which measures clearance 
of aggregated human serum albumin, has been shown to be useless in these 
evaluations. A large population of patients with Hereditary Angioedema 
have continued to be followed and evaluated. New therapy is being 
developed as is further understanding of the pathophysiology of this 
interesting "experiment of nature". 

CLINICAL ALLERGY AND HYPERSENSITIVITY SECTION 

Dr. Kirkpatrick' s laboratory has continued to study the biologic 
basis of cellular immunity. Continued studies of a large group of 
patients with mucocutaneous candidiasis has shown a proportion of 



20-4 



these patients have an abnormal ratio of helper T to suppressor T lymphocytes, 
suggesting a problem in the regulation of immunologic function. Dr. 
Kirkpatrick's group has isolated a new lymphokine termed E-RAF which 
appears to induce the maturation of null cells into E-rosette forming T 
cells. He has shown that there are several products which can be elaborated 
from lymphocytes which produces these effects and that certain kinds of 
patients with immunologic deficiency do not produce this factor. In Dr. 
Kirkpatrick's laboratory several controlled clinical trials of new 
agents for use in treatment of mucocutaneous candidiasis have been 
conducted including a control trial that has shown that clotrimazole 
troches are effective therapy for oral candidiasis. It has also been 
shown that transfer factor may induce more prolonged remissions from 
candidiasis in patients who receive this therapeutic agent. 

ALLERGIC DISEASES SECTION 

The Allergic Disease Section has continued to widen its investigations 
of the basic immunologic and physiologic events in immediate hypersensitivity 
reactions and in the pathophysiologic processes which take place in 
patients with a variety of allergic conditions. A number of areas are 
now being studied in detail and although the work is only in its infancy 
a wide variety of important leads have already been unearthed. One 
project involves characterization of mucous secretion and identification 
of the factors which control mucous secretion. This is quite important 
because excessive mucous secretion in such diverse diseases as asthma 
and cystic fibrosis play a major role in the pathology of the disease. 
Methods have been developed to study mucous secretion and the mucous 
products have now been identified and radiolabelled. The effect of 
drugs on mucous secretion is now under study. The site and control of 
human lung parenchyma as opposed to airway prostaglandin production has 
also been partially identified and the factors generated during the 
anaphylactic response which cause prostaglandin synthesis have been 
partially isolated. In another area of investigation the histologic 
responses to the injection of mast cell granules have been characterized 
both in rat and monkey skin and those factors responsible for eliciting 
the inflammatory response have been partially isolated and characterized. 

Detailed studies have been performed on the autonomic nervous 
system in patients with asthma and allergic rhinitis and a number of 
important points have been discovered. Among these is that patients 
with asthma and allergic rhinitis have significant impairment of beta 
adrenergic responsiveness and increased responsiveness to cholinergic 
stimuli. Patients with allergic rhinitis have normal response to alpha 
adrenergic stimulation while asthmatics have augmented response to 
alpha-adrenergic stimulation. These stimuli control the responsiveness 
and state of dilatation of the airways of the human lung and these 
observations are of direct relevance in our attempts to understand 
asthma. 



20-5 



BACTERIAL DISEASES SECTION 

Dr. John Gallin' s laboratory has continued to study various aspects 
of phagocytic cell function. Mechanisms of leukocyte activation by 
chemotactic factors have been studied using electrophysiologic techniques 
as well as techniques which probe cell surface charge and ultrastructure. 
Dr. Gallin' s group has shown that secretion of specific granules which 
may accompany chemotaxis is associated with increased cell adhesiveness 
and increased availability of chemoattractant receptors. Vigorous 
exocytosis is associated with depressed chemotaxsis, decreased availability 
of chemoattractant receptors, hydrolysis of the chemoattractant by 
secreted products and markedly increased cell adherence and aggregation. 
Human pyrogen has been shown to be a potent stimulator of neutrophil 
exocytosis and causes activation of the hexose monophosphateshunt . Dr. 
Gallin' s laboratory had previously shown that neutrophils can be divided 
into two populations, those with large numbers of Fc receptors and those 
with small numbers of Fc receptors. Recent work has shown that both 
sets of neutrophils have equal C3b receptor activity and that neither 
has a demonstrable C3d receptor. Biological differences however between 
the two sets of granulcytes have been clearly indicated. Thus, in vivo 
injection of endotoxin into patients or the hemodialysis of patients 
leads to loss from the circulation of those neutrophils with demonstrable 
Fc receptors and a marked increase in the percentage of granulocytes 
with no demonstrable Fc receptors. 

BIOLOGIC STRUCTURE SECTION 

Dr. Allen Rosenthal left the Laboratory of Clinical Investigation 
to assume the position of Director of Immunology of the Merck Sharpe and 
Dohme Company one year ago. At that time he had a number of fellows and 
a number of important research projects which are of interest to the 
Institute. During this year those projects were partially or entirely 
completed. 

It was shown that antiinsulin antibodies of exquisite antigenic 
specificity can be prepared and identified. Moreover, it was shown that 
the cellular immune response to the insulin antigen does not necessarily 
recognize the same antigenic groupings as the humoral response. Precise 
fine structural analysis of the actual amino acids recognized during the 
cellular response to insulin has been defined and initial experiments 
have begun on determining the role of histocompatibility groups on 
antigenic recognition of portions of the insulin molecule. This is a 
far ranging project which may ultimately give us a great deal of information 
about the biological basis of insulin allergy. Dr. Rosenthal intends to 
continue these investigations at the Merck Sharpe & Dohme Company. 

In all, this has been an impressive year in the Laboratory of 
Clinical Investigation. Both clinically and in terms of basic science 
great strides have been made. Our research groups find themselves with 
many more problems than we can possibly find time to investigate and we 
all look forward to future developments with great anticipation. 

20-6 



CLINICAL PARASITOLOGY SECTION 

The parasitic disease group has continued to extend its basic studies 
of Chagas ' disease, schistosome and filariasis. The association of absence 
of HLA tissue type-Al with chronic Chagas' disease was not confirmed by an 
expanded study of cases, but DR (B cell) typing will still be done on 
frozen cells. The diversity of HLA haplotypes encountered in this Brazilian 
patient population corroborates what anthropologists have said of this 
population - namely, extreme genetic mixing. Further study of different 
antigenic fractions of schistosome worms has disclosed one fraction to 
which antibody levels in patients correlate quite well with numbers of 
eggs being excreted, or intensity of infection. Evidence for serum in- 
hibitory factors and for suppressor cells has been found in filariasis 
patients characterized by unresponsiveness to filarial antigens. The role 
of immune complexes in filariasis patients is also being studied. Serum 
and cell factors were also found that contributed to antigen unresponsive- 
ness in patients with chronic schistosomiasis. 



20-7 



20-8 



Honors and Awards 

This has been a year in which the efforts of members of the Laboratory 
have been well recognized. Members of the Laboratory have been elected 
to many learned societies and now serve on the Editorial Board of many 
major journals. 

Specifically, Dr. Anthony Fauci was elected President of the American 
Federation for Clinical Research and received the Public Health Service 
Meritorious Service Award. Dr. Fauci joins Dr. Frank as a member of the 
Association of American Physicians. Dr. Frank was invited to give the 
plenary lecture at the Japanese Rheumatism Association Meeting in Japan in 
June of 1979 and Dr. Fauci gave the Stanislaus Jaros Memorial Lecture to 
the American Association for Clinical Immunology and Allergy in 1978. Dr. 
Frank was invited to speak at the plenary session of the Infectious Diseases 
Society Meeting in 1979. Dr. Michael Kaliner was elected to the American 
Society for Clinical Investigation bringing the number of members of that 
Society within the LCI to five. Dr. Jack Bennett was elected to the Council 
of the Infectious Diseases Society and Dr. Kirkpatrick was appointed to the 
Education Committee of the American Academy of Allergy. The American 
Academy of Allergy also appointed Dr. Fauci to the Postgraduate Education 
Committee. In addition, Dr. Gallin was appointed to the Program Committee 
of the American Association of Immunologists. Dr. Kwon-Chung was elected 
Chairperson of the Medical Mycology Division of the American Society for 
Microbiology and was also selected Society Lecturer for the British 
Mycopathological Society at the Annual Meeting in Birmingham, England. 

The Laboratory of Clinical Investigation is also well represented on 
Editorial Boards of major journals. Dr. Frank and Dr. Fauci are now both 
serving on the Editorial Board of the Journal of Clinical Investigation. 
Dr. Fauci serves as Section Head of the Editorial Board in Clinical 
Immunology of the Journal of Immunology and Dr. Gallin is a member of the 
Editorial Board. Dr. Fauci serves as a Section Head of the Editorial 
Board in Allergy and Immunology of the American Journal of Medicine. Dr. 
Frank serves on the Editorial Board of Blood and on the Editorial Board of 
the new Journal of Infectious Disease Reviews. Dr. Bennett serves on the 
Editorial Board of the Journal of Infectious Disease and the Journal, 
Antimicrobial agents and chemotherapy and on the Editorial Board of the 
Journal of Clinical Microbiology as well. Dr. Kirkpatrick serves on 
Editorial Boards of three journals, Thymus, The Journal of Allergy and 
Clinical Immunology, and the Journal of Cutaneous Pathology. Dr. Gallin 
serves on the Editorial Board of the Journal of Immunology and also of the 
new journal, Inflammation. Dr. Kaliner serves on the Editorial Board of 
the Journal of Allergy and Clinical Immunology. Dr. Fauci serves on the 
Editorial Board of the Journal of Immunopharmacology and on the Editorial 
Board of the American College of Physicians, Medical Knowledge. Self- 
Assessment, Audiocassett Program. Dr. Kwon-Chung serves on the Editorial 
Board of the Journal, Sabauraudia. Thus, many major journals in modern 
clinical immunology in infectious disease and clinical research are 
represented by members of the Laboratory. 



20-9 



20-10 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00043-14 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Immunology and Chemotherapy of Systemic Mycoses 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: J.E. Bennett 

OTHER: A.D. Hernandez 
D.K. Henderson 



Head, Clinical Mycology Section 
Guest Worker 



LCI NIAID 



COOPERATING UNITS (if any) 

S. Kim (FDA) 

A. Bhattacharjee (NIAMDD) 

P. Pizzo (NCI) 



LAB/BRANCH 



Laboratory of Clinical Investigation 



Clinical Mycology Section 



NSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS 

3 11/12 



PROFESSIONAL: 

3 H/12 



OTHER: 



2.0 



CHECK APPROPRIATE BOX(ES) 
Xj (a) HUMAN SUBJECTS 

□ (al) MINORS fj ( a 2) INTERVIEWS 



S (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The immunodominant groups on type A Cryptococcus neoformans were found to 
be the al,3 mannan backbone, particularly the 0-acetyl groups and, to a vari- 
able extent, the 3-glucuronyl side chain. 

Radioimmunoassay and ELISA have been developed to detect a specific 
neutral polysaccharide of Aspergillus f umigatus . 



20-11 



PHS-6040 
(Rev. IO-76) 



Project No. Z01 AI 00043-14 LCI 
Project Description : 

The general goals of the unit have been to study host defense in 
systemic mycoses and to improve diagnosis and treatment of these disorders. 

Objectives : 

1) Determine the role which antibody to C. neoformans plays in host 
defense; i.e., the cause and significance of absent antibody formation in 
most patients with the disease. 

2) Characterize the immunodominant groups which contribute to serotype 
specificity in C_. neoformans polysaccharides. 

3) Purify polysaccharides of Aspergillus fumigatus , devise assays for 
these polysaccharides and determine whether these antigens appear in body 
fluids during infection. 

Methodology, Results and Significance : 

1) Hapten inhibition radioimmunoassay has been used to explore further 
the immunodominant positions on C_. neoformans polysaccharides. Although 
individual sera do vary, antibody binding sites on type D polysaccharide in- 
clude the oil, 3 mannan backbone, particularly the 0-acetyl groups there, and, 
for occasional sera, the glucuronyl side chain. On type A, |3-glucuronyl 
mannan is one determinant. None of these determinants has proven immuno- 
dominant in type C; and it seems likely that larger, more complex structures 
are immunodominant. Lack of 0-acetyl groups and extensive substitution of 
the al,3 mannan backbone are the structural features of type C which account 
for these differences in immunodominance. (Bennett and Bhattacharjee) 

2) Young healthy volunteers were found to have lgM and lgG antibody to 
type A C_. neoformans polysaccharide. Antibody was significantly less common 
beyond 50 years of age, a time in life when cryptococcosis is more common. 
The majority of patients with cryptococcosis do not have anti-cryptococcal 
antibody during either illness or in later years. It remains to be deter- 
mined whether disease produces tolerance or inability to respond to the 
antigenic challenge reflects a predisposing cause of infection. (Bennett and 
Henderson) 

3) A neutral polysaccharide of Aspergillus fumigatus was tyraminylated 
and labeled with 12 ^I. From 40-50% of the radioactivity was precipitable 
with rabbit antisera. Although the polysaccharide has a fairly homogeneous 
pattern on Sephadex chromatography (mol. wt. 36,000 daltons), the portion of 
the polysaccharide not binding to antibody should be able to be removed by 
affinity chromatography. The final material can be used for structural 
analysis and to search for antigenemia in aspergillosis. 

The partially purified polysaccharide can be detected by ELISA down 
to 100 ng/ml. This assay also will be used to search for antigenemia in 



20-12 



Project No, Z01 AI 00043-14 LCI 

aspergillosis. (Bennett and Kim) 

4) Radioimmunoassay has found antibody against C^ albicans mannan in 
both normal volunteers and immunosuppressed patients, with no substantial 
differences between these groups. Small numbers of patients with fatal dis- 
seminated candidiasis have had antibody titers similar to the other two 
groups. 

Publications : 

1. Diasio, R.B., Lakings, D.E., Bennett, J.E.: Evidence for conversion of 
5-f luorocytosine to 5-f luorouracil in man: A possible factor in 
5-fluorocytosine clinical toxicity. Antimicrob . Agents Chemother . 14: 
903-908, 1978. 

2. Feldman, L., Lamson, M. , Gallelli, J.F. and Bennett, J.E.: Surveillance 
of nosocomial infections by antibiotic monitoring. J_. Amer . Med . Assoc . 
241:2806, 1979. 

3. Bennett, J.E.: Diagnosis and management of candidemia in the immuno- 
suppressed host. Scan . J_. Infect . Pis . Supp . 16:83-86, 1978. 

4. Mandell, G.M., Douglas, R.G., Bennett, J.E.: Principles and Practices 
of Infectious Disease . John Wiley and Sons, New York, N.Y., 1979. 

5. Segal, E., Berg, R. , Pizzo, P. and Bennett, J.E. : Detection of Candida 
antigen in the sera of patients with candidiasis by an ELISA inhibition 
technique. J. Clin . Microbiol . 10:116-118, 1979. 

6. Bennett, J.E., Dismukes, W.E., Duma, R.J., et al: A collaborative study 
comparing amphotericin B alone or combined with flucytosine in the treat- 
ment of cryptococcal meningitis. N_. Engl . J_. Med . 301:126-131, 1979. 



20-13 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl Al 00045-11 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Studies on the Interaction of Antibody and Complement on the Production 
of Immune Damage 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



M. Frank 



C. Hammer 

T. Gaither 

K. Katusha 

L. Renfer 

M. Santaella 

H. Gresham 

E. Brown 

S. Hosea 



Head, Clinical Immunology Section NIAID,LC| 
and Chief 

Senior Staff Fellow NIAID,LCt 

Resident Biologist NIAID.LCI 

Microbiologist NIAID.LCI 

Chemist NIAID,LCl 

Guest Researcher on IPA NIAID,LC:[ 

Medical Technologist NIAID,LCI 

Clinical Associate NIAID.LCI 

Fellow in Infectious Diseases NIAID,LCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Clinical Immunology Section 



INSTITUTE AND LOCATION 

NIH, NIAID, Bethesda, MD 20205 



TOTAL MANYEARS: 

5 4/12 



PROFESSIONAL: 

2 10/12 



3 6/12 



CHECK APPROPRIATE BOX(tS) 
J (a) HUMAN SUBJECTS 

□ (al) MINORS rj (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



(c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Techniques have now been developed in the laboratory for the large- 
scale purification of Clq, C4, C3, 5, 6, 7, 8, 9, and the C56 complex. 
New assays have been developed for the C3b inactivator and for the 
protein S1H and the role of these proteins in C3b inactivation has been 
further explored. Studies are ongoing on the characteristics of this 
interaction which leads to the destruction of the opsonically active C3b 
site on erythrocyte surfaces. Studies have also been conducted on the 
interaction of complement with bacteria and considerable progress has 
been made in studies of the role of complement in producing the clinical 
symptoms of infectious disease. 



20-1^ 



J HS-6040 
(Rev. 10-76) 



Project No. Z01 AI 0045-11 LCI 

Project Description: 

Objective : 

The objective of this program is the longterm study of the role of 
antibody and complement in the production of immune injury in vitro and 
in vivo . To this end the changes which were initiated last year in the 
Clinical Immunology Section have continued. A pronounced emphasis is 
continuing on protein purification and assay and on studies of protein 
interaction within the complement system with the goal of developing 
reagents suitable for pathophysiologic studies in patients. 

Methods Employed : 

The methods employed involved purification of complement components, 
their use in the sequential build-up of complement intermediates on cells, 
the identification of sera of patients with defined complement abnormalities 
so that these sera can be used as reagents, and the purification of immuno- 
globulins of various classes by standard techniques. Methods for titration 
of complement components have been developed in our laboratory, and these 
are in routine use. Methods for cultivation of various types of bacteria as 
well as for bactericidal assays have also been developed in the laboratory and 
methods for radiolabeling of bacteria. Disc gel polyacrylamide electro- 
phoresis as well as electrofocusing techniques have been developed in the 
laboratory and methods have been developed to prepare proteins of very high 
purity. Also large batch methods have been developed for the first time for 
the purification of the various complement proteins,,. Methods have also been 
developed for the radiolabelling of bacteria with I. 

Major Findings : 

Using methods initially developed for the purification of C3 , C5, C6, 
and C7 from separate plasma pools we have consolidated the purification 
protocol to allow for the isolation of most of complement proteins in bio- 
logically active, homogenous form from one large pool of human plasma. The 
basis of success of this purification scheme has rested on the ability to 
obtain a large number of selective highly resolved chromatographic pools 
early in the purification protocol in which biological activity has been 
stabilized. We have found that the use of specific protease inhibitors and 
agents which block complement activation is requisite for recovery of bio- 
logical activity of many of the complement proteins. We have demonstrated 
that in addition to the use of inhibitors in preservation of complement 
activity the ability to proceed rapidly through the first ion exchange 
steps yields improved recoveries of biologically active complement 
proteins. Using the biochemically homogeneous components we have begun 
to prepare monospecific antisera in goats and burros. These reagents 
are of indispensable value in clinical studies to determine antigenic 
levels in patients and to further study the interaction of the proteins 
and their biological activity. 

20-15 



Project No. Z01 AI 00045-11 LCI 

This year has also seen the completion of a new assay system which 
quantitates the functional activities of the alternative pathway control 
proteins glH and C3b inactivator. Homogeneous C3 preparations radio- 
labelled with I iodine are used to prepare cellular intermediates in 
the complement sequence and the use of these intermediates has allowed 
us to clarify the functions of glH, the C3b inactivator and a proteolytic 
serum enzyme involved in inactivation of the biologically important C3 
fragment C3b. The assay is based on the release of radiolabelled C3c 
which occurs only after all of these proteins have interacted with C3b 
bound to the cell membrane. The C3b cleavage fragments formed during 
inactivation of cellbound C3b have been characterized by SDS polyacrylamide 
gel electrophoresis. This analysis verified the C3b molecule undergoes 
two separate cleavages, first by the C3b inactivator and S1H and, 
second, by the proteolytic enzyme. The assay is presently being applied 
to study S1H and C3b inactivator activities in the sera of patients with 
a variety of illnesses. Experiments are currently being designed to 
study the inactivation of C3b bound to the surface of various strains of 
bacteria. The goal is to determine if there is relationship between 
virulence of various bacterial strains and the susceptibility of the 
bacterial bound C3b to inactivation by the control proteins. Preliminary 
studies have been performed using S. typhosa 0901 to establish the 
optimal conditions for C3 inactivation and C3 binding and for inactivation 
of the bound C3b by purified control proteins. Studies of interaction 
of bacteria and complement have proceeded with the development of new 
techniques for radiolabelling of bacteria. These studies will be extended 
to consideration of the role of complement in the development of the 
clinical signs and symptoms associated with bacterial infection. 

Significance to Biomedical Research 

At present it is clear that complement activation plays an essential 
role in protecting the individual from infection with microorganisms. It 
is also clear that unregulated complement activation is a crucial component 
of autoimmune disease and regularly leads to tissue injury. Very little is 
known about how the complement proteins function in disease states however. 
The studies underway in our laboratory at present will make it possible to 
explore each of these questions in a systemic manner. They represent the 
overcoming of formidable methodologic barriers and are essential to further 
progress in theses areas. 

Proposed Course : 

We plan to extend these studies to obtain a further understanding 
of the role of the proteins in the regulation of complement function in normal 
physiologic situations and in disease states. We plan to examine the 
role of control proteins in infectious states and in control against 
bacterial infection. In addition, we plan to determine the role of 
complement activation fragments in the development of clinical signs and 
symptoms of bacterial disease. We plan to further purify complement proteins 
and components so that we can study their metabolism and interaction 
in a variety of disease states. 

20-16 



Project No. AI 00045-11 LCI 
Publications 

1. Frank, M.M. : The Complement System in Host Defense and Inflammation. 
Rev . Infect . Pis . 1:3 483-501, 1979. 

2. Gaither, T.A. , and Frank, M.M. : Complement. In Todd, Sanford, Davidson, 
(Eds) : Clinical Diagnosis and Management by Laboratory Methods . 
Philadelphia, PA. W.B. Saunders Company, 16th Ed., 1979, pp. 1245-1261. 

3. Lawley, T.J. and Frank, M.M. : Complement. In Dermatologic Reviews . 
In press. 

4. Gelfand, M.C., Frank, M.M. , Green, I. and Shin, M.L. : Binding Sites 
for Immune Complexes Containing IgG in the Renal Interstitium. 
Clin . Immunol, and Immunopath. 13: 19-29, 1979. 

5. Gaither, T.A. , Hammer, C. and Frank, M.M. : Studies of the Molecular 
Mechanisms of C3b Inactivation and a Simplified Assay for 31H and 
C3b Inactivator. J_. Immunol . In press. 

6. Lawley, T.J., Moutsopoulos, H.M. , Katz, S.I., Theof ilopoulos , A.N. , 
Chused, T.M. and Frank, M.M. : Demonstration of Circulating Immune 
Complexes in Sjogrens Syndrome. J_. Immunol . In press. 

7. Macher, A.M., Bennett, J.E., Gadek, J.E., and Frank, M.M. : Complement 
Depletion in Cryptococcal Sepsis. J. Immunol. 120: 1686-90, 1978. 



20-17 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00646-11 LCI 



PERIOD COVERED 

October 1, 1978 to Setember 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Pathogenesis of Delayed Hypersensitivity 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



Charles H. Kirkpatrick 



Eskild A. Petersen 



A.I. 



>ata 



M. Adedin 
J.I. Gallin 
L.E. Greenberg 



Head, Clinical Allergy 
& Hypersensitivity Section 

Guest Worker 



Visiting Fellow, NIAID, LCI 

NICHD, LCI 
NIAID, LCI 
Bio. Lab. (Micro., NIAID, LCI 



COOPERATING UNITS (if any) 

J. Cook & A. Lewis, OSD, NIAID, S. Shama, Dermatology Branch, NCI, S. Gupta, 
Sloan-Kettering Cancer Center, P.G. Sohlne, Medical College of WI , D.W. Ailing 
OSD, NIAID. 



LAB/ BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Clinical Allergy and Hypersensitivity Section 



INSTITUTE AND LOCATION 

NIH, Bethesda, MD 20205 



TOTAL MANYEARS: 

4 9/1 2 



PROFESSIONAL: 
2 9/1? 



CHECK APPROPRIATE BOX(ES) 
£ (a) HUMAN SUBJECTS 

D (a1 ) MINORS □ (a2) INTERVIEWS 



HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The nature of the immunologic defect that predisposed certain patients to 
chronic and recurrent fungal infections such as mucocutaneous candidiasis has 
been further defined. Certain patients have abnormal ratios of Tu to TQ 
lymphocytes that are compatible with a problem with regulation of immunocyte 
function. 

A new lymphokine, E-RAF, has been defined. It seems to induce maturation of 
"null" cells into E-rosette-f orming T-cells. These observations indicate 
that "null" cells are not "null", but are pre- T-cells. 

Two therapies for chronic mucocutaneous candidiasis have been studied. A con- 
trolled clinical trial has shown that clotrimazole troches are effective thera- 
py for chronic oral candidiasis. Transfer factor from candida-sensitive donors 
may restore the cellular immune responses to Candida to these patients and 
prolong drug-induced remissions. 
20-18 

PHS-6040 
(Rev. 10-76) 



Project No. Z01 AI 00046-11 LCI 
Project Description : 
Objectives (sub-project A): 

1) Characterization of cellular immune responses in immunologically 
deficient patients with chronic infectious diseases; 

2) Studies of the pathogenesis of acquired and congenital immunologic 
deficiencies; 

3) Studies of lymphokine production by cells from normal and immuno- 
deficient subjects: 

4) Studies of developmental immunology including studies of the immuno- 
logic activities of human cord-blood lymphocytes and maturation of cell- 
mediated immune systems in Syrian hamsters. 

Methods Employed 

The major portion of our studies of cellular immune deficiency have been 
conducted in patients with chronic mucocutaneous candidiasis. Approximately 
60 patients with this syndrome have been evaluated. Evidence of immunologic 
deficiency has been found in 66% of the patients. 

The general model for the pathogenesis of cellular immunodeficiency that 
we presented in 1971 still serves as valid outline in which to conduct studies 
of host-defenses. According to his model, patients with candidiasis develop- 
ing during infancy or early childhood have a congenital basis for their 
immunologic deficits. Other patients are apparently normal until adult 
years when they develop candidiasis. In these patients, the immunologic ab- 
normalities are probably acquired. This problem has been under study 
and measurements of immuno- regulatory activities in candidiasis patients 
and the subpopulations of T-lymphocytes have been measured. 

In addition, the biological role of a lymphokine, E-rosette augmenting 
factor (E-RAF) , that was discovered by Dr. Agbata in our laboratory has 
been under study in patients with immunodeficiency syndromes both before 
and after they were treated with immuno-reconstitutive measures. 

The studies of development of immunocompetence by human and hamster 
lymphoid cells have continued. 

The collaborative studies (with J. I. Gallin) on the relationships be- 
tween cellular immunity and chemotaxis in patients with exceptional suscepti- 
bility to pyogenic and/or fungal infections have continued. The studies of 
effects of levamisole on chemotactic responses, lymphocyte function and resis- 
tance to infection have been extended and a controlled clinical trial has been 
designed. 



20-19 



Project No. Z01 AI 90046-11 LCI 
Two evaluations of chemotherapeutic agents for chronic candidiasis have 
been conducted. 

A major achievement in the laboratory has been the development of a mur- 
ine model for transfer factor. The system has been adapted for transfer of 
delayed-type hypersensitivity to soluble proteins and in vitro correlates 
of cellular immunity are being compared with the in vivo responses. 

Major Findings : 

The lymphokine, E-RAF, is produced by mitogen-stimulated cells and by 
antigen-stimulated lymphocytes if the cell donor has cellular immunity to 
the antigen. We now have evidence that antigen- induced E-RAF and mitogen- 
induced E-RAF have different molecular weights. It apparently functions 
by enhancing maturation of "null" cells into T-cells. 

E-RAF production has been studied in a patient with SCID both before 
and after thymus transplantation. Mitogen and antigen stimulated cells 
did not yield E-RAF before the transplant. By the third post-transplant 
day, mitogen-stimulated lymphocytes from the patient produced E-RAF. On 
the same day, the patients cells did not respond to mitogens with LIF pro- 
duction or lymphocyte transformation and he did not have normal numbers 
of circulating T-cells. 

Most candidiasis patients have normal numbers of circulating E-rosette- 
forming T-cells. However, the majority of patients have somewhat low 
numbers of T/'cells (containing the helper cells) and excessive numbers 
of T-cells (containing the suppressor cells). When the ratios of T/'/T/^ 
are calculated 6 of 16 patients had low values, suggesting an immunoregulatory 
defect in these patients. 

Human lymphocytes may develop suppressor activity when they are acti- 
vated with concanavalin-A. In contrast, human cord blood-lymphocytes are 
suppressive of responses by adult cells even without concanavalin activation. 
Fetal cells appear to be in a "suppressive" state under normal conditions. 

The work on ontogeny of lymphocyte function and resistance to oncogenesis 
with Ad 2-transformed cells and cell lines has been extended. All newborn 
hamsters are susceptable to these tumors until about 9 days of age. After this 
time, all animals are resistant. At about this age, spleen cells become 
responsive to concanavalin A; similar responses by blood lymphocytes do 
not appear until the fourth week of life. Of particular interest was the 
finding that spleen cells do not become responsive to alloantigens (in MLR) 
until 7-10 days after development of resistance to tumor induction. Other 
experiments have shown that the developmental delay is probably due to immature 
macrophages rather than lymphocytes. 

A controlled clinical trial showed that clotrimazole buccal troches were 
efficacious in treating chronic oral candidiasis. A study of the systemic 
antifungal, ketoconazole, has been started. 



20-20 



Project No. Z01 AI 00046-11 LCI 

The murine transfer factor system has been adapted to transfer of 
cell-mediated responses to ovalbumin and ferritin. The in vivo responses are 
compared to MIF production by antigen- stimulated cells. Studies with 
genetically-controlled antigens are underway. 

Significance 

1) These experiments and those presented in sub-project B (below) further 
define the relationship between cellular immune responses, inflammation and 
susceptibility to certain chronic infectious diseases. The relative contri- 
bution of individual lymphokines to cell mediated immune responses is unclear. 
For example, it is not known which or how many lymphokines are required to 
produce a "positive" skin test. It is also important to define the relationship 
of specific lymphokine production and "immunity" to infection with a micro- 
organism. E-RAF appears to induce maturation of "null" cells into E-rosette- 
forming T-cells. Thus, the "null" cells are probably pre-T cells. 

Proposed Course : These studies will be continued. 

Objectives (sub-project B) : 

1) Evaluation of immunologic reconstitution as a therapeutic adjunct for 
treating chronic infectious diseases; 

2) Identification and characterization of the components of dialyzable transfer 
factor and assessment of their functions in immunologic and inflammatory 
reactions; 

3) Investigation of transfer factor-like materials in laboratory animals. 

Methods Employed : 

The trial of transfer factor in candidiasis is being continued with the 
oldest participants being in the seventh year of treatment. These subjects 
receive transfer factor every four months. A companion study in which other 
immunodef icient candidiasis patients are being treated with transfer factor 
from either candida-sensitive or Candida- insensitive donors is underway. 

Major Findings : 

Thus far seven patients in whom remissions were induced with amphotericin 
are receiving transfer factor from candida-sensitive donors. In each case 
the patient has developed in vivo and in vitro evidence of cellular immunity 
to Candida. This indicates that the transfer factor was active and that trans- 
fer factor was capable of "correcting" the immunologic lesion in the patients' 
cells. Four of the patients have maintained positive skin responses to Candida 
and all of these patients have remained in remission for 3 to 8 years. These 
remissions are substantially longer than those expected with amphotericin alone. 
The three patients who failed to maintain cellular immunity to Candida also 
suffered relapses of cutaneous candidiasis. 

20-21 



Project No. Z01 AI 0,0046-11 LCI 

Five patients have received transfer factor from donors who do not have 
cellular immunity to Candida. All patients were treated with amphotericin to 
induce remissions. None of the recipients developed cellular immunity to 
Candida, an observation that suggests that transfer factor has immunologic 
specificity. Two of these patients have relapsed. The other three are in 
remission and have been for periods of 8 months to 3 years. 

In summary, all recipients of transfer factor (from either candida-sensi- 
tive or Candida- insensitive donors) have remissions that are longer than those 
seen with amphotericin B alone. It is important to note that the relapses 
occurred only in persons who did not achieve or maintain cell-mediated immunity 
to Candida. 

The differences between the numbers of remissions in the two groups 
is significant at the level of p=0.055. 

Publications : 



1. Kirkpatrick, C.H., Greenberg, L.E., Chapman, S.W., Goldstein, G. , Lewis, 
V.M. and Twomey , J.J.: Plasma thymic hormone activity in patients with 
chronic mucocutaneous candidiasis. Clin. Exp. Immunol. 34: 311-317, 1978. 

2. Kirkpatrick, C.H. : Transfer of Delayed Cutaneous Hypersensitivity With 
Transfer Factor. Cell . Immunol. 41: 62-71, 1978. 

3. Kirkpatrick, C.H. and Ailing, D.W. : Treatment of chronic oral candidiasis 
with clotrimazole troches. A controlled clinical trial. New Engl. J. 
Med. 299: 1201-1203, 1978. 

4. Togawa, A., Oppenheim, J.J. and Kirkpatrick, C.H. : Ability of dialysates 
containing transfer factor to induce lymphocyte activating factor by 
human mononuclear cells. Cell. Immunol. , 45 : - 133-141, 1979. 

5. Kirkpatrick, C.H. : Transfer of cellular immunity with transfer factor. 
J. Allergy Clin. Immunol. 63: 71-73, 1979. 

6. Agbata, A.I. and Kirkpatrick, C.H.: Release of E-rosette augmenting 
factor (E-RAF) following stimulation of human leukocytes with mitogens 
or antigens. J. Immunol . 122: 1080-1086, 1979. 

7. Kirkpatrick, C.H. and Windhorst, D.W. : Mucocutaneous candidiasis and 
thymoma. Am^ J. Med. 66:939-945, 1979. 

8. Petersen, E.A. and Kirkpatrick, C.H. : Nature and activities of transfer 
factor. Ann. N.Y. Acad. Sci. in press. 

9. Kirkpatrick, C.H. : Immune Deficiency Disorders, in Immunology II. 
(J. A. Bellanti, ed.), Saunders, Philadelphia, 1978, pp. 644-689. 



20-22 



Project No. Z01 AI 00046-11 LCI 

10. Kirkpatrick, C.H. and Greenberg, L.E.: Treatment of chronic mucocutaneous 
candidiasis with transfer factor, in Immune Regulators in Transfer Factor . 
(A. Khan, C.H. Kirkpatrick, and N.O. Hill, eds.), Academic Press, New York, 
1979, pp. 547-559. 

11. Kirkpatrick, C.H. and Sohnle, P.G. : Chronic Mucocutaneous Candidiasis, 

in Immunodermatology . (B. Safai and R.A. Good, eds.) Plenum Press, in press 

12. Kirkpatrick, C.H. : Transfer Factor, in CRC Critical Reviews in Clinical 
Laboratory Sciences , in press 

13. Cook, J.L., Lewis, A.M. and Kirkpatrick, C.H. : Age-related and thymus- 
dependent rejection of Adenovirus 2-transf ormed cell tumors in the 
Syrian hamster. Cancer Res. 39: in press, Sept. 1979 



20-23 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl AI 00047-10 LCI 



PERIOD COVERED 



October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (30 characters or less) 
Clinical Studies on Patients With Known or Suspected Parasitic Diseases. 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Pis: F.A.Neva Head, Clinical Parasitology Section, LCI, NIAID 

(dual position) 
I.E. Nash Senior Investigator, Clinical Parasitology Section, 

(dual position), LCI, NIAID 
E.A. Ottesen Senior Investigator, Clinical Parasitology Section, 

(dual position), LCI, NIAID 
D.J. Wyler Senior Investigator, Clinical Parasitology Section, 

(dual position), LCI, NIAID 
Others: R. D'A. Gusmao Visiting Fellow, LPD, NIAID 
T. Lawley Medical Investigator, NCI 

M.N. Lunde Research Zoologist, LPD, NIAID 
J.D. Berman Clinical Associate, LCI, NIAID 
D.M. Dwyer Research Microbiologist, LPD, NIAID 



cooperating units (if any) Federal University of Goias, Goiania, Brazil (Profs. J. 
Rezende and A. Rassi) ; Duke University, Division of Immunology (Dr. F. Ward); 
Federal University of Parana, Brazil (Dr. E. Ferreira) ; Tuberculosis Chemotherapy 
Center, Madras, India (Dr. S.P. Tripathy) ; Medical College of Madras, India 



LAB/BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Clinical Parasitology Section 



INSTITUTE AND LOCATION 

NIAID, Bethesda, Maryland 20205 



! TOTAL MANYEARS: 
5 3/12 



PROFESSIONAL: 

5 3/12 



I CHECK APPROPRIATE BOX(ES) 
'$%&) HUMAN SUBJECTS 

C (a1 ) MINORS H ( a 2) INTERVIEWS 



XX (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 *ords or less - underline keywords) 

The interesting association of absence of HLA tissue type- Al with chronic 
Chagas' disease was not confirmed by an expanded study of cases, but PR (B cell 



typing will still be done on frozen cells. The diversity of HLA haplotypes 
encountered in this Brazilian patient population corroborates what anthropo- 
logists have said of this population - namely, extreme genetic mixing. 

Further study of different antigenic fractions of schistosome worms has 
disclosed one fraction to which antibody levels in patients correlate quite 
well with numbers of eggs being excreted, or intensity of infection. 

Evidence for serum inhibitory factors and for suppressor cells has been 
found in f ilariasis patients characterized by unresponsiveness to filarial 
antigens. The role of immune complexes in f ilariasis patients is also being 
studied. Serum and cell factors were also found that contributed to antigen 
unresponsiveness in patients with chronic schistosomiasis . 



20-24 



3 HS-6040 
(Rev. 10- 



Project No. Z01 AI 00047-10 LCI 

Cooperating Units, cont'd: 

(Prof. K.V. Thiruvengadam) ; University of Kentucky Medical Center, 
Lexington, Ky. (Dr. J. Burke). 

Summary of work, cont'd: 

Evidence for multiplication of several species of leishmania within 
phagolysosomes was found using a system of macrophages derived from human 
monocytes . 

Project Description: 

This report covers the clinical investigations carried out by the 
four investigators in the Clinical Parasitology Section (Drs. Neva, Nash, 
Ottesen and Wyler) . Their experimental or non-clinical research is reported 
in the Annual Report from the Laboratory of Parasitic Diseases. Therefore, 
there will be some overlap in reporting and in publications listed. The 
Clinical Parasitology Section provides consultative service in clinical 
parasitic diseases to the Clinical Center and several members of the Section 
also participate with LCI Staff for clinical infectious diseases at the 
Clinical Center and at the Naval Medical Center. As in the past year, some 
of the studies were carried out in collaboration with investigators in 
India and in Brazil. 

Pathogenesis and Immunology of leishmaniasis. While there have been 
many in vitro studies of paras ite-macrophage interaction in leishmaniasis, 
human macrophages have not been used for such work. A system which would 
use human macrophages would obviously be a great advantage for research 
related to human disease with this parasite. Therefore, it was cf particular 
interest that a system involving macrophages derived from human monocytes 
could be developed in which leishmanial growth could be observed and demon- 
strated. The parasites were shown to be present and dividing within 
phagolysosomes, as happens in vivo . Intracellular multiplication in such 
cells was found with L. donovani and with L. tropica , the parasites 
associated with visceral and cutaneous leishmaniasis, respectively. Such 
an _in vitro system shows promise for studying effects of drugs as well as 
immunological events associated with leishmanial infections. (Wyler, Berman 
and Dwyer) . 

Possible influence of immune response on Chagas' disease. Collaborative 
studies with Brazilian colleagues were initiated recently on Chagas' disease 
as reported previously. In a preliminary investigation which involved 
mainly the study of blastogenic response to T^ cruzi antigens in patients 
with various forms of chronic Chagas' disease, tissue typing at the A, B, 
and C loci was also carried out. This preliminary study suggested that both 
positive and negative correlations with certain HLA specificities occurred 
in patients with the chronic disease. The most striking association was 
that none of 18 megaesophagus patients were A-l, while this type occurred 
in 23 percent of 44 seronegative controls and in 28 percent of individuals 
with infection but no disease, all from the same area of Brazil. Therefore, 



20-25 



Z01 AI 00047-10 LCI 

a larger study was carried out in the past year to include more cases and 
family members, and to freeze lymphocytes for B-cell and possible Dw typing 
as well. We have now studied larger groups of 40 to 50 in various clinical 
categories and the previous suspected HLA association is no longer present 
in typing at A, B and C loci. Therefore, we can only conclude that our 
sampling groups were not large enough for statistically significant results. 
The B-cell typing has not yet been done at the time this report was pre- 
pared. The geneticists and our collaborators interested in HLA are 
fascinated by the genetic diversity of the study population and believe 
they have found some new HLA specificities. But we are only sadder and 
wiser with the intricacies of relating HLA tissue types to parasitic 
diseases. (Neva, Gusmao, Ward and Brazilian collaborators). 

Clinical significance of schistosome antigens. While antigenic 
complexity of parasites makes immunologic studies more difficult, it also 
provides more opportunities to dissect and examine the immunologic signi- 
ficance of various antigenic components. Last year we reported that the 
immune response in patients to the polysaccharide gut antigen showed a 
characteristic time course, being highest in recent infections and falling 
rapidly with time. This antibody response, however, did not reflect or 
correlate with intensity of infection. Recent work which has focused upon 
individual fractions of the TCA-soluble material from whole worms has 
disclosed a relationship between response to one of these antigenic compon- 
ents and numbers of eggs excreted. This antigen fraction, a glycoprotein, 
is but one of several fractions that make up a crude antigen reported by 
others as correlating with intensity of infection. But the antibody 
response to this specific glycoprotein fraction did not change with time 
after infection. Thus, antibody response to various but specific antigens 
of parasites, such as schistosomes, may assist in clinical evaluation of 
patients with the infection. With the antigens just cited, antibody levels 
in a patient can indicate whether the infection is recent or more than a 
year in duration and some indication of how heavy the infection is. (Nash 
and Lunde) . 

Immune response to helminth infections. Recently completed studies 
of histamine release from cells of patients with tropical pulmonary 
eosinophilia, a form of filarial infection without circulating microfilariae, 
highlighted the specific nature of the IgE-mediated allergic reaction pro- 
voked by microf ilarial antigens upon contact with sensitized cells. But an 
equally if not more intriguing question is raised by this observation - 
namely, why is not histamine release going on all the time in vivo in those 
patients whose filariasis is associated with circulating microfilariae? 
There are indications that serum inhibitory factors are involved in this 
allergic hyporesponsiveness . Many of the patients with chronic filariasis 
and circulating microfilariae also exhibit both serum and adherent sub- 
populations of mononuclear blood cells that contribute to cell-mediated 
unresponsiveness to filarial antigens. (Ottesen) . 

The role of circulating immune complexes is also under study in patients 
with different clinical manifestations of filariasis. The Clq binding immune 
complexes that were demonstrated in patients with acute schistosomiasis 



20-26 



Z01 AI 00047-10 LCI 

are being studied further to better characterize antigenic make-up of these 
complexes. (Ottesen and Lawley) . 

Tests for specific antibody in strongyloidiasis . A long experience 
with persistent strongyloides infection in an immuno-def icient patient made 
it possible to obtain larval antigens from this parasite. Preliminary 
results suggested that such antigens might be useful for serologic tests 
in patients. Therefore, S_;_ stercoralis as well as other nematode larval 
antigens are being investigated for possible usefulness in the diagnosis 
of human strongyloides infection. (Neva and Burke) . 

Publications : 

1. Ottesen, E.A., Hiatt, R.A. , Cheever, A.W. , Sotomayor, and Neva, F.A.: 
The Acquisition and Loss of Antigen-Specific Cellular Immune Responsiveness 
in Acute and Chronic Schistosomiasis in Man. Clin. Exp. Immunol. 33 : 
38-47, 1978. 

2. Ottesen, E.A. and Weller, P.F.: Eosinophilia Following Treatment of 
Patients with Schistosomiasis mansoni and Bancroft's filariasis. J. Inf. 
Pis. 139: 343-347, 1979. 

3. Ottesen, E.A. , Neva, F.A. , Paranjape, R.S., Tripathy, S.P., 
Thiruvengadam, K.V. and Beaven, M.A. : Specific Allergic Sensitization to 
Filarial Antigens in Tropical Eosinophilia Syndrome. Lancet I: 1158-1161, 
1979. 

4. Nash, T.E., Ottesen, E.A. , and Cheever, A.W. : Antibody Response to a 
Polysaccharide Antigen Present in the Schistosome Gut. II. Modulation of 
Antibody Response. Am. J. Trop. Med. Hyg. 27: 944-950, 1978. 

5. Neva, F.A. and Ottesen, E.A. : Tropical (Filarial) Eosinophilia. New 
Eng. J. Med. 298 : 1129-1131, 1979. 

6. MacQueen, J.M., Ottesen, E.A. , Ottesen, C, Amos, D.B., and Ward, F.E.: 
HLA Histocompatibility Antigens in a Polynesian Population - Cook Islanders 
of Mauke. Tissue Antigens 13: 121-128, 1979. 

7. Lunde, M.N. , Ottesen, E.A. , and Cheever, A.W. : Serological Differences 
Between Acute and Chronic Schistosomiasis mansoni Detected by Enzyme Linked 
Immunosorbant Assay (ELISA) . Am. J. Trop. Med. Hyg. 28: 87-91, 1979. 

8. Collidge, E., Weller, P.F., Ramsey, P.G., Ottesen, E.A. , Beaver, P.C. 
and von Lichtenberg, F.C.: Zoonotic Brugia Filariasis in New England. 
Ann. Int. Med. 90: 341-343, 1979. 

9. Ottesen, E.A. : Modulation of the Host Response in Human Schistosomiasis 
II. Adherent Suppressor Cells which Inhibit Lymphocyte Proliferative 
Responses to Parasite Antigens. J. Immunol. (In press). 



20-27 



Z01 AI 00047-10 LCI 
Publications, cont'd: 

10. Ottesen, E.A. : Filarial Infection and the Host Response in Man. 
Paradoxes and Insights. In Escape from Immune Surveillance: The Interface 
Between Immune Mechanisms and Disease. D.B. Amos, R.S. Schwartz and B.W. 
Janicki, eds. Academic Press (In press). 

11. Ottesen, E.A. : Visceral Larva Migrans and Other Migratory Helminths 
of Man. In Principles and Practice of Infectious Disease, Mandell, G.I., 
Douglas, R.G. , and Bennett, J.E., eds. J. Wiley and Sons, New York 

(In press) . 

12. Lawley, T. J. , Ottesen, E.A. , Hiatt, R.A. and Gazze, L.A. : Circulating 
Immune Complexes in Acute Schistosomiasis. Clin. Exp. Immunol . (In press). 

13. Lunde, M.N and Ottesen, E.A.: Enzyme-linked immunosorbent assay (ELISA) 
for detecting IgM and IgE antibodies in human schistosomiasis. Am. J. Trop . 
Med. Hyg. (In press) . 

14. Cohen, S.G. and Ottesen, E.A.: "Eosinophils in immune function" in 
Cell Biology of Immunity and Inflammation, Oppenheim, J. , Rosenstreich, D. 
and Potter, M. , eds. Harvard Elsevier-North Holland, Inc. (In press). 

15. Nash, T.E.: Antibody response to a polysaccharide antigen in schisto- 
somialsi. I. Sensitivity and specificity. Am. J. Trop. Med. Hyg. 27 : 
939, 1978. 

16. Nash, T.E., Ottesen, E.A. , Cheever, A.W. : Antibody response to a 
polysaccharide antigen present in schistosome gut. II. Modulation of 
antibody response. Am. J. Trop. Med. Hyg. 27: 944-950, 1978. 

17. Neva, F.A. , Wyler, D.J. and Nash, T.E.: Cutaneous leishmaniasis - A 
case with persistent organism after treatment in presence of normal immune 
response. Am. J. Trop. Med. Hyg. 28: 467-471, 1979. 

18. Wyler, D.J.: Leishmaniasis. In Current Therapy (Conn, H., ed.) 
W.B. Saunders Co., Philadelphia, Pa. 

19. Wyler, D.J. and Miller, L.H: Plasmodium species (Malarial) (Chapter 
227). In Principles and Practices of Infectious Diseases (Mandell, G.L., 
Douglas, R.G., and Bennett, J.E., eds.) (In press). 

20. Wyler, D. J. : Cellular aspects of immune regulation in malaria. 
Bull. WHO . (In press). 

21. Wyler, D.J., Herrod, H. and Weinbaum, F.I.: Response of sensitized 
and unsentized human lymphocyte subpopulations to Plasmodium falciparum 
antigens. Infect. & Immunol. 24: 106, 1979. 



20-28 



Z01 AI 00047-10 LCI 

22. Wyler, D.J., Oppenheim, J.J. and Koontz, L.C.: The influence of 
malaria infection on the elaboration in vitro of soluble mediators by 
adherent mononuclear cells. Infect. & Immunol. 24 : 151, 1979. 

23. Wyler, D.J., Weinbaum, F.E. and Herrod, H. : Characterization of the 
in vitro proliferative responses of human lymphocytes to leishmanial 
antigens. J. Infect. Pis. 1979 (In press) 



20-28a 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00048-09 LCI 



PERIOD COVERED 

September 30, 1978 to October 1, 1979 



TITLE OF PROJECT (80 characters or less) 

The Pathophysiology of Autoimmune Hemolytic Anemia 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



M. Frank 



M. Hamburger 



Head, Clinical Immunology 
Section and Chief, LCI 

Clinical Associate 



LCI 
LCI 



NIAID 
NIAID 



cooperating units (if any) T> Lawley (Dermatology Branch, NCI/NIH) ; P. Plotz (Arthritis 
Branch, NIAMDD/NIH) ; H. Moutsopoulos, (NIDR) ; T. Chused, (NIAID); E. Franklin, 
(NYU School of Medicine); G. Sharp, (Univ. of Missouri School of Medicine). 



lab/branch 
T.^hnrat-nry of Clinical Investigation 



SECTION 

Clinical Immunology Section 



INSTITUTE AND LOCATION 

NIAID, NIH Bethesda, MD 



20205 



TOTAL MANYEARS: 

2 



PROFESSIONAL: 
2 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 
X 

D (al) MINORS [] (a2) INTERVIEWS 



(b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Me have continued to develop and extend the usefulness of our new technique for 
measuring IgG Fc receptors and complement receptors in various groups of patients 
with autoimmune disease . These techniques have proven useful both in diagnostic 
tests and for developing a further understanding of pathophysiologic processes. 
In the past year we have extended our studies to patients with mixed IgM, IgG 
cryoglobulins and have shown that these patients can be divided into two groups. 
The patients with Fc receptor defects are the ones who develop immune complex 
mediated renal disease. The patients with normal Fc receptor function do not 
appear to develop immune complex mediated renal disease. The follow-up studies 
have been performed in our patients with lupus erythematosus and it has been 
shown that as the patients improve the immune clearance defect also improves. 
Patients with mixed connective tissue disease have been studied and have been 
shown in general to be free of defects in immune clearance not withstanding the 
fact that they have circulating immune complexes. This is in keeping with 
previous observations that these patients do not develop immune complex mediated 
tissue injury. 20- Q 

PHS-6040 
(Rev. IO-76) 



Project No. Z01 AI 00048-09 LCI 

Project Description : 

Obj ectives : 

To define the pathophysiologic function of antibody and complement on 
the surface of red cells in patients with hemolytic anemia, and to use the 
findings which have been developed from this study to develop new tests for 
immunologically specific receptors function in patients with a variety of 
disease states. The tests developed for the study of autoimmune hemolytic 
anemia have been found to have wide application to other autoimmune and 
immunologic problems. 

Methods Employed : 

These studies examine the clearance of radiochromated erythrocytes 
from the circulation. The initial studies were performed in a guinea pig 
model, but all of the recent studies have been performed in human volunteers 
or in patients with various diseases. In the initial studies in humans, 
the clearance of erythrocytes coated with IgM isoagglutinin antibodies were 
examined. In later studies radiochromated erythrocytes were coated with 
highly purified human IgG anti-Rh antibodies and the survival of these cells 
was also studied. The third general set of clearance studies examined the 
clearance of radioactive albumin which had been aggregated. This is the 
standard method for determining RES clearance and can be used to determine 
both liver blood flow and the ability of the reticuloendothelial system to 
clear particulate materials. 

Major Findings : 

In past years, we identified the immunologically active protein frag- 
ments which, when deposited on red cells, are responsible for the clearance 
of those cells from the circulation. It was reported that receptors for these 
immunologically specific fragments were present on the membrane of macrophages 
which were responsible for removal of cells coated with these fragments from the 
circulation. It became clear that it was possible to study patients with a 
wide variety of diseases to determine whether their receptors for these immuno- 
logically active fragments were functioning normally in vivo . It has been 
known for years that the reticuloendothelial system (RES) plays a major role 
in removing foreign material from the circulation. These foreign materials 
include bacteria and parasites, as well as endogenously generated immuno- 
reactive materials such as immune complexes. It was suspected that defects 
in RES function might be important in both infectious and immunologic diseases. 
However, when this question was examined directly using the techniques available, 
no defects in RES function were noted in any disease state in which blood flow 
to the RES organs was normal. Until the present studies, the technique 
available for examination of RES function was that of study of clearance of 
aggregated albumin from the circulation. This study has led to the development 
of new techniques for evaluation of RES function. Using these new techniques 
developed within our unit we have shown that there are indeed diseases with 
defects in RES function if one examines this question looking at specific re- 

20-30 



Project No. Z01 AI 00048-09 LCI 

ceptors for immunologically active material. Thus receptors for comple- 
ment and immunoglobulin on a patient's macrophages will recognize these 
immunologically active materials coating foreign substances as they circu- 
late. 

The defects are quite specific for different sets of receptors. Our 
test utilizes the patient's own red blood cells. The cells are coated with 
antibody and/or complement fragments and are infused into the patient and 
their clearance is followed. Using this technique we have found that almost 
every patient with active lupus erythematosus has a major defect in Fc re- 
ceptor specific clearance although the clearance of aggregated materials is 
normal. We believe that it is quite likely that this clearance defect ex- 
tends to the clearance of the circulating immune complexes which occur in these 
patients. If such complexes are not cleared efficiently from the circulation, 
they can be deposited in glomeruli where they can cause immune damage. We 
find that as the disease improves, the clearance defect also tends to improve 
and the comlexes present in the circulation tend to disappear. However it is 
striking that most patients with lupus erythematosus have a persistence of 
their clearance defect for long periods of time, even on treatment. Our stud- 
ies in lupus erythematosus have already been confirmed by a group in London. 
These initial findings in lupus were published in the past year in the 
New England Journal of Medicine. 

Extensive studies have been conducted of Sjogren's Syndrome. These stud- 
ies demonstrated that patients with clearance defects in the presence of cir- 
culating immune complexes tend to develop peripheral manifestations of their 
Sjogren's Syndrome including autoimmune disease and extensive tissue injury. 
On the other hand patients who do not have clearance defects tend not to de- 
velop secondary manifestations of their Sjogren's Syndrome with peripheral 
tissue destruction even in the presence of circulating immune complexes. This 
material is in press in the Annals of Internal Medicine. We have now had an 
opportunity to analyze the circulating immune complexes in patients with 
Sjogren's Syndrome and we have shown that a very high proportion of patients 
have immune complexes and that these complexes are of multiple types. Moreover, 
the rheumatoid factor present in these patients is only one type of complex 
and is not responsible for many of the positive tests for immune complexes 
shown in the studies of the serum of these patients. 

In the past year with Dr. Edward Franklin and his group at NYU we 
have had an opportunity to study patients with IgM-IgG mixed cryoglobulinemia. 
These patients have been shown to fall into two groups with regard to 
the development of peripheral manifestation of disease in spite of the 
fact that all have circulating immune complexes. One group of patients 
has been shown to possess normal Fc receptor function and these patients 
do not develop certain severe peripheral manifestations of disease such 
as immune complex mediated glomerulonephritis. A second group of patients 
have been shown to have a clearance defect and it is these patients that 
have evidence of glomerulonephritis. Once again the usefulness of the 
clearance studies in determining the nature of the pathophysiologic process 
in this disease is clear. 

20-31 



Project No. Z01 AI 00048-09 LCI 

We have also studied a large group of patients with mixed connective 
tissue disease with Drs. Gordon Sharp and Harry Moutsopoulos. These 
patients tend not to have tissue deposition of immune complexes and exten- 
sive tissue damage although they have circulating immune complexes in 
quantity. Interestingly, our studies show little or no evidence of a 
clearance defect in these patients except for those who have many of 
the manifestations of systemic lupus erythematosus. The latter patients 
tend to resemble patients with lupus erythematosus in that they have anti-DNA 
etc. and interestingly these patients do have clearance defects. Finally, 
we have studied a group of patients with rheumatoid arthritis. These patients 
do not have striking Fc receptor clearance defects suggesting that much of 
the immunopathology that occurs is not systemic immune complex mediated 
injury but originates in the joints themselves. 

Significance to Biomedical Research 

These techniques allow for the complete reevaluation of reticuloendo- 
thelial system function in man. Already they are being widely applied. The 
use of these techniques has already allowed for the description of the first 
patients with receptor specific defects. C3b specific defects have been 
demonstrated in primary biliary cirrhosis and Fc receptor defects in a 
number of serious autoimmune diseases. These techniques provide a powerful 
new tool in the evaluation of these patient groups and provide a new approach 
to understanding the pathophysiologic basis of these diseases. 



Proposed Course : 

We intend to extend these studies to additional autoimmune diseases. 
Moreover, we intend to attempt to determine whether patients with various 
kinds of infectious disease may also have clearance defects which contribute 
to their inability to clear bacteria from the circulation. At the present 
time we are examining the possibility of studying patients with sickle cell 
anemia to determine whether these patients have a clearance defect that might 
help account for their propensity to develop overwhelming sepsis. 

Publications : 

1. Jones, E.A. , Frank, M.M. , Jaffe, C.J., and Vierling, J.M. : Primary 

Biliary Cirrhosis and the Complement System. Ann. Int. Med . : 
In press. 

2. Jaffe, C.J., Vierling, J.M. , Jones, E.A. , Lawley, T. and Frank, M.M. : 
Receptor specific clearance by the reticuloendothelial system in 
chronic liver diseases: Demonstration of defective C3b specific 
clearance in primary biliary cirrhosis. J. Clin. Invest . November 
1978: 62:1069-1077, 1978. 

3. Lawley, T. , Moutsopoulos, ELM. , Katz, S.I., Theof ilopoulos , A.N., 
Chused, T.M. , and Frank, M.M. : Circulating Immune Complexes in 
Sjogren's Syndrome. J . of Immun . In press. 

20-32 



Project No. Z01 AI 00048-09 LCI 



4. Frank, M.M. , Hamburger, M.I., Lawley, T.J., Kimberly, R.P., and 
Plotz, P.H.: Defective Reticuloendothelial System Fc Receptor 
Function in Systemic Lupus Erythematosus. N. Engl. J. Med . 
300: 518-523, March, 1979. 

5. Moutsopoulos, H.M. , Chused, T.M. , Mann, D.L. , Klippel, J.H., 
Fauci, A.S., Frank, M.M. , Lawley, T.J. and Hamburger, M.I.: 
Sjogren's Syndrome (Sicca Syndrome): Current Issues . An edited 
transcript of a Combined Clinical Staff Conference of the Clinical 
Center, Bethesda, Maryland, April 19, 1979. 

6. Hamburger, M.I., Moutsopoulos, H.M. , Lawley, T. and Frank, M.M. : 
A defect in Reticuloendothelial System Fc Receptor Function in 
Sjogren's Syndrome. Ann. Int. Med. In press. 

7. Hamburger, M.I., Gorevic, P., Franklin, E.C. and Frank, M.M. : The 
Function of the Reticuloendothelial System in Autoimmune Disease. 
Transaction of the Association of the American Physicians. 1979. 
In press. 

8. Frank, M.M. : The Function of the Reticuloendothelial System in 
Autoimmune Diseases. In. Immune Mechanisms in Renal Disease . 
Cummings, N. (Ed.) In press. 

9. Frank, M.M. : Reticuloendothelial Complement-Specific Clearance of 
Circulating Particles, p. 77-8 In Jones, E.A. (Moderator). Primary 
Biliary Cirrhosis and the Complement System. Ann. Intern. Med. 

90: 72-84, 1979. ' 

10. Frank, M.M. : The Role of Complement in the in vivo Destruction of 
Erythrocytes. In. Proceedings of the American Red Cross Eleventh 
Annual Scientific Symposium . In press. 

11. Frank, M.M. : The Role of the Reticuloendothelial system in the 
Clearance of Circulating Cells Coated with Antibody and Complement. 
In: Proceedings of the WHO Meeting on the Role of the Spleen in 
the Immunology of Parasitic Diseases . In press . 

12. Frank, M.M. , Atkinson, J. and Jaffe, C: The Role of Antibody and 
Complement in Immune Clearance of Erythrocytes in Man. In Clinical 
Aspects of the Complement System: International Symposium . Opferkuch, W. 
Rother, K. and Schultz, D.R. (Eds) Georg Thieme Publ., 1978, pp. 70-76. 



20-33 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00049-09 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Initiation and Regulation of Antigen Recognition 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



M.M. Frank 

J.T. Blake 
K.U. Cehrs 
K. Yokomuro 
J. Schroer 
J . Thomas 
R. Clark 



Acting Head, Biologic Structure 

Section 
Bio. Lab. Tech. (Micro.) 
Bio. Lab. Tech. (Micro.) 
Guest Worker 
Guest Worker 
Clinical Associate 
Clinical Associate 



LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 



COOPERATING UNITS (if any) 

None 



lab/branch 

Laboratory of Clinical Investigation 



SECTION 

Biologic Structure Section 



INSTITUTE AND LOCATION 

NIAID/NIH Bethesda, Maryland 20205 



TOTAL MANYEARS: 

5 11/12 



PROFESSIONAL: 

4 5/12 



1 6/12 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



(c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The interest of the Biologic Structure Section is to characterize the 
role of the macrophage in antigen recognition by T lymphocytes in the expres- 
sion and initiation of cell-mediated immunity in man and experimental animals. 
The Section has been interested in exploring the role of the major histocom- 
patibility complex in the genetic regulation of cell-cell interactions 



required for expression of cell mediated and humoral immunity. The Biologic 
Structure Section is also actively characterizing the physical state, 
localization, and fate of macrophage associated antigen. Additional studies 
of genetics of immune responsiveness to insulin done in man are assessed and 
compared to mouse and guinea pig. Determinant selection by macrophages 
functionally describes Ir gene control of antigen recognition by T lymphocytes 
in experimental animals. An understanding of the operation of a similar 
mechanism in man may have implications in the pathophysiology of insulin 
allergy in diabetics. 



20-34 



PHS-6040 

(Rev. IO-76) 



Project No. Z01 AI 00049-09 LCI 
Project Description : 
Objectives : 

1) To characterize in a quantitative and qualitative fashion the role 
of macrophages and lymphocytes in the development of cellular immunity. 

2) To study in experimental animals the mechanism of lymphocyte 
antigen recognition using defined antigens such as insulin. 

3) To study the cellular and molecular basis of the genetic control of 
immune responsiveness and the role of surface membrane determinants in cell 
cooperation. 

Methods Employed : 

Standard assays have been developed for: 

1) Determination of DNA synthesis of mouse and guinea pig lymphocyte 
populations . 

2) Preparation and isolation of partially purified populations of 
lymphocytes . 

3) Column purification of cells and proteins. 

4) Binding of lymphocytes to macrophage receptors. 

5) The uptake and fate in culture of soluble protein antigens. 

Major Findings : 

Dr. Alan Rosenthal left the NIH last year to assume a position as 
Director of Immunology of Merck, Sharp and Dohme. This year's efforts 
have been directed at completing projects of his fellows who were committed 
to an additional years tenure. 

Members of this Laboratory have begun to characterize seven anti- 
insulin antibody secreting cell lines prepared by cell fusion techniques in 
collaboration with Dr. K. Jin Kin. Several interesting results were 
obtained from these studies. An autoreactive antibody which binds to a 
mouse's own insulin was one of the seven isolated. Furthermore, the 
precise amino acid sequence of the binding site on the insulin molecule 
(region of A chain residues 8-10) of another anti-insulin fusion product 
was identified. This last antibody can distinguish beef insulin from pork 
insulin, rat insulin or other insulins which differ in amino acid sequence 
in this region of the insulin molecule. In collaboration with Dr. James W. 
Thomas, the kinetics, specificity and Ir gene control of the plaque forming 
cell response to insulin and TNP-insulin in the mouse were also completed 
and submitted for publication. 



20-35 



SMITHSONIAN SCIENCE INFORMATION 
PROJECT NUMBER (Do NOT use this 



EXCHANGE U.S. DEPARTMENT OF 

ioace) IHEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl AI 00050-09 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Clinical Studies of Complement Abnormalities of Man 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



M. Frank 



S. Hosea 
K. Katusha 
M. Santaella 



Head, Clinical Immunology 
Section and Chief LCI 

Clinical Associate 

Microbiologist 

Guest Researcher on IPA 



LCI 



NIAID 



LCI NIAID 
LCI NIAID 
LCI NIAID 



COOPERATING UNITS (if any) 

D.W. Ailing, OSD, NIAID, NIH) : D. Triantaphylapolis and M. Wickerhauser 
American Red Cross, Bethesda, MD 



lab/branch 

Laboratory of Clinical Investigation 



SECTION 

Clinical Immunology 



INSTITUTE AND LOCATION 

NIH/NIAID, Bethesda, MD 20205 



TOTAL MANYEARS: 

3 8/12 



PROFESSIONAL: 

3 2/12 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Hereditary Angioedema is an autosomal dominant disease characterized 
by abnormalities in CI esterase inhibitor . We have previously shown that 
danazol corrected the protein abnormality in patients with this disease. 
During this year we have further extended observations of the usefulness 
of danazol and danazol toxicity in a large patient population. We have 
begun to characterize the incidence of autoimmune diseases in this patient 
group as well. Further, we have extended our experience with the use of 
purified CI esterase inhibitor in the treatment of Hereditary Angioedema 
attacks and have shown that the therapy can abort these potentially life 
threatening attacks. 



20-36 



PHS-6040 
(Rev. IO-76) 



Project No. Z01 00050-09 LCI 

Project Description: 

Objectives : 

The general objective of this program has been to define the role of 
complement in human illness and to develop better methods of therapy for 
immune damage mediated by complement. As in the past year, major emphasis 
was placed on our studies with patients with hereditary angioedema. This 
illness, which carries a high mortality rate, is particularly useful for 
study since it can serve as a model for complement-mediated immune damage 
in man and as model for the study of genetically controlled autosomal domi- 
nant diseases. We were particularly interested this year in defining the 
usefulness and toxicity of danazol in a large patient population. We were 
also interested in extending our efforts to stop attacks of hereditary angio- 
edema after they have already begun. Danazol does not work, in this situation. 

Methods Employed : 

Purified complement components are prepared, and antisera to various 
complement components and component inhibitors are used for estimation of 
component levels in human sera and other body fluids. Double-blind thera- 
peutic studies are employed to test new methods of therapy. Patients on 
experimental drug protocols are followed to determine signs of toxicity. 

Major Findings : 

In the past years, we have developed and evaluated a new drug danazol 
for treatment of hereditary angioedema. Earlier we showed that this drug 
is markedly effective in treating this disease and is relatively non-toxic. 
The drug has the striking effect of causing patients with the deficient serum 
inhibitor protein to synthesize this protein so that levels of the protein in 
blood return to normal. With the restoration of normal levels of the protein 
in blood, the patient corrects his biochemical abnormalities and thereafter 
is free of symptoms. The long-term use of this new drug has never been 
studied in any patient population and it was essential to begin to define 
those situations in which the drug was useful or on the other hand was likely 
to cause problems. We also were interested in attempting to stop attacks of 
hereditary angioedema once they had begun. It takes days for danazol to have 
its effect. Patients who are not on danazol however, will often present to an 
emergency ward having severe angioedema which may be life- threatening. At 
the present time there is no safe method of treating such patients. The use 
of fresh, frozen plasma has been advocated, since fresh plasma has the protein 
which the patients are missing; however, fresh plasma also has complement 
components in it and the patients are depleted of these components at the 
time of an attack. It has been suggested that the infusion of complement 
components will make these patients much more ill before they improve. Thus, 
fresh plasma is not advocated for use in this disease. 



10-37 



Project No. Z01 00050-09 LCI 

Twenty-seven new patients were seen with Hereditary Angioedema in 1978 
and 8 additional new patients were seen in 1979. We now have 63 patients on 
danazol therapy, 7 on oxymethylone, 4 on EACA, 9 on methyltestosterone. We 
have developed considerable further experience with danazol toxicity and are 
now the leading group in developing methods to evaluate danazol toxicity. We 
are also now the largest group following patients with HAE on therapy and have 
begun to pull together our studies of the incidence of autoimmune disease in 
this interesting disease. We now have several patients with autoimmune thy- 
roiditis, glomerulonephritis, regional enteritis, ulcerative colitis, pan- 
creatitis, etc. We are initiating studies to assess how the complement de- 
ficiency state predisposes to autoimmune disease. 

Our infusion studies are also progressing in a highly satisfactory way, 
however, we are still having difficulty obtaining large quantities of the CI 
esterase inhibitor from the Red Cross. We have assisted the Red Cross in prep- 
aration and in evaluation of CI esterase inhibition. Moreover, we have now 
completed a series of infusions of purified CI esterase inhibitor into patients 
with HAE at times when they were not experiencing attacks and also have infused 
the protein into patients in the midst of attacks. These studies have shown 
that CI esterase inhibitor is quite benign and does not carry the risk of hep- 
atitis. When infused into patients free of clinical symptoms these patients 
experience an elevation of CI esterase inhibitor and a subsequent elevation 
of C4 levels as might be expected from pathophysiologic mechanism of this 
disease process. When infused into patients experiencing attacks of HAE, 
attacks, have been aborted in as short a period as 5 minutes and as long 
a period as several hours. It would appear that this will afford an 
excellent approach in emergency rooms for treating patients with life- 
threatening attacks of Hereditary Angioedema. 

Significance to Medical Research 

Our laboratory has been the focus for most of the recent therapeutic de- 
velopments in current treatment of this disease. Patients with hereditary 
angioedema experience a 25% mortality and have extensive morbidity from their 
constant attacks of the disease. As a result of our therapeutic interventions 
these patients lead a normal life. Moreover our development of drugs and 
our study of their mechanism of action has led to a far greater understanding 
of the genetic basis of the protein abnormality underlying the disease. At 
present, this disease represents one of the few genetically controlled dis- 
orders in which there has been biochemical correction and thereby cure of a 
severe illness. 

Proposed Course : 

We are continuing to evaluate the long term use of drugs in patients with 
this particular disease. We are also gradually increasing the number of other 
patients with other complement-related abnormalities which are under study. 
These include such diverse illnesses as autoimmune hemolytic anemia and 
systemic lupus erythematosus. 



20-38 



Project No. Z01 AI 00050-09 LCI 



Publications: 



1. Gelfand, J. A., Rosa, G.R., Conley, C.L., Allen, J.C., Reinhart , R. , 
Humphrey, R.L. and Frank, M.M. : Acquired CI Esterase Inhibitor Deficiency 
and Angioedema. Medicine 58: 321-28, 1979. 

2. Levinson, A.I., Summers, R.J., Lawley, T.J., Evans, R. , III, and 
Frank, M.M. : Evaluation of the Adverse Effects of Long-Term Hypo- 
sensitization. J_. Allergy Clin . Immunol . In press. 

3. Atkinson, J. P. and Frank, M.M. : The Complement System. In Parker, C. 
(Ed.): Clinical Immunol . Philadelphia, PA, W.B. Saunders Co. In press. 

4. Parillo, J.E., Lawley, T.J., Frank, M.M. , Kaplan, A. P. and Fauci, A.S.: 
Immunologic Reactivity in the Hypereosinophilic Syndrome. J_. Allergy 
Clin . Immunol . In press. 

5. Frank, M.M. : The Effect of Sex Hormones on a Complement Related 
Clinical Disorder, Hereditary Angioedema. Proceedings of the Kroc 
Foundation Meeting on Sex Factors, Steroid Hormones and the Host 
Response. Amacher, P. and Talal, N. (Eds.). In press. 

6. Gadek, J.E., Hosea, S.W., Gelfand, J. A. and Frank, M.M. : Response 
of Variant Hereditary Angioedema Phenotypes to Danazol Therapy: 
Genetic Implications. J. Clin. Invest . 64: 280-286, July, 1979. 

7. Frank, M.M. and Hosea, S.W.: Complement. In Cohen, A.S. (Ed.): 
The Science and Practice of Clinical Medicine . New York: Grune 
and Stratton, Inc. 1979, p. 393. 

8. Hosea, S.W. and Frank, M.M. : Differential Diagnosis of Hypocomple- 
mentemia. In Cohen, A.S. (Ed.): The Science and Practice of 
Clinical Medicine . New York: Grune and Stratton, Inc. 1979, p. 432. 

9. Lawley, T.J. and Frank, M.M. : Immune Complexes and Immune Complex 
Mediated Diseases. In Parker, C. (Ed.): Clinical Immunology . 

In press. 

10. Frank, M.M. , Gelfand, J. A., Sherins, R.J., Ailing, D.W., and 
Gadek, J.: The treatment of hereditary angioedema with danazol. 
In Clinical Aspects of the Complement System: International 
Symposium . Opferkuch, W. , Rother, K. and Schultz, D.R. (Eds.) 
Georg Thieme Publ. , 1978, pp. 134-138. 



20-39 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00051-07 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less 

Host Defense Mechanism Against Pseudomonas Infection in Normal and 
Immunosuppressed Hosts 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



R.P. Aduan 



E . Harvey 



Medical Officer 



Bio. Lab Tech. (Micro.) 



LCI NIAID 



LCI NIAID 



COOPERATING UNITS (if any) 

A.S. Levin, A.B. Deisserroth, and F.R. Applebaum (Pediatric Oncology Branch, 
NCI, NIH) 



LAB/BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Bacterial Disease Section 



INSTITUTE AND LOCATION 

NIAID, Bethesda, MD 20205 



TOTAL MANYEARS: 

2 3/12 



PROFESSIONAL: 

1 3/12 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



3 (c) NEITHER 



SUMMARY OF a/0RK (200 words or less - underline keywords) 

INACTIVE DURING CURRENT YEAR. TERMINATED. 



20-40 



PHS-6040 
(Rev. 10-76) 



SMITHSONIAN SCIENCE INFORMATION EXCHANG 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 000.54-07 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

The Etiology and Pathogenesis of Viral Gastrointestinal and Respiratory 
Tract Infections in Man 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 

OTHER: 



R. Dolin Head, Medical Virology Section LCI NIAID 

(until January 1, 1979) 

R. Berg Clinical Associate LCI NIAID 

R. Schooley Clinical Associate LCI NIAID 



COOPERATING UNITS (if any) 

A.Z. Kapikian (LID/NIAID/NIH) ; R.G.Wyatt (LID/NIAID/NIH) ; B.R. Murphy (LID/ 
NIAID/NIH) . 



lab/branch 

Laboratory of Clinical Investigation 



SECTION 

Medical Virology Section 



INSTITUTE AND LOCATION 

NIAID/NIH Bethesda, MD 



TOTAL MANYEARS 

6/12 



PROFESSIONAL: 

6/12 



CHECK APPROPRIATE BOX(ES) 
£ (a) HUMAN SUBJECTS 

D (aQ MINORS [] (a2) INTERVIEWS 



3 (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

TERMINATE THIS PROJECT 

Studies of the etiology and pathogenesis of viral gastrointestinal tract 
infections in man continued until the departure of the Principal Investigator. 



20-41 



PHS-6040 
(Rev. IO-76) 



Project No. Z01 AI 00054-07 LCI 
Project Description: 
Objectives : 

1) To investigate the pathogenesis of and host response to viral 
infections of the respiratory and gastrointestinal tracts in man. Experi- 
mentally induced disease in normal volunteers, as well as naturally occurring 
cases are studied. 

2) To define the biologic, immunologic, and epidemiologic properties 
of the etiologic agents of viral gastroenteritis in man. 

Methods Employed : 

Viral agents were obtained from naturally occurring outbreaks of 
gastroenteritis and respiratory infection. The pathogenesis and host 
responses to these agents were evaluated in in vitro systems and in 
volunteer studies. 

Major Findings: 

Characterization of the small 27-32nm gastroenteritis agents con- 
tinued in volunteer studies. Investigations of the cell-mediated immune 
responses to influenza vaccines were completed. 

Significance to Biomedical Research and the Program of the Institute : 

Viral respiratory and gastrointestinal tract infections are the most 
common disease experiences in American families and affect all age groups 
of a broad segment of population. As yet, there are no adequate control 
measures available. Identification and characterization of etiologic 
agents, as well as studies of the pathogenesis of these diseases in man, 
should form the basis for rational development of methods of prevention 
and treatment. 

Proposed Course : 

This study has been terminated upon the departure of the Principal 
Investigator. 



20-42 



Project No. Z01 AI 00054-07 LCI 
Publications : 

1. Dolin, R. : Antiviral compounds in viral infection of the gastrointesti- 
nal tract. In Buchanan, R. , Galasso, G. and Merigan, T. (Eds.): 
Antivirals in Man . In press. 

2. Dolin, R. : Viral gastroenteritis. In Beeson, P.B., McDermott, N. 
and Wyngaarden, J.B. (Eds.) Textbook of Medicine 15th Edition, 
Philadelphia, Pa., W.B. Saunders. In press. 

3. Dolin, R. : Norwalk-like agents of viral gastroenteritis. In Mandell, 
G. , Douglas, R.G., Bennett, J.E. (Eds.) Principles and Practice of 
Infectious Diseases , New York, Wiley and Sons. In Press. 

4. Reichman, R.C., Pons, V.G., Murphy, B.R. , Caplan, E.A. and Dolin, R. : 
Cell mediated cytotoxicity following influenza infection and vaccination 
in humans. J. Med. Virol . In press. 

5. Pons, V.G., Reinertsen, J.R., Steinberg, A.D. and Dolin, R. : Decreased 
cell mediated cytotoxicity against virus-infected cells in systemic 
lupus erythematosus. J. Med. Virol. In press. 



20-43 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00055-07 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Regulation of the Immune Response in Man and Experimental Animals 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Head, Clinical Physiology Section LCI NIAID 
Deputy Clinical Director NIAID 

Biologist LCI NIAID 

Clinical Associate LCI NIAID 

Guest Worker LCI NIAID 

Senior Investigator LCI NIAID 

Clinical Associate LCI NIAID 

Clinical Associate LCI NIAID 

Clinical Associate LCI NIAID 

Biologist LCI NIAID 



PI: 


A. 


S. Fauci 


Other: 


C. 


Burch 




T. 


Cupps 




A. 


Dimitriu 




B. 


F. Havnes 




P. 


Katz 




H. 


C. Stevenson 




D. 


Volkman 




G. 


Whalen 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Clinical Physiology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 



6 5 /i: 



PROFESSIONAL: 



4 5 /12 



CHECK APPROPRIATE BOX(ES) 
3 (a) HUMAN SUBJECTS 

□ (al) MINORS H ( a 2) INTERVIEWS 



□ (b) HUMAN TISSUES 



• underline keywords) 

and immunoregulat 



gulatory events associated with triggering 



SUMMARY OF WORK (200 ^ords or less • 

The precise mechanisms 
of human B lymphocytes following non-specific and antigen induced stimulation havd 
been delineated. Functionally distinct as well as overlapping immunoregulatory 
.-ymphocyte subsets have been characterized. The precise abnormalities of immuno- 



logic reactivity have been characterized in several immunologically mediated 
diseases such as systemic lupus erythematosus, Sjogren's syndrome, acute infec- 
tious mononucleosis, sarcoidosis and tuberculosis. For the first time, an anti - 
: -diotypic antibody has been produced which specifically blocks the function of 
iiuman B cells bearing the corresponding idiotype. Hybridoma lymphocyte cell lines 
have been established which are secreting antibody which binds specifically to 
functionally distinct subsets of human blood lymphocytes. Counter-current 
centrifugation techniques have been adapted which result in 95% and greater purity 
and yield of monocytes from human blood. These cells have been characterized 
functionally and structurally. Modulation of lymphocyte subsets by a number of 
clinically relevant pharmacologic agents have been defined. Clinical therapeutic 
studies have yielded extraordinarily favorable results in the cytotoxic drug 
Approach to the systemic necrotizing vasculitides ■ 



PHS-6040 

10-76) 



20-44 



Project No. Z01 AI 00055-07 LCI 
Project Description 
Obj ectives : 

1) To delineate the mechanisms of regulation of immunologic reactivity 
by purified subpopulations of human lymphoid cells. To examine the precise 
functional capabilities of these individual lymphoid cell subpopulations as 
well as the mechanisms whereby they maintain the normal homeostatis of 
positive and negative signals which regulate normal immunologic reactivity. 

2) To determine the role of these lymphoid cell subpopulations in the 
pathogenesis of immunologically mediated diseases such as the vasculitides , 
hypersensitivity diseases, granulomatous diseases, the spectrum of auto- 
immune diseases, as well as infectious diseases characterized by aberrations 
of immunologic reactivity. In particular, to delineate the alterations of 
various subpopulations of cells by mechanisms such as a) modulation of cell 
surface receptors (for example, Fc receptors) by immune complexes, cryo- 
globulins or other serum factors; b) activation of cell subsets by viruses 
(such as Epstein-Barr) virus); and c) depletion, proliferation, or redistri- 
bution of cell subsets among lymphoid compartments. In this regard, to 
determine the role of these mechanisms in the abnormalities of immunoregulation 
characteristic of these diseases. 

3) To develop hybridoma cell lines secreting monoclonal antibodies 
directed against stable lymphoid cell surface antigens which define func- 
tionally distinct subpopulations of immunoregulatory cells. 

4) To determine the role and mechanisms of anti- idiotypic antibodies 
in the regulation of human B cell responses in normal immune reactivity and 
in diseases characterized by the production of abnormal serum components 

(M components) . 

5) To investigate the cellular interactions, molecular, and biochemical 
events associated- with triggering of various mononuclear cell subpopulations 
and to determine the relationship between selective triggering via various 
cell surface receptors and the kinetics of expression of various functional 
capabilities of the given cell populations. 

6) To continue to develop and further perfect culture and assay systems 
for primary in vitro activation of human B lymphocytes to antibody production 
following polyclonal activation or specific antigenic stimulation. 

7) To develop an in vitro model of autoreactivity by selective 
triggering or depletion of immunoregulatory cell subpopulations in order to 
define more precisely the primary or secondary role of altered immunoregula- 
tion in autoimmune diseases. 



20-45 



Project No. Z01 AI 00055-07 LCI 

8) To delineate the mechanisms whereby recognition of self molecules 
(such as la-like determinants) plays a role in non-self antigenic recog- 
nition. In this regard, to determine the role of autoreactivity in the 
normal immunologic homeostasis in man and to characterize the aberrancies of 
this otherwise normal level of autoreactivity which are associated with 
pathologic autoimmunity and altered immune surveillance in certain neo- 
plastic diseases, particularly those of the lymphoprolif erative type. 

9) To develop methodologies of fractionating purified populations of 
human peripheral blood monocytes and T cell subsets by elutriation centri- 
fugation techniques in order to avoid pre-activation of the respective 
populations which invariably occurs with positive selection methodologies. 

10) To induce, purify and characterize soluble factors from purified 
subpopulations of human lymphocytes and monocytes which are involved in the 
regulation of immunologic reactivity. 

11) To delineate the precise mechanisms whereby immunosuppressive agents 
such as corticosteroids and cytotoxic drugs affect the immune responses in 
man specifically examining their selective effects on subpopulations of 
lymphocytes and monocytes with regard to their circulatory kinetics, cellular 
interactions, activation, and expression of in vivo and in vitro functional 
capabilities. In addition, to correlate the selective effects of corticoster- 
oids and cytotoxic drugs on these various parameters with the suppression of 
disease activity in various inflammatory and immunogically-mediated disease 
states in man, and by doing to aim at a greater understanding and therapeu- 
tic specificity in the clinical use of these agents. Furthermore, to study 
the mechanisms whereby manipulations of immune reactivity as described above 
affect host defenses against infection, tumor surveillance and propensity 
towards autoimmune states. 

12) To study the functional capabilities of eosinophils and mononuclear 
cells in the idiopathic hypereosinophilic syndrome in order to elucidate the 
mechanisms whereby these cells cause invasion of tissue and subsequent tissue 
damage in this disease. To use this information to gain a greater under- 
standing of the functional properties of eosinophils in normal states, various 
diseases and to determine their sensitivity to various therapeutic regimens. 

13) To extend the concept of immunoregulation to the effect of the immune 
system on other cell types. Specifically, to study the mechanisms of immuno- 
regulation by lymphoid cell subpopulations on eosinophil and neutrophil 
kinetics and function in the hypereosinophilic syndrome and certain neutro- 
penic states respectively. 

14) To continue to study the spectrum of the vasculitides in man from a 
mechanistic, pathophysiologic, clinical and therapeutic standpoint. 

Methods Employed : 

1) The predominant theme of this laboratory is the delineation of the 

20-46 






Project No. Z01 AI 00055-07 LCI 

mechanisms of immunoregulation in normal human immune reactivity and in diseases 
characterized by abnormalities of immunoregulation. 

In this regard, a major component of the methodology relates to the 
fractionation, identification and purification of immunoregulatory lymphoid 
cell subpopulations and the _in vitro culturing of these cells to determine 
their diverse functional capabilities. Subpopulations of cells are identified 
predominantly by surface markers and functional capabilities are assessed by a 
variety of in vitro assays. We have focused on the relationship between various 
in vivo and in vitro triggering signals and the subsequent _in vivo expression of 
direct and immunoregulatory functional capabilities. This has been focused 
predominantly in the area of activation, proliferation and differentiation of 
human B lymphocytes to antibody producing and secreting cells. In this regard, 
we have been utilizing a unique model of human B cell function with was 
originally developed in this laboratory. It is a system of primary in vitro 
stimulation of bone marrow derived (B) lymphocytes by polyclonal activation 
as well as specific antigenic stimulation with subsequent measurement of 
single cell antibody production by a direct hemolysis-in-gel plaque forming 
cell (PFC) assay. We have further extended this system to now measure indirect 
(IgG) PFC. In addition, we also have developed a system for the measurement of 
Ig secreting cells of all classes by use of staphylococcal protein A (SPA) 
coated target erythrocytes together with class specific developing antisera. 

Using these models, we have developed a system for the selective generation 
of suppressor cells or helper cells capable of modulating In vitro antibody 
production. In addition, we have produced soluble mediators of B cell function 
in man and have characterized the cell types of their origin. 

Other assays employed to delineate the functional capabilities of 
lymphoid cells are the _in vitro blastogenic responses to mitogenic and 
antigenic stimulation, elaboration of various soluble mediators, cell 
mediated cytotoxicity against various autologous, allogeneic and xenogeneic 
targets. Various cytotoxicity assays include spontaneous, mitogen dependent, 
and antibody dependent cellular cytotoxicity. A particular aim is to identify 
and characterize the abnormalities of lymphocyte or monocyte subpopulations 
involved in immunologically mediated diseases to determine the primary or 
secondary nature of these abnormalities and their relationship to the altered 
state of immune reactivity, as well as to create _in_ vitro models of altered 
immunoregulation by selectively triggering or manipulating immunoregulatory 
subpopulations of cells. 

2) Highly sensitive assays (enzyme linked immunosorbent - ELISA) have 
been developed and are being employed to measure the in vitro production of 
specific antibody following in vivo immunization of human subjects as well 
as primary in vitro antigenic stimulation. 



20-47 



Project No. Z01 AI 00055-07 LCI 

3) Antisera against purified subpopulations of human lymphoid cells 
have been developed in order to identify, isolate, characterize, inhibit 
functional capabilities or deplete these functionally distinct subsets from 
various cell suspensions. 

4) In relationship to #3, hybridoma cell lines have been developed 
which are producing large quantities of monoclonal antibodies directed 
against functionally distinct lymphocyte subsets. 

5) Recently developed elutriation centrif ugation techniques are being 
employed to isolate purified (greater than 95%) populations of human monocytes 
from peripheral blood, thus avoiding adherence techniques. 

6) Methods to fractionate purified populations of eosinophils (90- 
98% pure) from human blood have been developed and are being employed. 

7) Corticosteroids are administered to normal volunteers and patients 
requiring this drug and selective effects of these agents on the compart- 
mentalization and functional capabilities of monocytes and lymphocyte 
subpopulations are determined. In addition, the differential effects of 

in vitro corticosteroids and _in vitro irradiation on various lymphoid cell 
functional capabilities such as triggering, proliferation, differentiation, 
antibody production and secretion are being studied. 

8) A subject's lymphoid cells are labeled with chromium and reinfused 
back into their circulation to determine the effects of disease states and 

of chemotherapeutic agents on the viability and circulation patterns of 
these cells. 

9) Bone marrow aspirates are performed in normal subjects and patients 
with various diseases prior to, during and following therapy. Purified 
populations of lymphoid cells are obtained from these bone marrow cell 
suspensions by various separation techniques in order to delineate the 
immunocompetence of bone marrow lymphoid cells in normal man, as well as 
the alteration of compartmentalization and function of these cells in 
untreated and treated disease states. 

Major Findings and Significance to Biomedical Research and the Program 
of the Institute : 

A number of significant findings in human immunobiologic research have 
emerged from this laboratory over the past year. These have been in several 
general areas including mechanisms of triggering of human B lymphocytes; 
immunoregulation of B and T cell function; identification and purification 
of functionally distinct immunoregulatory and effector lymphocyte and monocyte 
subpopulations in normal individuals and a characterization of the aberrations 
of such subsets in certain disease states; and finally studies involving the 



20-48 



Project No. Z01 AI 00055-07 LCI 

pathogenesis and therapeutic approach to diseases characterized by abnormalities 
of immunologic reactivity. Specific findings of particular relevance include: 

1) Major advances have been made in the delineation of the mechanisms of 
activation of human B lymphocytes. Mitogen induced and antigen specific in 
vitro assays of B cell function have been originally developed and are now 
being extended in this laboratory. These include the first polyclonally in- 
duced and antigen induced direct and indirect hemolytic plaque forming cell 
(PFC) assay for human B cells. In addition, a highly sensitive enzyme linked 
immunosorbent (ELISA) assay has been developed to detect minute quantities 

of supernatant Ig and specific antibody from ^n vitro cultures of human lympho- 
cytes. These innovative assay systems are being used to delineate the complex 
mechanisms of immunoregulation of human B cell function in normal individuals 
and in disease states, as will be described below. Representatives from 
numerous laboratories throughout the world have visited our laboratory this 
year to learn these unique methodologies, and have been successful in instituting 
these systems in their own respective laboratories. Of particular note in this 
regard is the fact that in 1978 an international workshop on human B cell 
function was organized and directed by the Head, Clinical Physiology Section, 
LCI, NIAID. The purpose was to bring together the world's leading investigators 
in this area and compile a state of the art publication with guidelines for 
furture research. This led to the extremely well received book "Antibody 
Production in Man. In Vitro Synthesis and Clinical Implications" Edited by 
A. S. Fauci and R. E. Ballieux, Academic Press, New York, 1979. 

2) Precise delineation of immunoregulatory lymphocyte subpopulations in 
normal individuals has been accomplished. We have previously reported the 
original description of a delicate balance between helper and suppressor cell 
subpopulations in the modulation of normal B cell immunologic reactivity. In 
addition, we have demonstrated that certain otherwise normal individuals are 
hypo-responders in these _in vitro B cell systems, and that this hyporesponsive- 
ness resulted directly from overly active or triggered suppressor cells. We 
have now extended these studies to precisely characterize the identity and 
nature of this naturally occuring suppressor T cell. 

In addition, we have characterized the precise identity, kinetics and 
mechanisms of action of the mitogen (Con-A) induced suppressor cell in 
normal humans . 

3) Applying the abovementioned systems of immunoregulation we have charact- 
erized the primary and secondary immunoregulatory abnormalities in a number of 
immunologically mediated diseases. 

We have demonstrated the presence of altered proportions and absolute 
numbers of immunoregulatory T cell subpopulations in patients with systemic 
lupus erythematosus (SLE) . In addition, we have demonstrated a deficiency of 
suppressor cell function in SLE patients. In this regard, an in vitro model of 
pre-triggering suppressor cells by activation with immune complexes has been 
developed to support the hypothesis of immune complex associated alterations 



20-49 



Project No. Z01 AI 00055-07 LCI 

of immunoregulation in SLE and related syndromes. We have defined the spectrum 
of relative alterations of immunologic reactivity by demonstrating the presence 
of mild hyperreactivity of B cells associated with reversible alterations of 
immunoregulatory T cell subsets in Sjogren's syndrome (SS) without other 
associated connective tissue disorders; modest to severe B cell hyperreactivity 
with irreversible alterations of immunoregulatory T cell subsets in SS 
associated with mild SLE; and severe B cell hyperreactivity together with 
alterations and depletions of immunoreguatory T cell subsets in frank SLE. 

Of major inportance is the fact that we have developed an anti-idiotypic 
antibody against the surface IgM and serum monoclonal IgM peak of a patient 
with chronic lymphocytic leukemia whose leukemic B cell clone is secreting 
monoclonal antibody against SRBC determinants. The anti-idiotypic antibody 
reacts with the patient's B cell surface IgM and serum IgM but not with normal 
B cell surface IgM or serum IgM. Furthermore, the anti-idiotypic antibody 
blocks the spontaneous and mitogen induced secretion of anti-SRBC antibody by 
the patient's B cells but not by normal B cells. This is the first demonstration 
in man of the inhibition of B cell function by anti-idiotypic antibody and 
has obvious important implications in the understanding of the role of the 
idiotype network in the regulation of human immunologic reactivity. 

We have demonstrated the presence of spontaneously activated B cells 
in Epstein-Barr virus (EBV)-induced acute infectious mononucleosis (IM) . We 
have further shown that this is due to the direct triggering of B cells by EBV via 
surface receptors. We have characterized the atypical lymphocyte in IM and 
have demonstrated for the first time the emergence of suppressor T cells in 
acute IM and their subsequent disappearance in the convalescent state. This 
has important implications in the suppression of B cell outgrowth in IM which 
may be an important mechanism of containment of disease activity and prevention 
of evolution into a B cell neoplasm. 

In addition, we have demonstrated the alteration of immunoregulatory T 
cell subsets as well as the presence of adherent suppressor monocytes in 
sarcoidosis and tuberculosis. 

4) Because of the potential relationship between certain of the abovementioned 
autoimmune diseases and aberrancies of normally occurring autoreactivity , we 
investigated the mechanisms of regulation of autologous and allogeneic reactivity 
in the models of the autologous and allogeneic mixed lymphocyte reactions (MLR) . 
In addition, we have defined the relative stimulatory and responder capabilities 
of various lymphocyte and monocyte subpopulations in the MLR. The relationship 
between the autologous and allogeneic MLR and the development of suppressor and/or 
cytotoxic T cells was described. In this regard, the presence of a potent 
adherent suppressor cell of the autologous MLR was demonstrated in sarcoidosis. 
Furthermore, the modulation of the autologous and allogeneic MLR in normal sub- 
jects by adherent cells, prostaglandins, irradiation and in vitro and in vivo 
corticosteroids has been delineated. 



^0-50 



Project No. Z01 AI 00055-07 LCI 

5) Because of the potential role of cytotoxic cells mediating either antibody 
dependent cellular cytotoxicity (ADCC) or natural killer (NK) activity in 
immunologically mediated disease, we have precisely delineated the ADCC and NK 
capabilities of multiple heterogeneous subpopulations of T cells, null cells 
and monocytes. In these studies, we have demonstrated the overlapping and 
distinct cytotoxic capabilities of these heterogeneous lympohoid cell subsets. 

6) We have adapted a unique cell fractionation technique called elutriation 
(counter-current centrifugation) for the purpose of purifying human blood 
monocytes. This technique provides a major advance in our ability to carefully 
and precisely study the human monocyte. 

This technique has the advantage over the current adherence techniques of 
monocyte isolation of negatively selecting the human monocyte in excellent 
purity (95%) and excellent yield (95%). The yield of this procedure is so 
great, that up to 1.5 billion monocytes have been purified from the blood of 
a single normal human at one time. In addition, this technique has proved to 
provide much more effective monocyte depletion of mononuclear cell suspensions 
than the current technique of depleting adherent cells on sephadex G-10 columns 
since elutriation does not remove non-monocyte adherent cells (such as B cells) . 
Thus, we have been able to characterize large numbers of purified human monocytes 
with regard to their intracytoplasmic enzyme activity. We have also examined 
monocytes with scanning and transmission electron microscopy for the details 
of their cytoskeleton assembly. In addition, we have placed large numbers 
of these cells into culture to examine how these parameters are altered 
when these cells mature into macrophages. We have been able to examine the 
evolution of these functions on a daily basis for one week using a single 
normal individual's monocytes. Human monocytes have been examined for their 
ability to perform the interrelated functions of phagocytosis and killing in 
an ADCC system. We have shown that while monocytes have improved phagocytic 
function after being placed in culture, they have diminished killing capability 
in ADCC. Of interest is that we are the first group to demonstrate spontaneous 
killing of monocytes against human red cells and that this function improves 
when monocytes mature into macrophages. Hence, for the first time, human 
monocytes in a highly purified, non-activated state have been precisely 
characterized. 

7) We have characterized functionally distinct subpopulations of T cells on 
the basis of relatively unstable surface receptors such as the Fc receptor 
for IgG or IgM (T or T ). In addition, we have developed and characterized 
a rabbit anti-human T cell antiserum against stable cell surface components. 

Most importantly, we are among the first few laboratories in the world to 
have produced and fully characterized a variety of mouse lymphocyte hybrid 
cell lines which are producing virtually unlimited quantities of antibodies 
which bind to a number of distinct as well as overlapping subpopulations of 
human peripheral blood mononuclear cells. In particular, antisera have 
been produced which bind to a functionally distinct human peripheral blood 
T cell subset. In addition, another antibody is being produced which binds 
specifically to human blood monocytes. 



20-51 



Project No. Z01 AI 00055-07 LCI 

8) In line with our interest in the pharmocologic modulation of the immuno- 
regulatory apparatus in man, we have studied and characterized the selective 
and differential effects of a number of agents directly on effector lympho- 
cye subsets as well as on functionally distinct immunoregulatory lymphocyte 
subsets. These agents include corticosteroids, cyclophosphamide, azathioprlne, 
irradiation, cyclic nucleotides, and prostaglandin inhibitors. A graded 
differential sensitivity of B cells, more than suppressor T cells, more than 
helper T cells was demonstrated to cyclophosphamide, azathioprine and irrad- 
iation. Cyclic AMP directly inhibited suppressor T cells, while prosta- 
glandin inhibitors blocked prostaglandin mediated suppression of certain 
subsets of adherent suppressor cells. Corticosteroids had the most complex 
effects with selective and differential effects on the circulatory kinetics 
and functional capabilities of T and B cell subsets. 

We have also delineated the relationship of corticosteroid receptors on 
lymphocyte subsets (with regard to receptor density, binding affinity and 
dissociation constant) and the differential effects of _in vivo and in vitro 
corticosteroids on these subsets. 

9) We have continued our clinical studies of a number of diseases of estab- 
lished or suspected immunologic mediation. These comprise virtually the entire 
spectrum of vasculitis including Wegener's granulomatosis, systemic necrotizing 
vasculitis (polyarteritis nodosa type), hypersensitivity vasculitis, Takayasu's 
arteritis, temporal arteritis, and lymphomatoid garnulomatosis . In addition, 
we are studing idiopathic midline granuloma, sarcoidosis, granulomatous 
hepatitis, juvenile rheumatoid arthritis, Weber-Christian panniculitis, a 
heterogeneous group of acquired immunodeficiency diseases, and host defense 
defects (chronic granulomatous disease, Chedick-Higashi syndrome) , and a large 
number of patients with the idiopathic hypereosinophilic syndrome. The above 
patient groups are under our primary care. In addition, we are carrying on 
collaborative studies with others in SLE, Sjogren's syndrome, mixed connective 
tissue disease, rheumatoid arthritis, chronic lymphocytic leukemia, acute 
infectious mononucleosis, tuberculosis, and Cogan's syndrome. 

In addition to carrying on extensive investigations delineating the mech- 
anisms of altered immunologic reactivity in these diseases, the results of which 
are described above, we have established treatment protocols for several of these 
disorders. Major contributions have resulted from these and dramatic long-term 
remissions and even cures have been established by us in several of these 
formerly fatal diseases by the use of chronic low dose cytotoxic agents (usually 
cyclophosphmide) , particularly in the severe systemic necrotizing vasulitides. 
These results are either recently published or in press. In particular, we 
continue to prospectively follow the largest group of patients with Wegener's 
granulomatosis in the world and have established a greater than 90% remission 
rate with the use of cyclophosphamide. In addition, we have most recently 
published similar striking results with cyclophosphamide in systemic necrotizing 
vasculitis of the polyarteritis nodosa group. 



20-52 



Project No. Z01 AI 00055-07 LCI 

In addition, we are prospectively following the largest group of patients 
in the world with the idiopathic hypereosinophilic syndrome and have effected 
striking remissions by the use of hydroxyurea which has been shown to prevent 
and/or halt the eosinophilic myocardopathy which is the major source of mor- 
bidity and mortality in this disease. 

Given the striking therapeutic results mentioned- above, our group has 
been the major referral center for these diseases and the therapeutic 
protocols which we have established are now being adopted and employed 
successfully world-wide. 

Proposed Course : 

These projects will continue along the lines which have been described. 

Publications : 

1. Fauci, A. S.: Immunosuppressive and ant i- inflammatory effects of 
glucocorticoids. In Baxter, J. D., Rouseau, G. G. (Eds.): Mechanisms 
of Glucocorticoid Hormone Action . Springer -Verlag, New York- 
Heidelberg-Berlin. In press. 

2. Fauci, A. S.: Midline granuloma, In Textbook of Medicine , Fifteenth 
Edition. P. B. Beeson, W. McDermott, J. B. Wyngaarden, Editors. 

W. B. Saunders and Co., Philadelphia, 1979, pp. 220-221. 

3. Fauci, A. S.: Wegener's granulomatosis. In Textbook of Medicine , 
Fifteenth Edition. P. B. Beeson, W. McDermott, J. B. Wyngaarden, 
Editors, W. B. Saunders and Co., Philadelphia, 1979, pp. 218-220. 

4. Fauci, A. S.: Familial mediterranean fever. In Textbook of Medicine . 
Fifteenth Edition, P. B. Beeson, W. McDermott, J. B. Wyngaarden, 
Editors. W. B. Saunders and Co., Philadelphia, 1979, pp. 2055-2057. 

5. Hunninghake, G. W. , Haynes , B. F. , Parrillo, J. E., and Fauci, A. S.: 
Comparison of the relative effector cell capabilities and proportions 
of cells bearing various surface markers in human tonsil and peripheral 
blood mononuclear cells. Clin. Exp. Immunol. 32:186-191, 1978. 

6. Reddick, R. L, Fauci, A. S., Valsamis, N. P., and Mann, R. B.: 
Immunoblastic sarcoma of the central nervous system in a patient with 
lymphomatoid granulomatosis. Cancer . 42:652-659, 1978. 

7. Blitzer, B. L. , Weiss, G. B., Osbaldiston, G. W. , Markham, R. B., 
Aamodt, R. , Berk, P. D., Wolff, S. M. , and Fauci, A. S.: Early idiopathic 
hemochromatosis with absent stainable bone marrow iron stores. 
Gastroenterology . In press. 



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Project No. Z01 AI 00055-07 LCI 

8. Katz, P., and Fauci, A. S.: Inhibition of polyclonal B cell activation 
by suppressor monocytes in patients with sarcoidosis. Clin. Exp. Immunol. 
32:554-562, 1978. 

9. Fauci, A. S.: Vasculitis. In Clinical Immunology , C. W. Parker, Editor, 
W. B. Saunders and Co., Philadelphia. In press. 

10. Dimitriu, A., and Fauci, A. S.: Activation of B lymphocytes. IX. 
Modulation of antibody production by products of activated macrophages. 
J. Immunol. 120:1818-1823, 1978. 

11. Fauci, A. S.: Granulomatous hepatitis. In Principles and Practice of 
Infectious Disease , G. L. Mandell, R. G. Douglass, Jr., J. E. Bennett, 
Editors. John Wiley and Sons, New York. In press. 

12. Parrillo, J. E., and Fauci, A. S.: Comparison of the effector cells in 
human spontaneous cellular cytotoxicity and antibody-dependent cellular 
cytotoxicity: Differential sensitivity of effector cells to in vivo 
and _in vitro corticosteroids, Scand. J. Immunol. 8:99-107, 1978. 

13. Fauci, A. S.: Mechanism of action of glucocorticosteroids . In Annual 
Reports in Medicineal Chemistry , Vol. 13, F. H. Clarke, Editor. Academic 
Press, New York, 1978, pp. 179-183. 

14. Gelfand, J. A., Hurley, D. H. , Fauci, A. S., and Frank, M. M. : The role 
of complement in experimental disseminated candidiasis. J. Infect. 
Pis. , 138:9-16, 1978. 

15. Fauci, A. S.: Mechanisms of the immunosuppressive and antiinflammatory 
effects of glucocorticosteroids. J. Immunopharmacol . 1:1-25, 1978. 

16. Fauci, A. S., Haynes , B. F., and Katz, P.: Drug-induced T and B 
lymphocyte dysfunction. In Infections Complicating the Abnormal Host , 
M. H. Grieco, Editor. Yorke Medical Books, New York. In press. 

17. Haynes, B. F., and Fauci, A. S.: Activation of human B lymphocytes. X. 
Heterogeneity of concanavalin A generated suppressor cells of the pokeweed 
mitogen induced plaque forming cell response of human peripheral blood 
lymphocytes. J. Immunol . 121:559-565, 1978. 

18. Hunninghake, G. W. , and Fauci, A. S.: Suppression of the generation of 
human Con A-induced cytotoxic effector cells by Con A-activated suppressor 
cells. J. Immunol. 120:1828-1831, 1979. 

19. Fauci, A. S., Pratt, K. R. , Whalen, G.: Activation of human B lymphocytes. 
VIII. Differential radiosensitivity of subpopulations of lymphoid cells 
involved in the polyclonally-induced PFC responses of peripheral blood 

B lymphocytes. Immunology. , 35:715-720, 1978. 



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Project No. Z01 AI 00055-07 LCI 

20. Doppman, J. L. , Dunnick, N. R. , Girton, M. , and Fauci, A. S.: Bile duct 
cysts secondary to liver infarcts: Experimental production by small 
vessel hepatic artery occlusion and clinical correlation. Radiology . 
130:1-5, 1979. 

21. Fauci, A. S., Steinberg, A. D. , Haynes , B. F., and Whalen, G.: Immuno- 
regulatory aberrations in systemic lupus erythematosus. J. Immunol. 
121:1473-1479, 1978. 

22. Aduan, R. P., Fauci, A. S., Dale, D. C, Herzberg, J. H. , and Wolff, 
S. M. : Factitious fever and self- induced infection. A report of 32 
cases and a review of the literature. Ann. Intern. Med. 90:230-242, 1979. 

23. Editor: Ricci, M. , Fauci, A. S., Arcangeli, P., and Torzuoli, P. 
(Eds.): Developments in Clinical Immunology . London, Academic Press, 
1978. 

24. Fauci, A. S., Haynes, B. F., Pratt, K. R., and Whalen, G.: Regulation 
of PWM-induced plaque forming cell responses of human peripheral 
blood lymphocytes. In Ricci, M. , Fauci, A. S., Arcangeli, P., and 
Torzuoli, P., (Eds.): Developments In Clinical Immunology . London, 
Academic Press, 1978, pp. 23-29. 

25. Parrillo, J. E., Fauci, A. S., and Wolff, S. M. : Therapy of the 
hypereosinophilic syndrome. Ann. Intern. Med. 89:167-172, 1978. 

26. Katz, P., Haynes, B. F., and Fauci, A. S.: Alterations of T lymphocyte 
subpopulations in sarcoidosis. Clin. Immunol. Immunopathol. 10:350-354, 
1978. 

27. Fauci, A. S., and R. E. Ballieux (Eds.): Antibody Production in Man : 
In Vitro Synthesis and Clinical Implications. Academic Press, Inc., 
New York, 19 79. 

28. Fauci, A. S., and Haynes, B. F.: Pokeweed mitogen induced plaque-forming 
cell responses of human peripheral blood lymphocytes: Regulation of B 
cell triggering. In Antibody Production in Man : In Vitro Synthesis and 
Clinical Implications. Edited by A. S. Fauci and R. E. Ballieux. Academic 
Press, Inc., New York, 1979, pp. 17-34. 

29. Haynes, B. F., Katz, P., and Fauci, A. S.: Effect of hydrocortisone on 
the kinetics and function of peripheral blood immunoregulatory cells in 
man. In Antibody Production in Man : In Vitro Synthesis and Clinical 
Implications. Edited by A. S. Fauci and R. E. Ballieux. Academic Press, 
Inc., New York, 1979, pp. 291-302. 

30. Katz, P., Haynes, B. F., and Fauci, A. S.: Aberrant regulation of B 
cell function in immunologically mediated diseases: Systemic lupus 
erythematosus and sarcoidosis. In Antibody Production in Man : In 
Vitro Synthesis and Clinical Implications. Edited by A. S. Fauci and 
R. E. Ballieux. Academic Press, Inc., New York, 1978, pp. 351-366. 

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Project No. Z01 AI 00055-07 LCI 

31. Fauci, A. S., and R. E. Ballieux: Human B cell function: Recent 
advances, unanswered questions, and future directions. In 

Antibody Production in Man : In Vitro Synthesis and Clinical Implications. 
Edited by A. S. Fauci and R. E. Ballieux. Academic Press, Inc., New 
York, 1979, pp. 393-396. 

32. Haynes, B. F., and Fauci, A. S.: Wegener's granulomatosis. In Current 
Ocular Therapy , F. T. Fraumf elder and F. Hampton Roy, (Eds.), W. B. 
Saunders Co., Philadelphia, PA, 1979. In press. 

33. Haynes, B. F. , and Fauci, A. S.: Cogan's syndrome. In Current Ocular 
Therapy , F.T. Fraumf elder and F. Hampton Roy (Eds.), W. B. Saunders 
Co., Philadelphia, PA, 1979. In press. 

34. Haynes, B. F., and Fauci, A. S.: Diabetes insipidus associated with 
Wegener's granulomatosis successfully treated with cyclophosphamide. 
N. Engl. J. Med . , 299:764, 1978. 

35. Five, M. W. , G. H. Mundinger, and A. S. Fauci: Diagnostic and 
therapeutic aspects of the surgical approach to Wegener's granulomatosis. 
J. Thor. Cardiovasc. Surg ., 1978. In press. 

36. Kornblut, A. S., J. E. Gadek, A. S. Fauci, and S. M. Wolff: Head and 
neck manifestations of histocytic medullary reticulosis. The Laryngoscope , 
LXXXVIII: 1596-1602, 1978. 

37. Katz, P., and A. S. Fauci: The effects of corticosteroids on 
immuno regulation in sarcoidosis. Cell. Immunol . , 42:308-318, 1979. 

38. Katz, P., B. F. Haynes, and A. S. Fauci: Lack of generation of killer 
cells in the mixed lymphocyte reaction between mitogen stimulated and 
unstimulated autologous lymphocytes. J. Immunol. , 121:1998-2001, 1978. 

39. Fauci, A. S., F. F. Haynes, and P. Katz: The spectrum of vasculitis: 
Clinical, pathologic, immunologic and therapeutic considerations. 
Ann. Intern. Med. , 89:660-676, 1978. 

40. Dimitriu, A. and A. S. Fauci: Activation of human B. lymphocytes. XI. 
Differential effects of azathioprine on B lymphocytes and lymphocyte 
subpopulations regulating B cell function. J. Immunol . 121:2335-2339, 
1978. 

41. Hunninghake, C. W. and A. S. Fauci: Pulmonary manifestations of the 
collagen vascular diseases. Am. Rev. Resp. Pis . , 119:471-503, 1979. 

42. Gallin, J. I., R. E. Elin, R. T. Hubert, A. S. Fauci, M. A. Kaliner, 
and S. M. Wolff: Efficacy of ascorbic acid in the Chediak-Higashi 
syndrome: Studies in humans and mice. Blood , 53:226-234, 1979. 

43. Parrillo, J. E. and A. S. Fauci: Mechanisms of glucocorticoid action 
on immune processess. Ann. Rev. Pharmacol. Toxicol. , 19:179-201, 1979. 

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Project No. Z01 AI 00055-07 LCI 

44. Haynes , B. F. and Fauci, A. S.: Mechanisms of corticosteroid action 
on lymphocyte subpopulations . IV. Effects of in vitro hydrocortisone 
on the generation and function of mitogen- induced and naturally 
occuring suppressor cells in man. Cell. Immunol . , 44:157-168, 1979. 

45. Haynes, B. F., Katz, P., and Fauci, A. S.: Mechanisms of corticosteroid 
action on lymphocyte subpopulations. V. Effects of in vivo hydrocortisone 
on the kinetics of mitogen-induced and naturally occuring suppressor 
cells in man. Cell. Immunol ., 44:169-178, 1979. 

46. Haynes, B. F., R. T. Schooley, J. E. Grouse, C. R. Payling-Wright , R. 
Dolin, and A. S. Fauci: Characterization of thymus-derived lymphocyte 
subsets in acute Epstein-Barr virus induced infectious mononucleosis. 
J. Immunol. , 122:699-702, 1979. 

47. Fauci, A. S.: Human B cell function in a polyclonally induced plaque 
forming cell system. Cell triggering and immunoregulation. 
Immunological Rev. , . In press. 

48. Clark, R. A. F., J. G. Gallin, and A. S. Fauci: Effect of in vivo 
prednisone on ±n vitro eosinophil and neutrophil adherence and 
chemotaxis. Blood . 53:633-641, 1979. 

49. Dimitriu, A., and A. S. Fauci: Differential sensitivity of human 
lymphocyte subpopulations to azathioprine. Transplant. Proc. 11:878- 
881, 1979. 

50. Katz, P., R. A. Goldstein and A. S. Fauci: Immunoregulation in 
tuberculosis: Alteration of T lymphocyte subpopulations and 
presence of suppressor monocytes. J. Infect. Pis. In press. 

51. Parrillo, J. E., T. J. Lawley, M. M. Frank, A. P. Kaplan and A. S. Fauci: 
Immunologic reactivity in the hypereosinophilic syndrome. J. Allergy 
Clin. Immunol. In press. 

52. Fauci, A. S., T. Murakami, D. D. Brandon, D. L. Loriaux, and M. B. 
Lipsett: Mechanisms of corticosteroid action on lymphocyte subpopulations, 
VI. Lack of correlation between glucocorticosteroid receptors and the 
differential effects of glucocorticosteroids on T cell subpopulations. 
Cell. Immunol. In press. 

53. Stevenson, H. D. and A. S. Fauci: Activation of human B lymphocytes. 
XII. Differential effects of _in vitro cyclophosphamide on human 
lymphocyte subpopulations involved in B cell activation. Immunology . 
In press. 



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Project No. Z01 AI 00055-07 LCI 

54. Fauci, A. S.: Mechanisms of altered immunoregulation in systemic lupus 
erythematosus and Sjogren's syndrome. Proc. Soc. Exp. Biol. In press. 

55. Fauci, A. S.: Wegener's granulomatosis, lymphomatoid granulomatosis, 
and related diseases. CME Medicine . 1(09) :l-6, 1979. 

56. Parrillo, J. E. and A. S. Fauci: Necrotizing vasculitis, coronary 
angiitis, and the cardiologist. Amer. Heart J. In press. 

57. Dimitriu, A., B. F. Haynes , and A. S. Fauci: Activation of human B 
lymphocytes. XIV. Characterization of the percursor of the pokeweed 
mitogen- induced anti-sheep red blood cell plaque forming cell. 

J. Immunol. In press. 

58. Katz, P., D. W. Ailing, B. F. Haynes, and A. S. Fauci: Association of 
Wegener's granulomatosis with HLA-B8. Clin. Immunol. Immunopathol. 

In press. 

59. Kingry, K. R. and A. S. Fauci: Activation of human B lymphocytes. The 
in vitro polyclonal B cell activator-induced plaque-forming cell system. 
La Ricerca . In press. 

60. Fauci, A. S.: Immunoregulation by human lymphocyte subpopulations . In: 
Regulatory T Lymphocytes , B. Pernis and H. J. Vogel (Eds.), Academic 
Press, New York. In press. 

61. Parrillo, J. E. , J. S. Borer, W. L. Henry, S. M. Wolff and A. S. Fauci: 
The cardiovascular manifestations of the hypereosinophilic syndrome. 
Prospective study of 26 patients with review of the literature. 

Amer. J. Med. In press. 

62. Fauci, A. S., P. Katz, B. F. Haynes, and S. M. Wolff: Cyclophosphamide 
therapy of severe systemic necrotizing vasculitis. N. Engl. J. Med. 

In press. 

63. Fauci, A. S.: Assays for suppressor cells. In: Manual of Clinical 
Immunology . N. R. Rose and H. Friedman (Eds.), ASM publication. 

In press. 

64. Fauci, A. S., B. F. Haynes, and G. Whalen: Multiple subpopulations of 
lymphoid cells regulating human B cell function. In: Cell Biology and 
Immunology of Leukocyte Function , M. Quastel (Ed.), Academic Press, 
New York, 1979, pp. 485-488. 

65. Haynes, B. F, G. S. Eisenbarth, and A. S. Fauci: Human lymphocyte 
antigens: Production of a monoclonal antibody which defines functional 
thymus-derived lymphocyte subsets. Proc. Natl. Acad. Sci. (USA) . In 
press . 



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Project No. Z01 AI 00055-07 LCI 

66. Haynes, B. F., R. T. Schooley, C. R. Payling-Wright , J. E. Grouse, 

R. Dolin, and A. S. Fauci: Emergence of suppressor cells of immunoglo- 
bulin synthesis during acute Epstein-Barr virus-induced infectious 
mononucleosis. J. Immunol. In press. 

67. Katz, P., and A. S. Fauci: Autologous and allogeneic intercellular 
interactions: Modulation by adherent cells, irradiation; in vitro 
and in vivo corticosteroids. J. Immunol. In press. 

68. Fauci, A. S.: Vasculitis: New insights amid old enigmas. Amer . 
J. Med. In press. 



20-59 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00056-06 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Biochemical Pathways of Mediator Release and Mechanism of Tissue Injury 
in Allergic Diseases 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



OTHER: 



A. P. Kaplan 



Head, Allergic Diseases Section 



M.A. 


Kaliner 


Senior Investigator 


J.I. 


Gallin 


Senior Investigator 


R.J. 


Mandle, Jr. 


Guest Worker 


G.G. 


Miller 


Clinical Associate 


H.L. 


Meier 


Chemist 


R.E. 


Thompson 


Chemist 


A.L. 


Weinstein 


Guest Worker 



LCI NIAID 

LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 
LCI NIAID 



OPERATING UNITS (if any) _ 

Z. Horakova Senior Investigator 

S. Katz Acting Director, Dermatology Branch 

R. Sigler Fellow, Walter Reed Army Hospital 



NHL I 
NCI 



LAB/BRANCH 

Laboratory of Clinical Investigation 



;ection 

Allergic Diseases Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, MD 20205 



TOTAL MANYEARS: 

5 7/12 



PROFESSIONAL: 

3 7/12 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS C (*2) INTERVIEWS 



G (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 
INACTIVE DURING CURRENT YEAR. TERMINATED. 



20-60 



PHS-6040 
(Rev. 10-76) 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZQ1 AI 00057-06 LCI 



PERIOD COVERED 



October 1. 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Basic Studies on Pathogenic Fungi 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



K.J. Kwon-Chung 



Senior Investigator 



LCI NIAID 



OTHER: J.E. Bennett Head, Clinical Mycology Sec, 
Itzhack Polacheck Visiting Fellow 



COOPERATING UNITS (if any) 

A.K. Bhattacharjee (NIAMDD) 



lab/branch 



Laboratory of Clinical Investigation 



Clinical Mycology Section 



INSTITUTE AND LOCATION 

NTATT) , NTH r Bethesda. Maryland 20205 



TOTAL MANYEARS: 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
□ (a) HUMAN SUBJECTS 

G (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



3 (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The interests and proposed course of our unit are to cover basic and 
applied aspects of various pathogenic fungi including their morphology, taxon- 
omy, pathology, epidemiology, biochemistry and genetics. Topics of present 
interest include: 1) structure of the capsular polysaccharide of serotype D 
of Cryptococcus neoformans , 2) metabolic pathway of creatinine in Cryptococcus 
neof ormans and C^. bacillisporus , 3) nuclear status of basidiospores in the 
sexual state of C_. neoformans , and 4) distribution of a_ and a. mating types 
among clinical and natural isolates of C_. neoformans and C_. bacillisporus . 



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PHS-6040 
(Rev. 10-76) 



Project No, Z01 AI 00057-06 LCI 

Project Description : 

Objectives : 

Objectives of the project cover both basic and applied aspects of vari- 
ous pathogenic fungi, including their morphology, taxonomy, pathogenicity, 
life cycle, biochemistry and genetics. The topics of present interest 
include: 

1) Chemical structure of capsular polysaccharide of serotype D of 
Cryptococcus neof ormans . 

2) Metabolic pathway of creatinine in C. neoformans and C. bacil- 
lisporus . 

3) Nuclear status (cytogenetics) of basidiospores in the sexual state 
of jC. neoformans . 

4) Distribution of a_ and a_ mating types among clinical and natural 
isolates of C^ neoformans and C_. bacillisporus . 

Methods Employed : 

1) Growth and the Preparation of the Cell-free Extracts : Cells of 
C. neoformans and C_. bacillisporus were grown on minimal medium containing 
creatinine or ammonium sulfate as the nitrogen source. The cells were har- 
vested, washed and suspended in phosphate buffer (0.05 m) , and broken by 
using glass beads and voltex mixture. The supernatant obtained after 10 min. 
breakage was used as cell-free extracts. 

2) Metabolic Product of Creatinine : The cell-free extract was incu- 
bated with radiolabled creatinine. The radiolabled metabolites were de- 
tected by thin layer chromatography on silica gel with phenol-ethanol-water 
as the developing solution. The product was identified by autoradiographic 
technique. 

3) Enzyme Assay : A very sensitive assay method was developed for 
creatinine deiminase by using the reaction mixture containing radiolabled 
creatinine buffer and cell-free extract. The enzyme reaction was stopped by 
adding acetic acid. The reaction mixture was shaken with cation exchanger 
Dowex-50 and then filtered. Under this condition, the substrate was found on 
the filter and the radioactivity of the filtrate represented the enzymatic 
product. 

Genetic Analysis : Crosses of isolates with appropriate genetic 
markers were made on hay infusion agar. The progeny from such crosses were 
isolated by micromanipulation and the genetic markers were analyzed. 

Production of Capsular Polysaccharide : Cultures were grown on 
Sabouraud dextrose broth, and cells were killed by adding buffered formalin 



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Project No. Z01 AI 00057-06 LCI 

and removed by centrifugation, The supernatant was treated with sodium ace- 
tate, acetic acid and 95% ethanol to precipitate polysaccharide. The poly- 
saccharide was dissolved in distilled water and treated with sodium acetate 
and acetic acid. The solution was deproteinated with chloroform and butanol. 
The aqueous solution was treated with sodium acetate, acetic acid and etha- 
nol. The precipitate was redissolved in distilled water and reprecipitated 
with 95% ethanol. The polysaccharide was chromatographed on a column of 
DEAE-cellulose using 0.01 M phosphate buffer, pH 7.3, and a linear salt 
gradient of 0-1 M NaCl. The 80% of material was eluded at a salt cone. 0.2 M, 
and this material was dialized and freeze dried. 

Chemical Structure of the Polysaccharide : Gel filtration, ultra- 
centrifugation, paper chromatography, hydrolysis by 1 N HC1, methylation, 
periodate oxidation and chromium trioxide oxidation methods were used to 
analyze the chemical structure of the polysaccharide. 

Major Findings and Significance to Biomedical Research and the Program of 
the Institute : 

1) The most significant series of developments in this laboratory over 
the past year with important implications to biomedical research have been 
the series of studies which have shown that the etiologic agent of crypto- 
coccosis is two distinct species of genus Cryptococcus rather than a single 
species, C. neoformans , as has been believed for the past 80 years. We have 
utilized genetical, biochemical, chemical and epidemiological aspects to 
reveal the distinct characteristics of the two species, C_. neoformans and 

C. bacillisporus . 

2) Significant Findings in the Area of Biochemistry : 

a) The demonstration of two different regulatory mechanisms for 
the synthesis of creatinine deirainase between C. neoformans (serotype 
A-D) and C_. bacillisporus (serotype B-C) . One of the best known natural 
reservoirs of the etiologic agent of cryptococcosis is pigeon droppings. 
The widely accepted view is that pigeon droppings contain high concen- 
tration of creatinine, and it serves as a selective medium for the 
growth of cryptococcal pathogens. We reported in the past that only 
two serotypes (A,D) of Cryptococcus neoformans were found in the pigeon 
droppings throughout the world. Also reported was that serotype B-C 
utilized creatinine better than A-D and the natural reservoir of the 
B-C remained unknown. We described the serotype B-C as a distinct 
species, C. bacillisporus . Metabolic pathway of creatinine is known in 
several species of bacteria but not in fungi. Our study demonstrated 
that the creatinine metabolism in the cryptococci involved one step 
resulting in methylhydantoin and ammonia. The enzyme responsible for 
this degradation was identified as creatinine deiminase and was found 
to be inducible in both species. However, the enzyme synthesis was 
regulated by the presence of ammonia in C_. neof ormans but not in C_. 
bacillisporus . This difference may be due to their ecological 
difference. 



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Project No. Z01 AI 00057-06 LCI 

b) An extremely sensitive assay method for creatinine desimidase 
was developed during this study by using autoradiogram. 

3) Findings in the Area of Genetics : We have further extended our 
previous studies on the cytogenetics of basidiospore formation in Filobasi- 
diella neoformans (C_. neoformans ) . Using two stable genetic markers, nuclear 
status of spore chains was clarified. Nuclear inclusion in the spore chains 
was found to be at random. Also demonstrated was the formation of diploid 
spores which go through meiosis during the blastospore formation. 

4) Findings in the Area of Epidemiology : Survey revealed that the 
mating type a_ is predominant among natural and clinical isolates of Crypto- 
coccus neoformans regardless of the serotype. The ratio of a_ and a. type was 
about 40:1 among 105 natural isolates and 30:1 in 233 clinical isolates. 

5) Findings in the Area of Immunochemistry : Capsular polysaccharide 
of Cryptococci contains antigenic determinants defining their serotypes. We 
extended the study on the chemical structure of serotype D of C_. neoformans . 
The major sugars were found to be similar to that of C_. bacillisporus but 
contained 0-acetyl group which was not found in C_. bacillisporus . Also found 
was that the substitution of mannose backbone by xylose and glucose was sim- 
pler than serotype A of C_. neoformans and also isolates of C^. bacillisporus . 

Proposed Course : 

The biochemical and genetical aspects of 5FC resistant strains and 
phenoloxidase negative strains of C. neoformans will be studied. The chemi- 
cal structure of capsular polysaccharide of serotype B, C. bacillisporus , 
will be studied. 

Publications : 

1. Kwon-Chung, K.J. and Bennett, J.E.: Distribution of a_ and a_ mating types 
of Cryptococcus neoformans among natural and clinical isolates. Am. J_. 
Epidemiol . 108:337-341, 1978. 

2. Kwon-Chung, K.J., Bennett, J.E. and Theodore, T.S.: Cryptococcus bacil- 
lisporus sp . nov. : Serotype B-C of Cryptococcus neoformans . Int ■ ^J. 
Syst . Bacteriol . 28:616-620, 1978. 

3. Young, N.A., Kwon-Chung, K.J., Kubota, T.T. and Jennings, A.E.: Dissem- 
inated infection by Fusarium monilif orme during treatment for malignant 
lymphoma. J. Clin . Microbiol . 7:589-594, 1978. 

4. Bhattacharjee, A.K., Kwon-Chung, K.J. and Glaudemans, C.P.J. : On the 
structure of the capsular polysaccharide from Cryptococcus neoformans 
serotype C. Immunochemistry 15:673-679, 1978. 

5. Kwon-Chung, K.J.: Comparison of Sporothrix schenckii isolates obtained 
from fixed cutaneous lesions with isolates from other types of lesions. 



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Project No. Z01 AI 00057-06 LCI 
J. Infect . Pis . 139:424-431, 1979. 

6. Bhattacharjee, A.K., Kwon-Chung, K.J. and Glaudemans. C.P.J. : On the 
structure of the capsular polysaccharide from Cryptococcus neof ormans 
serotype C II. Immunochemistry . In press. 

7. West, B.C. and Kwon-Chung, K.J.: Mycetoma caused by Microsporum 
audouinii . Am . J_. Clin . Path . In press. 

8. Kwon-Chung, K.J. and West, B.C.: Mycetoma caused by dermatophytes. 
First Int. Sym. Mycetoma, Venezuela, 1978. In press. 

9. Kwon-Chung, K.J.: Serotypes, epidemiology and the sexual life cycle of 
Cryptococcus neof ormans . British Mycopathological Soc . Report, 1979. In 
press. 

10. Bhattacharjee, A.K., Kwon-Chung, K.J. and Glaudemans, C.P.J. : On the 
structure of the capsular polysaccharide from Cryptococcus neof ormans 
serotype D. Carbohydrate Research . In press. 



20-65 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00058-06 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (30 characters or less) 

The Pathogenesis and Chemotherapy of Herpesvirus Infections 
in Man 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: R. Dolin Head, Medical Virology Section 
(until Jan 1, 1979) 

S.E. Straus Senior Investigator, MVS 
(since Jan 1, 1979) 

Other: R.Schooley Clinical Associate 
R. Berg " 



LCI NIAID 



COOPERATING UNITS (if any) 

P. Howley (LP, NCI) and R. Whitley (Cooperative Antiviral Study Group) 



LAB/BRANCH 



Laboratory of Clinical Investigation 



Medical Virology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

2 



PROFESSIONAL: 

1-1/2 



1/2 



CHECK APPROPRIATE BOX(ES) 
5§ (a) HUMAN SUBJECTS 

D (al) MINORS □ (a2) INTERVIEWS 



(b) HUMAN TISSUES 



Q (c) NEITHER 



SUMMARY OF «I0RK (200 words or less - underline keywords) 

The pathogenesis and natural history of herpesvirus infections in man are being 
investigated. Patients are being identified, followed clinically and diagnosed 
definitively by virus isolation. Studies of patients with infectious mononu- 
cleosis have documented a variety of effects of the causative agent, the EB 
virus, upon the target B lymphocyte. The development and nature of T-cell 
mediated immune responses to the EBV infected B cells have been characterized. 
The results of initial trials of adenine arabinoside therapy of serious herpes- 
virus infections have been substantiated. The tools are being developed to 
study the biology and molecular epidemiology of varicella-zoster virus 
infection. 



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PHS-6040 
(Rev. IO-76) 



Project No. Z01 AI 00058-06 LCI 

Project Description ; 
Objectives : 

1) To determine the pathogenesis and natural history of herpesvirus 
infections in both normal and immune compromised humans. 

2) To evaluate the efficacy of antiviral agents in the treatment of 
human herpesvirus infection. These infections include both minor and serious 
diseases produced by herpes simplex types 1 and 2, varicella-zoster and EB 
virus. 

Methods Employed : 

1) Patients with suspected herpesvirus infections are referred to the 
Medical Virology Section from the Clinical Center, the National Naval Medical 
Center, local hospitals and practicing physicians. The patients are evalu- 
ated and appropriate specimens are collected for virus isolation and sero- 
logic testing. 

2) Virus isolation is performed in primary or continuous human or 
simian cell lines. The viruses are identified by the appearance of typical 
cytopathic changes and typed by immunof luorescent procedures where required. 

3) In selected cases, the viral DNA is purified, digested by restric- 
tion endonucleases and analyzed by agarose gel electrophoresis. This pro- 
vides epidemiologic information regarding the transmission of the virus and 
may provide structural correlates of viral pathogenicity. 

4) The clinical efficacy of adenine arabinoside, adenine arabinoside 
monophosphate, acycloguanosine and ribavirin is being investigated in the 
treatment of herpesvirus infections. This unit is collaborating in a 
NIAID-sponsored multicenter trial evaluating adenine arabinoside in the 
treatment of herpes simplex encephalitis, herpes zoster infection of the 
immune compromised host, and chronic mucocutaneous herpes simplex infection. 

Major Findings : 

1) EB Virus Infection. 

a) Studies of cell-mediated immune responses in patients with in- 
fectious mononucleosis have progressed. T lymphocytes from patients 
with antibody to EBV are capable of suppressing lymphoblastoid trans- 
formation of EBV infected autologous lymphocytes. The acquisition of 
this suppressive capacity is absent early during primary clinical in- 
fection, develops after the first several days and remains present for 
life. This T-cell function is independent of humoral antibody and is 
not HLA restricted. 



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Project No. ZQ1 AI 00058-06 LCI 

b) EBV has been found to be a potent in vitro polyclonal activator 
of B cell function as manifested by induction of a plaque forming cell 
response and production of intracytoplasmic antibody. The PFC response 
is not T cell dependent but may be modulated by helper and/or suppressor 
T cells when present. 

c) Lymphocytes from patients with infectious mononucleosis are 
hyporesponsive to polyclonal stimulation by EBV. 

d) Polyclonal stimulation of B cells requires successful infection 
by live EB virus. Not all virally infected B cells, however, produce 
immunoglobulin, suggesting that cellular control mechanisms may play a 
role in the expression of EBV induced B lymphocyte activation. 

2) Clinical trials of systemically administered adenine arabinoside in 
herpesvirus infections. 

a) Herpes simplex encephalitis: The results of the earlier small 
collaborative study wherein adenine arabinoside (AEA-A) was demonstrated 
to substantially reduce mortality in herpes simplex encephalitis have 
been confirmed. An additional 78 patients have been treated with ARA-A 
in the nationwide trial, with a fatality rate of 31%, quite comparable 
to the 28% incidence reported in the first study. Additional trials 
will be initiated within the next several months to compare two new 
promising agents, adenine arabinoside monophosphate (ARA-AMP) and 
acycloguanosine, with ARA-A. 

b) Varicella-zoster infections: Patients will continue to be en- 
rolled for the next few months into the collaborative trial of ARA-A 
for VZ infections of immunocompromised patients. The earlier prelimi- 
nary trial suggested that the drug speeds clearing of virus from vesi- 
cles and healing of lesions. The present trial is aimed at defining 
whether treatment initiated within the first 72 hours of infection is 
capable of preventing dissemination of virus and/or subsequent post- 
herpetic neuralgia. 

3) Characterization of nucleic acid from varicella-zoster virus: 
VZV DNA purified from clinical isolates is digested with restriction endo- 
nucleases and analyzed by agarose gel electrophoresis. Highly labeled DNA 
will be prepared by nick translation and used in sensitive reassociation 
kinetic analyses to compare the DNA from different isolates. 

Significance to Biomedical Research and the Program of the Institute : 

Herpesviruses produce substantial morbidity and mortality each year. 
Normal adults and children suffer individual or recurrent infections caused 
by members of the herpesvirus family. These infections range from rather 
benign afflictions to those such as herpes simplex encephalitis which when 
untreated causes death in 60-70% of individuals. Immune compromised indi- 
viduals, of which there are an ever increasing number and variety, are 



20-68 



Project No. Z01 Al Q0058-Q6 LCI 

especially susceptible to herpes infections. The above studies have ex- 
panded current knowledge of the natural history and pathogenesis of these 
infections. The therapeutic trials in which we collaborate have been the 
first to clearly demonstrate efficacy of antiviral compounds for these types 
of infections. These results have been encouraging and pave the way for 
future studies with even more potent antiviral drugs. 

Proposed Course : 

With the recent departure of the former principal investigator, the 
direction of this laboratory will undergo some changes. It is expected that 
a more molecular approach will be applied to the analysis of the herpesvirus 
infection with particular emphasis on herpes simplex and varicella zoster 
infections. The mechanism of virus latency and reactivation will be explored. 
Sensitive DNA reassociation analyses using highly labeled VZ DNA probes will 
be performed in an effort to detect the presence of viral sequences in the 
genome of tissues obtained at autopsy from patients with a known history of 
zoster infection. 

Clinical trials with antiviral agents will continue. Within the next 
year, the NIAID sponsored collaborative study group will initiate trials of 
ARA-AMP and acycloguanosine. These agents hold particular promise because 
they are minimally toxic, are able to attain higher tissue levels and appear 
significantly more potent than ARA-A. In addition, this laboratory will at- 
tempt to initiate within the next several months a trial of topical therapy 
of herpes genital infection with ribavirin, another promising new agent. 
This compound has been found in uncontrolled use to be extremely effective 
in superficial herpes simplex infection. Its true merits must be defined by 
placebo controlled trials. 

Publications : 

1. Whitley, R.J., et al: Adenine arabinoside therapy of biopsy proved 
herpes simplex encephalitis. N^. Engl . J_. Med . 297:289, 1977. 

2. Whitley, R.J., et al: Adenine arabinoside therapy of herpes zoster in 
the immunosuppressed: NIAID Collaborative Antiviral Study. N. Engl . ^J. 
Med . 294:1193, 1976. 

3. Haynes, B.F., Schooley, R.T., Grouse, J.E., Payling-Wright , C.R. , Dolin, 
R. and Fauci, A.S.: Characterization of thymus-derived lymphocyte sub- 
sets in acute Epstein-Barr virus-induced infectious mononucleosis. 

J. Immunol . 122:699-702, 1979. 

4. Schooley, R.T., Haynes, B.F., Grouse, J.E., Payling-Wright, C.R., Fauci, 
A.S. and Dolin, R.: Quantitative assessment of suppression of Epstein- 
Barr virus induced B-lymphocyte outgrowth. Manuscript in preparation. 

5. Schooley, R.T., Haynes, B.F., Grouse, J.E., Payling-Wright, C.R., Fauci, 
A.S. and Dolin, R. : Mechanism of EBV-induced B-lymphocyte activation. 
Manuscript in preparation. 

20--69- 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE U.S. DEPARTMENT OF 
PROJECT NUMBER (Do NOT use this space) HEALTH, EDUCATION, AND WELFARE 

PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00154-04 LCI 



PERIOD COVERED 

October 1, 1978 to September 30. 



1979 



TITLE OF PROJECT (80 characters or less 

Immunologic, Neurophysiologic , Biochemical and Cellular Events in Immediate 
Hypersensitivity 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



Michael A. Kaliner 



Senior Investigator 



LCI NIAID 



OTHER: 


J. 


Shelhamer 




L. 


Steel 




H. 


Oertel 




M. 


Donlon 




S. 


Wescott 




G. 


Myers 




P. 


Davis 



Clinical Associate LCI NIAID 

Staff Fellow LCI NIAID 

Visiting Fellow LCI NIAID 

Guest Worker LCI NIAID 

Bio. Lab. Tech. (Biol.) LCI NIAID 

Bio. Lab. Tech. (Biol.) LCI NIAID 



Clinical Associate 



PMB NIAMD 



COOPERATING UNITS (if any) 

Walter Reed Army Medical Center (R. Evans, R. Summers, L. Smith and R. Sigler) 



LAB/BRANCH 

Laboratory of Clinical Investigation 



SECTION 

Allergic Diseases Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, MD 20205 



TOTAL MANYEARS: 
6 1/2 



PROFESSIONAL: 
4 1/2 



OTHER: 
2 



CHECK APPROPRIATE 30X(ES) 
Lx(a) HUMAN SUBJECTS 

□ (al) MINORS Q (a2) INTERVIEWS 



H (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Human asthma and rhinitis involve the excessive secretions of mucus. 



Cultured 



human airways incorporate radiolabeled molecules permitting monitoring of 
mucous secretion. Human lung mucus has been characterized biochemically and 
the immunologic as well as neuropharmacologic control of mucous secretion 
defined. The site and control of human lung parenchyma versus airway 
prostaglandin production has been identified and the factors generated by 
anaphylaxis causing prostaglandin synthesis isolated. The histologic responses 
to rat mast cell granules have been characterized both in rat and monkey skin 
and the factors responsible for eliciting the inflammation isolated and 
characterized. The relationship between calcium influx and rat mast cell 
degranulation has been analyzed as has the response of mast cells to histamine 
stimulation. Finally, the neuropharmacologic responsiveness of subjects with 
asthma, allergic rhinitis and cystic fibrosis has been studied. 



20-70 



PHS-6040 
(Rev. 10-76) 



Project No. Z01 Al 00154-04 LCI 

Project Description: 

Objectives : 

The goals of the Allergic Diseases Section are to expand our knowledge 
of all aspects of immediate hypersensitivity reactions. Our particular ex- 
pertise and focus are defining the biochemical events which accompany and 
control the triggering of allergic reactions, the neurophysiologic mechanisms 
which potentially modulate these events, the characterization of the mediators 
of anaphylaxis and identification of the responses of relevant target tissues 
to these mediators. 

We are concentrating on three primary areas: 1) Employing human lung 
tissue, we study "asthma in a test tube" in order to understand the immuno- 
logic triggers, biochemical concomitants and neurophysiologic controls of 
mediator release; the identification of additional mediators of anaphylaxis 
and the study of mucous secretion. 2) Rat peritoneal mast cells are utilized 
as a pure source of effector cells which may be readily studied in vitro 
and may amplify as well as initiate new areas of interest in relationship 
to the human lung model and 3) Clinical allergic asthma is being evaluated 
in an extensive protocol designed to expand our understanding of the neuro- 
physiologic mileau of allergic individuals as compared with cystic fibrosis 
and normal subjects. 

Methods Employed : 

1. Human lung model: Lungs removed, usually for cancer resection, almost 
always contain large amounts of normal, functioning lung tissue. We obtain 
these lungs in cooperation with the surgical and pathology departments of 
all the major hospitals in the Bethesda area. The lung tissue is washed, 
fragmented and replicated into 200 mg samples and subsequently incubated in 
dilutions of allergic serum for 2 hours at 37 °C or 18 hours at 25 °C. After this 
"sensitization" period, the serum is removed and the fragments challenged 
with allergen. The interaction of the allergen with tissue (mast cell) 
bound IgE induces the release of histamine, slow-reacting substance of ana- 
phylaxis (SRS-A), eosinophil chemotactic factor of anaphylaxis (ECF-A) , and 
prostaglandin E and F (PGE and PGF ) . These mediators may be assayed 
on the isolated terminaiportion of the guinea pig's ileum (histamine, SRS- 
A) , chemotactic chambers (ECF-A) or by radioimmunoassay (PGE , PGF 2a ). 

We have been determining alterations in the tissue levels of cyclic 
AMP and cyclic GMP after treating lung with various agonists in order to 
define the role these nucleotides play in modulating the release of mediators. 
Cyclic AMP may be measured by either a protein kinase binding assay or radio- 
immunoassay while cyclic GMP is determined by radioimmunoassay after acety- 
lation or succinylation. 

3 
Mucous glycoproteins are monitored by incorporating H-glucosamine in- 
to the mucous glands during an 18 hour incubation period. Secretions are 
dialyzed against 6M urea, filtered over sepharose 2B, and analyzed by ion 

20-71 



Project No. Z01 AI 00154-04 LCI 

exchange chromatography. Mucous glycoproteins may also be hydrolyzed by 
NaBH, or be focused on a sucrose gradiant in the presence of ampholytes. 

2. Rat mast cells: Rat peritoneal and pleural mast cells are obtained from 
freshly sacrificed male Sprague-Dawley rats by lavage of these cavities. The 
peritoneal cells include 5% mast cells (or approximately 500,000 to 1 million/ 
animal) . The cells are used either in mixed cell suspensions or after purifi- 
cation by centrifugation into albumin cushions. Mast cells granules are 
isolated with intact perigranular membranes by sonication and centrifugation 
through sucrose cushions. Granules free of membranes are obtained by osmotic 
lysis of mast cells. 

Calcium studies with rat mast cells involve incubation of cells with 
4o 

Ca, followed by rapid centrifugation through silicone oil into water. Hista- 
mine released. into the upper layer is assayed by an automated fluorometric 
assay while Ca associated with the cells spun into the bottom layer is 
quantitated by scintillation. 

3. Clinical asthma study: The methods employed in this study are covered 
in detail in the clinical protocol and include: a) measurement of cutaneous 
blood flow by xenon disappearance; b) pupillary responses as measured by 
a binocular pupillometer ; c) airway obstruction as measured by flow-volume 
curves with and without helium inhalation; d) serum cyclic nucleotide re- 
sponses as measured after intravenous isoproterenol administration. 

Major Findings : 

1) Human Lung 

a) The response of human and guinea pig peripheral lung preparations to 
histamine stimulation was compared with airway smooth muscle preparation from 
the same species. Histamine induced both PGE and PGF ? from both parenchymal 
preparations, only PGE from human airways and both PGE and PGF. from guinea 
pig airways. In order to analyze the mechanism of histamine- induced prosta- 
glandin synthesis, the responses to KCL at membrane depolarizing concentrations 
and the muscle stimulant carbachol were studied. Both guinea pig and human 
airway preparations were equivalently stimulated with KCL or carbachol while 
neither of these agents stimulated lung parenchyma. Therefore, histamine, 
through H-l receptor stimulation, generates prostaglandin synthesis from lung 
by direct stimulation of parenchymal cells and by causing muscle contraction 

in the airways. 

b) Human airways can be maintained in organ culture for at least 96 hours. 
The mucous secreting cells take up radiolabeled sugars, amino sugars, amino 
acids and sulfate and incorporate these labels into newly synthesized glyco- 
proteins. The glycoproteins produced can be differentiated by size 
(fraction A is > 7,000,000 daltons while fraction B is 400,000 daltons) on 

gel filtration and carbohydrate: protein ratios (fraction A=70:30; fraction 
B=20:80; wt:wt). However, both molecules elute from anion exchange columns at 
the same salt concentration, each has an identical isoelectic focusing point, 
each has an identical constituent sugar composition, each has similar amino 
acids constituting the protein core and the size of the protein core 
of each molecule is the same. Thus, human airway submucosal glands 
synthesize two similar but distinct glycoproteins. 

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Project No. Z01 AI 00154-04 LCI 

Employing quantitation of non-dialyzable glycoprotein radiolabeled with 
3H-glucosamine to monitor mucous secretion, the influence of immunologic 
challenge as well as several neurohornomal agonists on mucous secretion was 
analyzed. Reversed and direct anaphylaxis of airways increased mucous 
secretion. As supernatants rich in the various mediators released during 
anaphylaxis were also effective in increasing mucous release, we determined 
which mediator was responsible. Histamine acting through H-2 receptor stimu- 
lation was found to cause mucous secretion, as did PGF , PGF , PGD ? and 
PGA ? . PGE„ and thromboxane B ? were ineffective. Muscarinic stxmulation of 
mucous glands by methacholine~augmented mucous release as did alpha adrenergic 
stimulation. Beta adrenergic stimulation was ineffective. Therefore, several 
mediators of anaphylaxis (including histamine and several prostaglandins) 
and several neurophormones (cholinergic and alpha adrenergic agonists) are 
capable of increasing mucous secretion from human airways. 

c) Prostaglandin formation by human lung after anaphylaxis may in 
part be attributed to histamine. However, about 50% of the prostaglandin 
formation is independent of histamine. Several factors released from 
human lung during anaphylaxis are able to cause guinea pig lung parenchymal 
preparations to produce thromboxane B„ , PGF,, and PGE. We have characterized 
these factors and now recognize that they consist of two major components. 
The larger is retained on a DM5 filter (> 5000 MW) , and separates on a 
Sephadex G-150 column into two factors with apparant molecular weights 
of >200,000 and 55000 daltons. The second component filters through DM5 
but is retained by UM05 filters (MW = <5000 but >5000), filters on Sephadex 
G25 with an apparant molecular weight of 1500, adheres to DE52 in 0.001 
of NH.HC0 , pH7.8, and elutes in one major peak at 0.1M NH HCO . Thus, 
human lung undergoing anaphylaxis generates at least three molecules in addi- 
tion to histamine which are capable of causing prostaglandin synthesis. Fur- 
ther purification and biologic characterization of these molecules are 
underway. 

2) Rat Mast Cells 

Mast Cell granules injected into the skin of rats and monkeys were 
found to cause significant inflammatory reactions. Granules isolated with 
intact perigranular membranes caused a polymorphonuclear-rich infiltrate apparent 
in 2 hours which peaked by 8 hours. Subsequently, an intense mononuclear 
infiltrate replaced the PMN's and peaked at 24-48 hours. Granules isolated 
free of membranes and washed free of elutable mediators injected into skin 
caused a somewhat less pronounced polymorphonuclear infiltrate and a somewhat 
greater mononuclear infiltrate. The responsible factors were found primarily 
in close association with the granule matrix. In fact, granule preparations 
washed free of elutable mediators were found to have three separable factors 
capable of inducing these infiltrates. These factors may be separated on 
filters - the two larger factors are retained on a UM10 filter while the small 
factor filters through UM10 but is retained by a UM05 filter. The 
larger molecules have apparent molecular weights of 200,000 and 70000 daltons 
respectively on gel filtration through Sephadex G-200 . The smaller molecule 
chromatographs on Sephadex G-25 with a apparent MW of 1250. The smaller 
molecule binds to both anion and cation exchange columns and elutes as 
one major peak in a salt gradient. The larger molecules may be precipitated 

20-73 



Project No. Z01 AI 00154-04 LCI 

by 10% ethyl alcohol but not by (NH.)-SO,. Therefore, there are three 
factors constituent within the mast cell granule which elicit delayed 
inflammatory responses in rat skin. Further characterization of the 
structure and biologic activity of these molecules is underway. 

b) Initiation of rat mast cell secretion involves transmembrane move- 
ment of extracellular calcium. The equilibration with extracellular calcium 
in resting mast cells is extremely rapid, being completed within 60 sec 

at 37 °C in the presence of 0.8 mM calcium. Analysis of calcium movement 
into the rat mast cell undergoing a secretory event reveals that uptake 
is increased within 5-10 sec, only 50% is removed by subsequent exposure 
to EGTA and the time course of calcium uptake continues after granule 
release is completed. Calcium uptake was initiated by phosphatidyl 
serine (PS) added to the bathing medium in the absence of histamine 
release. As PS augments antigen- induced histamine release, the calcium 
translocation observed may relate to this property. ATP (10 mM) 
inhibits mast cell degranulation and chelates calcium; at ImM, ATP 
induces release and stimulates calcium uptake. ATP does not require 
hydrolysis to ADP or AMP for this response and there is insufficient ATP 
released by the mast cell to suggest that secreted ATP has any influence 
on the release reaction. Further characterization of the role which 
calcium plays in degranulation by mast cells is ongoing. 

c) Rat mast cell cyclic AMP and cyclic GMP levels are important in so 
far as these levels determine the capacity of mast cells to undergo degranu- 
lation. We are completing an analysis of factors responsible for changes 

in mast cell cyclic nucleotides and have found that histamine fails to 
change the mast cell levels. This observation suggests that the mast cell 
which is a major synthesis and storage site for histamine is either desensi- 
tized to histamine or has no functional histamine receptor sites. 

3) Clinical Studies 

The autonomic responses of asthmatic subjects have been compared with 
those of subjects with allergic rhinitis and normal controls. Alpha adrenergic 
function was assessed by pupillometry and cutaneous vascular responses. In 
both instances, the subjects with asthma were significantly more sensitive 
than both allergic rhinitis and normal control subjects. Beta adrenergic 
function has been analyzed by the cardiovascular and cyclic AMP responses to 
intravenous isoproterenol in these subjects. Patients with either asthma or 
allergic rhinitis have significant impairment of both beta adrenergic responses 
as compared with controls. Parasympathetic function has been analyzed by 
pupillometry, sweat responses and bronchial challenge. Again, patients with 
either allergic rhinitis or asthma demonstrate the same degree of exaggerated 
responsiveness in comparison with normal controls. Therefore, asthmatics are 
selectively more responsive to alpha adrenergic stimulation while both asthmatics 
and subjects with allergic rhinitis have increased cholinergic and dimished 
beta adrenergic responses. 



20-74 



Project No. Z01 AI 00154-04 LCI 

Subjects manifesting reproducibly positive allergic skin tests but who 
are asymptomatic have also been examined. These subjects who are "pre-allergic" 
have reduced beta adrenergic, hyper-responsive cholinergic and normal alpha 
adrenergic responses much as the subjects with allergic rhinitis. Taken 
together these data indicate that reduced beta adrenergic responsivity is 
associated with the atopic state as is excessive cholinergic responsiveness. 
However, excessive alpha adrenergic responses are seen only in asthmatics. 

More recently, we have completed an examination of 11 young adult 
subjects with cystic fibrosis and 9 parents ( obligate heterozygotes) of cystic 
fibrosis children. The CF subjects were significantly more sensitive than 
asthmatics in regards to both alpha adrenergic and cholinergic responsive- 
ness and equally abnormal in regards to depressed beta adrenergic responses. 
Only 3 of the CF's were concomitantly allergic and these 3 were relatively 
less abnormal than the other 8. Therefore, the autonomic abnormalities un- 
covered may be found in diseases other than allergy and may relate to glandular 
(mucus) secretion rather than muscle spasm. 

The obligate heterozygotes were, as a group, abnormal in both alpha 
adrenergic and cholinergic responses although much less so than the affected 
children. This observation suggests that the hyper-responsiveness is inherited 
as a primary defect rather than appearing as a secondary phenomena. 

Significance to Biomedical Research and the Program of the Institute 

Increased understanding of the mechanisms of immediate hypersensitivity 
and its controls may eventually allow improved therapy of the 44 million 
Americans with allergic rhinitis, the 9 million with asthma and the 22 
million with urticaria. The model systems employed permit sophisticated 
analysis of many of the aspects involved _in vivo . Unquestionably, our 
analysis of mucous secretion will enable us to approach clinical problems 
of bronchorrhea (asthma, cystic fibrosis, chronic bronchitis) with a 
greater understanding as well as permit _in vitro analysis of new modalities 
of therapy. 

The observation that mast cell granules may elicit delayed inflammatory 
responses may provide the mechanisms for explaining certain long- 
observed but hitherto poorly-understood clinical problems such as: the 
epithelial destruction accompanying asthma, the chronic inflammation 
associated with perennial rhinitis, the hyper-reactive airways disease occur- 
ing in asthma and delayed responses seen with allergy skin tests. Isolation 
of a low molecular weight molecule eliciting this late phase response implies 
potent chemotactic properties and should allow studies of the cellular mechanisms 
of chemotaxis employing a biologically-relevant molecule. 

Characterization of prostaglandin generating factors elaborated by 
human tissues undergoing biologic processes (allergic responses) 
will permit identification of the role these factors play in a variety 
of diseases including asthma. Further, purification of these factors 
will permit a detailed analysis of the mechanisms of prostaglandin 
synthesis. 

20-75 



Project No. Z01 AI 00154-04 LCI 

Finally, analysis of the autonomic responsiveness of allergic and 
cystic fibrosis patients enables us to approach these diseases with a 
unique appreciation of the capacity of neurophysiologic processes to 
modulate and complicate these diseases. Clearly the abnormalities 
uncovered have therapeutic implications in both the CF and asthma populations. 

In summary, the Allergic Diseases Section is approaching immediate 
hypersensitivity at several levels with interests in both clinical and basic 
areas. This approach is providing a mileau in which results are rapidly 
transmitted from the laboratory bench to the clinic and from which a 
finer appreciation of disease processes is evolving. The direction in 
which the lab is headed should continue to translate basic observations 
to enhanced clinical practice. 

PUBLICATIONS 

1. Raphael, G.D., Henderson, W.R. , and Kaliner, M. : Isolation of 
membrane bound rat mast cells. Exp. Cell Res . 115:428-431, 1978. 

2. Kaliner, M. : Human lung tissue and anaphylaxis: The effect of 
histamine upon the immunologic release of mediatos, Am. Rev. Resp . 
Pis. 118: 1015-1022, 1978. 

3. Gallin, J. I., Elin, R.J., Hubert R.T., Fauci, A.S., Kaliner, M.A. , 
and Wolff, S.M. : Efficacy of ascorbic acid in the Chediak-Higashi 
Syndrome: Studies in humans and mice. Blood . 53: 226-234, 1979. 

4. Platshon, L., and Kaliner, M. : The effect of the immunologic 
release of histamine upon human lung cyclic nucleotide levels and 
prostaglandin generation. J Clin . Invest . 62:1113-1121, 1978. 

5. Henderson, W.R., and Kaliner, M. : Mast cell granule peroxidase: 
Location, secretion and SRS-A inactivation. J_. Immunol . 122:1322-1328, 
1979. 

6. Henderson, W.R. , Shelhamer, J.E. , Reingold, D.B., Smith, L.J., Evans, 
R. , and Kaliner, M. : Alpha adrenergic hyperresponsiveness in 
asthma - analysis of vascular and pupillary responses. N. Engl . J. 
Med. , 300: 642-647, 1979. 

7. Metcalfe, D.D., Corash, L.M. and Kaliner, M. : Type II arylsul- 
fatases of human platelets: identification and characterization. 
Immunology, in press. 

8. Kaliner, M. : Human Lung tissue and anaphylaxis: Cyclic nucleotide 
response and prostaglandin synthesis. 12th Symposium of Collegium 
Internationale Allergologicium. Karger Press. In Press. 



20-76 



Project No. Z.01 AI 00154-0.4 LCI 

9. Steel, L. , Platshon, L. , and Kaliner, M. : Prostaglandin generation 
by human and guinea pig lung tissue. J_;_ Allergy , Clin . Immunol . , 
in press. 

10. Kaliner, M. : Prostaglandins and allergic inflammation. Elsevier Press. 
In press. 

11. Kaliner, M. : Mast cell derived mediators and bronchial asthma, in press. 



20-77 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl Al 00155-04 LCI 



PERIOD COVERED 

October 1, 1977 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Phagocyte Cell Function 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI: John I. Gall in 

Other: Steven Whited 
John Davis 
Anthony S. Fauci 
Charles H. Kirkpatrick 

Bruce Seligmann 
Deborah H. Cotton 
Mark Fletcher 
Haig Donabedian 
Michael M. Frank 



AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 



Head, Bacterial Disease Section 

Clinical Associate 

Guest Worker 

Head, Clinical Physiology Section 

Head, Clinical Allergy and 

Hypersensitivity Section 
Staff Fellow 
Clinical Associate 
Clinical Associate 
Clinical Associate 
Chief, Laboratory of Clinical 

Investigation 



LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 


LCI 


NIAID 



cooperating UNITS (if anykr. David Ailing (NIAID, NIH) , E. K. Gall in (Div. Exp. Hem., 
Armed Forces Radio. Biol. Res. Inst.), E. B. Cramer (Dept. Anat., Downstate Med. 
Ctr.), M. Klempner and B. Babior (Dept. Med., Tufts-New Engl. Med. Ctr.), E. 
Schiffmann (NIDR, NIH) 



LAB/BRANCH 

Laboratory of Clinical Investigation 



bacterial Disease Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 



TOTAL MANYEARS: 

4 1/12 



PROFESSIONAL: 

4 1/12 



OTHER: 




CHECK APPROPRIATE BOX(ES) 
g (a) HUMAN SUBJECTS 



S (b) HUMAN TISSUES 



□ (c) NEITHER 



i2) INTERVIEWS 



SUMMARY OF WORK (200 words or less - underline keywords) , , . 

The mechanism of leukocyte activation by chemotactic factors has been studied 
using electrophysiology , fluorescent probe , surface charge and ultrastructural 
techniques. 

Studies assessing the mechanism of modulating leukocyte locomotion indicate 
that limited secretion of specific granules , which accompanies chemotaxis , is 
associated with increased cell adhesiveness and increased availability of chemo - 
attra ctant receptors . Vigorous exocytosis is associated with depressed chemo- 



taxis, decreased availability of chemoattractant receptors, chemoattractant 
hydrolysis by secreted products and markedly increased cell adherence and aggre- 
gation . Human pyrogen has been shown to be a potent stimulator of neutrophil 
exocytosis and activation of the hexose monophosphate shunt. 

Studies of the two populations of neutrophils we had identified previously in- 
dicate that during the neutropenia that follows in vivo endotoxin or hemodialy- 
sis , a subpopulation of neutrophils with poorly demonstrable Fc receptors is the 
predominant neutrophil left in the circulation. Clinical studies assessing the 
effect of pharmacologic agents on the neutrophil subpopulations in normal sub- 
jects, patients with recurrent infection and host defense defects are underway. 



PHS-6040 
(Rev. 10-76) 



20-78 



Project No. Z01 Al 00155-04 LCI 
Project Description 
Objectives : 

1) Study the mechanism of leukocyte chemotaxis. 

2) Study the phenomenon of deactivation of leukocyte chemotaxis and to relate 
this to the pathophysiology of leukocyte dysfunction syndromes. 

3) Develop new techniques to quantitate the chemotaxis and the spreading of 
living phagocytes. 

4) Study the role of cations in leukocyte activation with particular emphasis 
on the utilization of indirect probes of membrane potential to study the 
relationship between changes in membrane potential and initiation of neutro- 
phil motility, phagocytosis, degranulation and superoxide generation. 

5) Study neutrophil subpopulations in normal subjects and in patients with 
neutrophil dysfunction. 

6) Study the mechanism of neutrophil margination and aggregation at capillary 
beds prior to cell migration into tissues. 

7) Study the mechanism of mobilization and secretion of neutrophil lysosomal 
granules, study the relationship of the secretory process to neutrophil 
function and dysfunction and to study how neutrophil secretory products may 
influence homeostasis. 

8) Characterize and define the mechanism of abnormal neutrophil chemotaxis 
following thermal injury. 

9) Study the role of fibroblast secretory products in the inflammatory 
response. 

10) Study the effects of pharmacologic agents on leukocyte function in vitro 
and in vivo . 

11) Assess the chemotactic activity of histamine for eosinophils in vivo. 

Methods Employed 

Peripheral blood leukocytes were separated from heparinized whole blood by 
dextran sedimentation and Hypaque-Ficoll separation. Chemotaxis was evaluated 
using a radioassay employing Cr labeled leukocytes and a double micropore 
filter system or a single filter measuring the distance migrated into the filter 
by the population of responding cells. Cell adherence was measured by quanti- 
tating the number of leukocytes adhering to plastic surfaces or to nylon wool 
columns. Phagocytosis, bactericidal capacity, nitroblue tetrazolium dye re- 
duction, hexose monophosphate shunt activity and superoxide generation were 

20-79 



Project No. Z01 Al 00155-04 LCI 
measured using standard techniques. PMN receptors for the chemoattractant 
f-met-leu-phe were quantitated as published previously. 

The electrophysiology of cultivated human macrophages was studied using 
standard intracellular recording techniques. The fluorescent probe dipentylo- 
xacarbocyanine and the radioisotope trimethylphosphonium were used as indirect 
probes of membrane potential changes in neutrophils. Leukocyte surface charge 
was measured with a Zeiss cytopherometer . Intracellular calcium was localized 
in human neutrophils by the intracellular precipitation of pyroantimonate anion 
followed by microprobe analysis and studies with the metal chelators (EDTA and 
EGTA) . 

The cytoskeleton of polymorphonuclear leukocytes was studied during 
conditions of chemotaxis and chemokinesis using 0.45 ym micropore filters. 
These small filters impede leukocyte migration but permit pseudopod penetration 
and a fixed orientation of leukocytes is thereby established. Orientation of 
living cells was monitored by well established techniques . Secretion of leuko- 
cyte granule enzymes were monitored by standard spectrophotometric assays. 
Polymorphonuclear leukocyte granules were separated and fractionated using 
sucrose gradient techniques and various enzyme markers were utilized to identify 
granule types. 

Neutrophils were fractionated into subpopulations based on their ability 
to rosette IgG coated erythrocytes. In some studies, in normal human volunteers, 
the effects of intravenous endotoxin on neutrophil subpopulations was examined. 

Major Findings 

1. Technologic advances have been made for quantitating leukocyte chemo- 
taxis, and bactericidal activity. Using an Optomax image analyzer the amount 
of time required to quantitate the number of cells migrating a distance into a 
micropore filter or the number of bacteria on a pour plate has been reduced by 
90%. In addition, the image analyzer has made possible a quantitative assay 
of leukocyte spreading. Preliminary studies indicate the analyzer will make 
possible the tracking of moving cells thereby providing quantitative information 
on initial rates of locomotion (Ailing) . 

2. During chemotaxis in vitro and exudation in vivo neutrophils secrete 
their granule contents and this secretion is preferential for specific granules. 
It has been shown that this secretory event modulates chemotactic responsiveness. 
With limited exocytosis there is increased chemoattractant binding to the cell 
membrane and this increased binding of chemoattractants increases availability 

of chemoattractant receptors without altered affinity of binding. Preliminary 
data indicates the increased receptor availability is related to translocation 
of specific granule membrane, which contains the chemoattractant receptors, to 
the cytoplasmic membrane. This may provide the mechanism for membrane 
turnover during sustained locomotion. Following vigorous exocytosis both chemo- 
taxis and cell orientation in a gradient of chemoattractant are inhibited. 
This inhibition is related to decreased binding of a chemotactic factor to the 
cell. The latter has been associated with decreased peptide affinity and 
increased hydrolysis of chemoattractant by granule products secreted extracellu- 

20-80 



Project No. Z01 Al 00155-04 LCI 
larly (Fletcher, Schif fmann) . 

3. Exocytosis of neutrophil granules in vitro increases neutrophil 
adhesiveness and aggregation. This has been related to neutralization 

of the net negative surface charge of the cell membrane and facilitation of 
cell-cell and cell-surface adhesion. Treatment of neutrophils with neuraminidase 
or poly-1-lysine also increases their adhesiveness without causing degranulation. 
In addition, submembranous deposition of cations (calcium) at the leading edge 
of cells in a gradient of chemoattractant was observed and speculated to facil- 
itate fusion of intracellular granules with the cytoplasmic membrane. In this 
regard, it is of interest that the specific granules, which are most accessible 
to extracellular release, are more negatively charged than the azurophil granules 
(Gallin, Cramer) . 

4. Using fluorescent cyanine dyes and tritiated trimethyl phosphonium 
ions chemoattractants and degranulating stimuli were shown to stimulate membrane 
potential changes in neutrophils. The potential changes in neutrophils are 
similar to those observed in macrophages using direct intracellular recording 
techniques. The data using degranulating stimuli indicate low concentrations 

of degranulating stimuli increase f-met-leu-phe receptor efficacy for eliciting 
membrane potential changes as monitored with a fluorescent dye. Vigorous secre- 
tion inhibits receptor efficacy. These latter observations support and extend 
the data on the effect of degranulating stimuli on chemoattractant receptor 
availability and on chemotactic responsiveness. In addition, the data suggest 
that alteration of neutrophil plasma membrane ion permeability or the concen- 
tration of intracellular ions by degranulating stimuli modulates the locomotory 
responsiveness of the cell. 

5. Assessment of chemoattractant elicited membrane potential changes using 
indirect probes indicates that neutrophils from patients with chronic granulo- 
matous disease have a major abnormality. Neutrophils from other patients with 
chemotactic defects did not show any such abnormality (Seligmann) . 

6. Highly purified leukocyte pyrogen causes selective secretion of human 
neutrophil specific granules in vitro and rabbit neutrophils in vivo . In 
addition, pyrogen stimulates neutrophil nitroblue tetrazolium dye reduction, 
hexosemonophosphate shunt activation and superoxide generation (Klempner) . 

7 . The acquired defect of neutrophil locomotion seen following thermal 
injury precedes sepsis and was shown to be a function of burn wound area and 
to be linearly related to the degree of lysosome lost from the neutrophil. A 
rabbit model for thermal injury has been developed to enable further study of 
this acquired chemotactic defect (Davis) . 

8. Twenty minutes following intravenous administration of E. coli endo- 
toxin to normal volunteers, during the period of neutropenia, the predominant 
circulating neutrophil is a "subpopulation" without readily demonstrable IgG 
(Fc) receptors. Plasma obtained at the time of neutropenia increases neutro- 
phil adhesiveness. In related studies, twenty minutes after initiating hemo- , 
dialysis in patients with chronic Schizophrenia, also during a period of neutro- 



20-81 



Project No. Z01 Al 00155-04 LCI 

penia, the remaining circulating neutrophils are significantly enriched with a 
subpopulation of neutrophils with poorly demonstable Fc receptors. Plasma 
obtained at this time during hemodialysis increased the adhesiveness and 
aggregation of control leukocytes (Klempner, Cotton). 

9. Hydrocortisone sodium succinate reversibly inhibits adherence of IgG 
sensitized erythrocytes to human peripheral blood neutrophil monolayers suggest- 
ing hydrocortisone interferes with the availability of the neutrophil Fc recep- 
tor for binding (Klempner) . 

10. Normal human neutrophils were separated into two populations based 
on their ability to rosette human IgG coated erythrocytes and tested for their 
to rosette complement-coated erythrocytes. Both neutrophil populations formed 
rosettes with complement-coated erythrocytes equally well. Moreover, neither 
population of cells displayed those complement receptors felt to be markers of 
immature granulocytes (Whited, Frank) . 

11. Administration of prednisone to normal human subjects inhibits in 
vitro neutrophil and eosinophil adherence to nylon wool. In vivo prednisone 
also inhibits eosinophil but not neutrophil chemo taxis (Fauci) . 

12. Collection of human neutrophils by filtration leukapheresis for sub- 
sequent intravenous administration results in functional changes of the neutro- 
phils attributable to degranulation and secretion of granule contents. Col- 
chicine pretreatment of filtration leukapheresis donors significantly reduces 
these adherence induced changes (Wright) . 

13. Human fibroblasts cultured in vitro secrete at least two chemoattrac- 
tants which, based on their elution from G-75 Sephadex chromatography columns, 
have molecular weights of greater than 150,000 and less than 5,000 daltons . 
These attractants appear distinct from collagen and other previsouly described 
chemotactic factors. They are protein in nature and attract both polymorpho- 
nuclear leukocytes and monocytes. Elaboration of this material in vitro is 
inhibited by colchicine (10 M) and hydrocortisone sodium succinate (1 mM) 
(Gallin) . 

14. In studies of patients with recurrent pyrogenic infections, a patient 
with abnormal neutrophil chemotaxis whose neutrophils are missing a membrane 
glycoprotein and impaired neutrophil spreading has been identified (Babior) . 

Significance to Biomedical Research and the Program of the Institute 

The accumulation of leukocytes at inflammatory, immune and allergic sites 
is critical for appropriate responses. Understanding the physiologic basis 
for events regulating these processes will provide the basis for therapeutic 
manipulation. 

One of the first steps in leukocyte mobilization from the blood stream is 
increased cell adhesiveness to the endothelium. This is followed by local 



20-8^ 



Project No. Z01 Al 00155-04 LCI 

leukocyte aggregation and then diapedesis. Our finding that limited degranu- 
lation and exocytosis of intracellular granules markedly enhances these events, 
together with the observation that human pyrogen initiates these processes, 
provides a clue as to what controls margination and then migration of leuko- 
cytes from the blood stream. The related observation that vigorous degranu- 
lation in vitro inhibits chemotaxis suggested that some acquired chemotactic 
defects relate to excessive degranulation in_ vivo . In support of this is our 
finding that following thermal injury the severity of the acquired chemotactic 
defect is linearly related to the amount of intracellular lysozyme released. 
Clinically, it is of interest that the degranulation and defective chemotaxis 
precedes the severe and often lethal pyrogenic infections that follow thermal 
injury. 

In related studies we confirmed that neutrophil specific granules contain 
lactoferrin and showed this is secreted when cells are incubated with pyrogen. 
Lactoferrin irreversibly binds iron which is then sequestered in the reticu- 
loendothelial system. Another specific granule component, B „ binding protein, 
may effect B „ related events when secreted from the neutrophil. Thus, the 
potential regulatory role of neutrophil products on a number of systems is 
interesting and currently under investigation. 

Two observations from our studies of the mechanism of the phenomena of 
degranulation, cell adhesiveness and chemotaxis may be particularly important 
to the cell biology of chemotaxis and perhaps relevant to clinical studies as 
well. We have shown that leukocyte adherence, aggregation and secretion may 
be under the modulating influence of electrostatic forces. Development of 
techniques for controlling these forces in vivo may have potential clinical 
use. Our data that neutrophil specific granules are a potential source of new 
cytoplasmic membrane and chemoattractant receptors is intriguing in terms of 
understanding the basis for membrane turnover during chemotaxis. These studies 
need to be extended and explored further. 

The studies of the effect of steroids on PMNs are interesting and have 
shown that prednisone iri vivo inhibits both neutrophil and eosinophil adherence 
and eosinophil chemotaxis. Hydrocortisone sodium succinate also blocks the 
expressability of neutrophil Fc receptors and inhibits chemotactic factor induced 
changes of neutrophil and macrophage membrane potential. These data contribute 
to our understanding of the physiologic basis for steroid action on these cells. 

The use of fluorescent carbocyanine dyes to measure membrane potential is 
emerging as a rapid test of cell reponsiveness that appears to be as reliable 
and easier to use than other indirect probes of membrane potential. The demon- 
stration of a severe abnormality of chronic granulomatous disease neutrophils 
using this test may provide a new simple rapid diagnostic test for this disease. 
In addition, the implications that the observed abnormality in chronic granu- 
lomatous disease reflects abnormal ion flux studies, perhaps related to abnormal 
activation mechanisms provides new insights into the nature of the defect. 

The continuation of our studies of neutrophil subpopulations has indicated 
that one population of cell (PMNs with readily demonstrable Fc receptors) is 

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Project No. Z01 Al 00155-04 LCI 

particularly available for margination. We are presently extending these studies 
of neutrophil subpopulations to explore their kinetics of circulation, and 
their functional role. In addition, the effect of various disease states and 
pharmcologic agents on these two neutrophil populations in man and in animals 
is under study. 

Prosposed Course 

We plan to continue some of the studies outlined above. 

Publications 

1. Wright, D. G., Kauffmann, J. C, Terpstra, G. K., Graw, R. G., 
Deisseroth, A. B., and Gallin, J. I. Mobilization and exocytosis of 
specific (secondary) granules by human neutrophils during adherence to 
nylon wool in filtration leukapheresis . Blood . 52:770-782, 1978. 

2. Wright, D. G., Ungerleider, R. S., Gallin, J. I., and Deisseroth, A. B. 
Pretreatment of leukaphersis donors with colchicine. Blood . 52:783- 
792, 1978. 

3. Gallin, J. I., Wright, D. G., and Schiffmann, E. Role of secretory 
events in modulating human neutrophil chemotaxis. J. Clin. Invest. 
62:1364-1374, 1978. "~ " 

4. Klempner, M. S. and Gallin, J. I. Inhibition of neutrophil Fc 
receptor function by corticosteroids. CI in . Exp . Immunol . 34:137- 
143, 1978. 

5. Gallin, J. I., Elin, R. J., Hubert, R. T., Fauci, A. S., Kaliner, M. A., 
and Wolff, S. M. : Efficacy of ascorbic acid in the Chediak-Higashi syn- 
drome: Studies in humans and mice. Blood . 53:226-234, 1979. 

6. Clark, R. A. F., Gallin, J. I. and Fauci, A. S. Effects of in vivo 
prednisone on in vitro eosinophil and neutrophil adherence and chemo- 
taxis. Blood . 53:633-641, 1979. 

7. Sobel, J. D. and Gallin, J. I.: Polymorphonuclear leukocyte and 
monocyte chemoattractants produced by human fibroblasts. J. Clin. 
Invest. 63:609-618, 1979. 

8. Gallin, J. I. The Compromised Host. In P. B. Beeson and W. McDermott 
(Eds.): Textbook of Medicine, 15th Edition , pp. 145-153, 1979. 

9. Wright, D. G. and Gallin, J. I. Secretory responses of human neutro- 
phils: Exocytosis of specific (secondary) granules by human neutrophils 
during adherence in vitro and during exudation in vivo . J . Immunol . 
123:285-294, 1979. , 



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Project No. Z01 Al 00155-04 LCI 

10. Cramer, E. B. and Gallin, J. I.: Localization of submembranous 
cations to the leading end of human neutrophils during chemotaxis. 
J. Cell Biol. In Press. 

11. Gallin, J. I., Gallin, E. K., and Schiffmann, E. The Mechanism of 
Leukocyte Chemotaxis. In Proceedings of the International Congress of 
Inflammation . Raven Press, In Press. 

12. Gallin, E. K., Seligmann, B. and Gallin, J. I. Alteration of macrophage 
and monocyte membrane potential by chemotactic factors. In R. Van Furth 
(Ed.): Mononuclear Phagocytes . Martinus Nijhoff B. V., The Hague. 

In Press. 

13. Seligmann, B. and Gallin, J. I. Secretagogue modulation of the response 

of human neutrophils to chemoattractants : studies with a membrane potential 
sensitive cyanine dye. Molecular Immunology . In Press. 

14. Whited, S. C. and Gallin, J. I. Neutrophil chemotaxis . Interna. 
J. Dermatol. In Press. 

15. Davis, J. M. and Gallin, J. I. The Neutrophil . The Cell Biology of 
Immunity and Inflammation . Ed. by Oppenheim, J. J., Rosenstreich, D. L. 
and Potter, M. Elsevier North-Holland. In Press. 

16. Klempner, M. S., Dinarello, C. A., Henderson, W. R. and Gallin, J. I. 
Stimulation of neutrophil oxygen-dependent metabolism by human leuko- 
cytic pyrogen. J. Clin. Invest. In Press. 

17. Gallin, J. I. The Cell Biology of Leukocyte Chemotaxis. In. 
G. Weissmann Ed. Handbook of Inflammation . New York, Elsevier 
North Holland. In Press. 

18. Schiffmann, E. and Gallin, J. I. Biochemistry of Phagocyte Chemotaxis. 
In E. R. Stadtman Ed. Cellular Regulation . New York, Academic Press. 
In Press. 



20-85 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl AI 00188-01 LCI 



PERIOD COVERED 



October 1, 1978 to September 30, 1979 



TITLE OF PROJECT 



characters or less 



Rapid Diagnosis of Infections by Enzyme-Linked Immunoadsorbent Assays 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Stephen E. Straus 
Other: Richard A. Berg 



Senior Investigator 
Medical Virology Section 

Clinical Associate 



LCI NIAID 



COOPERATING UNITS (if any) 

J.E. Bennett (LCI, NIAID), R. Brown (LCI, NIAID), P. Pizzo (LPO, NCI), 
B. Murphy (LID, NIAID), and S. Rennard (LDBA, NIDR) 



LAB/BRANCH 



Laboratory of Clinical Investigation 



Medical Virology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARSi 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
□ (a) HUMAN SUBJECTS 

C (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Enzyme-linked immunoadsorbent assays have been developed to detect influenza A 
hemagglutinin, Candida cell wall mannan, human adenoviruses, herpes simplex 
virus, and antibodies to pneumococci. Current efforts are designed to augment 
the sensitivity of the assays and establish their utility in clinical and 
research applications. 



20-86 



PHS-6040 
(Rev. 10-76) 



Project No. Z01 AI 00188-01 LCI 
Project Description : 
Objectives : 

1) To develop an assay which will rapidly, simply and reliably detect 
Candida antigenemia, with the aim of identifying those patients who may 
benefit from early therapy with amphotericin B. 

2) To develop a rapid assay for influenza infection in order to iden- 
tify individuals and patient groups which may benefit from amantadine 
chemoprophylaxis . 

3) To develop rapid, more sensitive assays to detect adenoviruses and 
herpes viruses in body fluids and in research specimens. 

4) To develop a convenient assay for the development of antibodies 
directed against specific strains of pneumococci. 

5) To detect group A streptococcal polysaccharide in throat swabs of 
patients with pharyngitis. The rapidity of the test would eliminate the 
1-2 day wait for identification by routine microbiologic methods. 

Methods Employed : 

Either direct or competitive assays are employed. Briefly, the desired 
antigen is adsorbed to the surface of plastic microtiter wells. Specific 
antisera is added and binds to the antigen where present. Development of 
the antigen-antibody reaction is detected by addition of a second enzyme- 
linked antibody directed at the first antibody. The reaction is detected 
colorimetrically by addition of a chromogenic substrate specific for the 
enzyme. 

Major Findings : 

1) Candida: An ELISA has been developed which detects mannan at the 
nanogram level. A limited retrospective clinical trial has been completed. 
Briefly, sera of patients with proven disseminated candidiasis and those 
without candidiasis were tested in a blinded fashion. The assay discerned 
significant differences in level of Candida mannan between the groups. 

2) An ELISA capable of detecting less than 1 hemagglutinin unit of 
influenza A virus has been developed. The assay is currently being tested 
in experimental infections in ferrets. 

3) Adenoviruses: An ELISA has been developed to reliably detect all 
of 26 types of human adenoviruses tested thus far. Modifications of the 
assay are being attempted which would permit virus typing. 

4) Herpesviruses: ELISA tests for both types of herpes simplex are 
being developed using monoclonal type specific antisera. 



20-87 



Project No. Z01 AI 00188-01 LCI 

5) Pneumococci: A successful assay is currently being tried in 
experimental pneumococcal infection of guinea pigs. A pilot study has been 
completed in which the ELISA was used to quantitate the antibody response to 
pneumococcal polyvalent vaccine in six volunteers. The assay results cor- 
related very highly with results obtained by standard radioimmunoassay. 

6) Streptococci: An assay using monoclonal specific antisera is being 
developed. The test thus far is insufficiently sensitive. 

Significance to Biomedical Research and the Program of the Institute : 

Rapid assay for a variety of antigens and antibodies has broad appli- 
cability to both clinical and research areas. The tests currently being 
developed will speed up, simplify, and reduce the cost of detecting infec- 
tions. At the same time, they will permit clinical decisions regarding 
therapy to be soundly based at an earlier time in the course of an illness 
than is currently practiced. 

Proposed Course : 

Once the ELISA tests are fully developed and their sensitivity and 
specificity defined, they will be examined prospectively with patient ma- 
terial. Their ability to correctly identify infectious agents will be 
tested by comparison with the results of traditional methods. Assays for 
viral agents will also be applied to routine testing of research materials 
generated in the study of virus pathogenesis and treatment. 

Publications : 

1. Weiner, M.H., Yount, W.J.: Mannan antigenemia in the diagnosis of 
invasive Candida infections. J_. Clin . Invest . 58:1045, 1976. 

2. Voller, A., et al: Microplate enzyme immunoassays for the immuno- 
diagnosis of virus infections. In N.R. Rose and H. Friedman (eds.) 
Manual of Clinical Immunology . ASM, Washington, D.C., 1976, p 506. 

3. Yolken, R.H., et al: Measurement of rotavirus antibody by an enzyme- 
linked immunoadsorbent assay blocking assay. ^J. Clin . Microb . 8:283, 
1978. 

4. Segal, E., Berg, R.A., et al: Detection of Candida antigens in sera of 
patients with candidiasis by an ELISA test. J_. Clin - Microb. In press. 

5. Berg, R.A. , et al: Type-specific pneumococcal antibody measurement with 
enzyme-linked immunoadsorbent assay. In preparation. 



10-88 



SMITHSONIAN SCIENCE INFORMATION EXCHANC 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00189-01 LCI 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Clinical and Biochemical Studies of Human Enteral Adenovirus 
Infections 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



S.E. Straus 



Senior Investigator 
Medical Virology Section 



LCI NIAID 



COOPERATING UNITS (if any) 

A.Z. Kapikian (LID, NIAID) 

H.S. Ginsberg (Columbia University) 



LAB/BRANCH 

Laboratory of Clinical Investigation 



Medical Virology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 



PROFESSIONAL: 
1 



CHECK APPROPRIATE BOX(ES) 
8 (a) HUMAN SUBJECTS 



S (b) HUMAN TISSUES 



(c) NEITHER 



□ (a1 ) MINORS 



a2) INTERVIEWS 



SUMMARY OF WORK (200 words or less - underline keywords) 

Enteral adenoviruses are a recently defined group of agents which cause gastro- 
enteritis in children. They differ from other known human adenoviruses in their 
inability to be propagated in tissue culture. Adenoviruses recovered from pa- 
tients will be examined for their relation to known respiratory adenoviruses by 
serologic, DNA hybridization, restriction endonuclease and protein electrophor- 
esis studies. The mechanism of restriction of growth of these agents will be 
examined by nucleic acid hybridization, immuno fluorescent microscopy and AAV 
helper studies. Attempts will be made to grow the viruses in tissue culture 
using adenovirus transformed cell lines and a series of early and late tempera- 
ture sensitive mutants of adenovirus 5 as helpers. Enteral adenovirus-induced 
diarrheal illness will be studied in normal volunteers. The features of the 
illness, virus shedding and immune resolution will be examined. 



20-89 



PHS-6040 
(Rev. 10-76) 



Project No. Z01 AI 00189-01 LCI 
Project Description : 

Objectives : 

1) To define the clinical spectrum of illness induced by enteral adeno- 
viruses in both naturally occurring and experimental infections. 

2) To define the biochemical properties of these adenoviruses and their 
relation to other respiratory adenoviruses. 

3) To develop methods of promoting the growth of these agents in tissue 
culture so that further studies will be simplified. 

Methods Employed : 

1) Source of viral agents : Enteral adenoviruses will be obtained from 
specimens collected during natural infections at Children's Hospital and 
identified as enteral adenoviruses by their EM appearance, reactivity in 
ELISA tests, and their fastidious behavior in standard tissue culture lines. 

2) In vitro culture system : Standard primary and continuous human and 
simian, adult and fetal tissues will be employed for attempted virus isola- 
tion. A variety of methods will be employed to promote the efficient repli- 
cation of these viruses in tissue culture. Stool specimens will be inocu- 
lated into an adenovirus transformed cell line (293) with the hope that the 
integrated viral sequences will complement the presumably restricted func- 
tions of the enteral viruses. Similar complementation studies will be at- 
tempted using early and late temperature-sensitive mutants of adenovirus 
type 5 which have been developed at Columbia University. Simian cells will 
be coinfected with SV40 and enteral adenoviruses to determine whether the 
helper function that SV40 provides for respiratory adenoviruses in monkey 
cells extends to these agents as well. 

3) Biochemical Studies : The mechanism of restricted growth in tissue 
culture will be examined by performing DNA-DNA and DNA-RNA hybridizations on 
infected cell extracts using in vitro labeled DNA probes. Immunofluorescence 
microscopy will indicate whether adenovirus T antigen or late proteins are 
being synthesized in the restricted system. The expression of early adeno- 
virus function will be assessed by testing for helper activity for adeno- 
associated viruses. 

4) Clinical Studies : After rigorous safety testing, enteral adenovirus- 
containing stool filtrates will be innoculated into normal adult volunteers. 
The ability of these agents to reproduce the typically mild diarrheal illness 
will be explored. The illness will be characterized by close clinical obser- 
vation, virologic and serologic studies of blood, stool and respiratory 
secretions, and electron microscopic examination of stools. 



20-90 



Project No. Z01 AI 00189-01 LCI 
Major Findings : 

Our laboratory has to date developed the tools necessary to perform 
these studies. We are now proficient with the maintenance of the required 
cell lines, the growth and replication of respiratory adenoviruses, labeling 
and extraction of viral nucleic acids and proteins, nucleic acid hybridiza- 
tion, restriction endonuclease analysis, SDS-PAGE, immunof luorescent micros- 
copy, HA, HI, neutralization and ELISA tests. We are currently awaiting our 
first stool specimen. 

Proposed Course : 

Detailed investigation of the viral pathogenesis and host defense 
mechanisms will depend upon our ability to generate substantial quantities 
of virus and reproduce the clinical illness in volunteers. Since the other 
major viral diarrhea agents (rotaviruses, parvovirus-like agents) can not be 
grown in the laboratory, our success with these enteral adenoviruses may 
provide the model for the study of human viral gastroenteritis. 

Publications : 

1. Flewett, T.H., et al.: Epidemic viral enteritis in a long-stay 
children's ward. Lancet 1:4, 1975. 

2. Richmond, S.J., et al.: An outbreak of gastroenteritis in young 
children caused by adenoviruses. Lancet 1:1178, 1979. 

3. Dolin, R. : Viral infections of the gastrointestinal tract. In 
G.J. Galasso, et al. (eds.) Antiviral Agents and Viral Diseases . 
Raven Press, New York, N. Y. , 1979, p. 289. 

4. Wadell, G. : Classification of human adenoviruses by SDS-polyacrylamide 
gel electrophoresis of structural polypeptides. Intervirology 11:47, 
1979. ' 



20-91 



LABORATORY OF IMMUNOGENETIGS 

1979 Annual Report 

Table of Contents 



Z01-AI 



Project Number Page 

Summary Report 21-1 

00166-02 Chemical Characterization of Rabbit 21-5 

Histocompatibility Antigens 

0016 7-02 Mass Spectrometry and Sequence Analysis 21-8 

of Polypeptides 

00168-02 Structure of Rabbit Immunoglobulin and 21-11 

Antibody Heavy and Light Chains 

00169-02 Primary Structural Analysis of Murine 21-13 

Transplantation Antigens 

00170-02 Structural Studies on Precursors to 21-17 

Immunoglobulin Molecules 

00171-02 Genetic Studies on Rabbit Immunoglobulins 21-19 
and Other Serum Proteins 

00172-02 Carbohydrate and Glycoprotein Antigens of 21-22 
Microbial Cell Walls 

00173-02 Nonallelic Expression of Genes Encoding Rabbit 21-24 
Immunoglobulins 

00180-01 Monoclonal Antibodies Directed Against Cell 21-27 
Surface Proteins 

00191-01 Immunoregulation of T Lymphocytes - The Role 21-29 
of Anti-idiotype and Immune Complexes 



Annual Report of the Laboratory of mmunogenetics, National Institute of 
Allergy and Infectious Diseases, NIH, October 1, 1978 - September 30, 1979 

Summary Report 

The Laboratory of Immunogenetics continues to conduct research aimed 
at elucidation of the genetic basis of immunologic function. Studies on 
genetic markers of immunoglobulins have concerned the expression of latent 
allotypes. In the past year progress has been made on structural 
determination of molecules bearing latent allotypes and more recently, 
interspecies cell hybrids (mouse-rabbit) are being utilized as a source of 
mRNA which will be used to initiate molecular biological studies which 
hope to elucidate the genetic basis for latent allotypes. Studies on the 
structure of histocompatibility molecules continue; the 280 residue papain 
fragment of the murine K molecule is near completion and intense 
structural analysis of molecules from this and other mouse MHC haplotypes 
have begun. In additon, progress has been made on studies of rabbit 
histocompatibility antigens this year. Other areas of active interest 
include studies of cell surface molecules using hybridoma reagents which 
have been applied with some effectiveness in our H-2 studies. Initially 
the hybridoma antisera will be directed against T-cells from the rabbit. 
Work on the control of the immune response by T-cell components has 
recently been initiated and this will involve a chemical and biological 
approach to this problem. 

Recent additions to the LIG staff include Dr. Lee Maloy a Biochemist 
from Case Western Reserve University and Dr. Thomas Folks who joins us 
from the Naval Medical Research Unit in Bethesda. Dr. John Sogn has been 
given the position of Chemist, GS-13 after serving a year and one-half as 
Senior Staff Fellow in the Laboratory. Dr. Blair Fraser, a Staff Fellow 
left to accept a permanent position with the Bureau of Biologies. In 
addition to the permanent personnel our program has been greatly enriched 
by participation of students from various part-time and summer programs. 

Research Accomplishments 

Rabbit Histocompatibility Antigens . Products of the rabbit 
histocompatibility genes were isolated from a rabbit lymphoid tumor cell 
line (RL-5) following growth in culture with radioactive amino acids. 
Molecules were obtained from detergent lysates and glycoprotein fractions 
were isolated by affinity chromatography using immobilized lentil lectin. 
Chromatography on purified sheep anti-rabbit beta-2-microglobulin yielded 
a 43,000 dalton fraction which was noncovalently associated with 
beta-2-microglobulin. Amino terminal sequence analysis allowed assignment 
of 28 of the amino terminal 35 residues. These data revealed 89% homology 
between this RLA-11 product and the human HLA-b7 molecule. Similar 
comparison with the mouse H-2K histocompatibility antigen yielded a 
homology of 82%. Although, beta-2-microglobulin is associated with 
histocompatibility antigens of several types (K,D, and L in the mouse) in 
other species only a single molecular species could be detected in our 
studies even though beta-2-microglobulin was the basis upon which our 
molecule was isolated. The extent of similarity between the rabbit and 
other histocompatibility cmplexes is currently under investigation. 

21-1 



Mass Spectrometric Analysis of Peptides . Immunogenetic studies require 
fast, reproducible and sensitive methods for amino acid sequence analysis 
of proteins which are not intrinsically radiolabelled. One possibility to 
attain this goal is mass spectrometric-gas chromatograhic (MS-GC) 
techniques. Work in the past year has included studies on a method which 
involves digestion of polypeptides into dipeptides from the amino terminus 
and identification of these dipeptides by computer aided MS methodology. 
More recently, the use of an enzyme dipeptidyl carboypeptidase (DCP) , has 
been investigated for use with these techniques. This enzyme cleaves 
dipeptides from the carboxy terminus. It is anticipated that this enzyme 
will be extremely useful either as an adjunct to the DAP digestions, or as 
a method to complement normal Edman degradation. Model compounds under 
study at present include rabbit and mouse Ig heavy chains as well as rat 
beta-2-microglobulin. 

Murine Transplantation Antigens . Gene products from murine major 
histocompatibility locus are involved in diverse functions which are 
associated with immune recognition and reaction. Our structural studies 
thus far, have dealt with classical transplantation antigens encoded at 
the K and D loci. Studies using radiochemical methodology have allowed 
assignment of nearly 280 residues to the K glycoprotein molecule. Data 
indicate a high degree of homology to human histocompatibility antigens 
and furthermore show that the differences between these molecules are not 
spread over the molecule but rather cluster in discrete areas. In 
addition to the K molecule, primary structural analysis is being carried 
out on the D , D and L molecules. In addition, the determination of the 
primary structure of murine beta-2-microglobulin, a molecule found in 
association with major histocompatibility antigens, is near completion. 
It is anticipated that knowledge of the primary structure of these 
molecules will aid in an understanding of their function and mechanism of 
action. The major histocompatibility antigens are deeply involved in 
allograft rejection and in the response of T lymphocytes to altered cell 
surface antigens. 

Precursors of Immunoglobulin Molecules . When certain protein products are 
formed by _in vitro translation using mRNA the products yielded are larger 
than those found for the normal secreted products. The amino terminus of 
certain of these proteins may be extended by as many as 30 amino acids. 
These precursor sequences have been reported for immunoglobulin light and 
heavy chains as well as a number of hormones and secreted enzymes. The 
present studies are directed toward a study of the immunoglobulin 
precursor molecules of the rabbit. This study will attempt to correlate 
the variable region extension with the constant region allotypes of the 
rabbit L chains. The source of mRNA, which in the past has proved 
troublesome, will be hybrid cell lines which secrete a single heavy or 
light chain of the rabbit. 

Rabbit Latent Allotypes . Rabbit immunoglobulin allotypes are antigenic 
determinants which were thought to be inherited as autosomal codominant 
allele. This notion has been challenged by observations of low 
concentrations of allotypes which were not detected by qualitative tests 



21-2 



nor predicted by parental genotypes. Detailed structural analyses have 
shown that latent allotypes isolated from the sera of pedigreed rabbits 
are indistinguishable from the normal allotypes. Current research focuses 
on hybridoma cells maintained in culture that are able to synthesize 
chains with rabbit allotypes. RNA which encodes the immunoglobulin chains 
secreted by these cells will be isolated and cDNA probes will be prepared 
in order to carry out molecular biological studies aimed at elucidation of 
the basis for inheritance of latent allotypes. Other studies in this area 
have included measurement of the clearance of normal and latent allotypes 
in animals using doubly radiolabelled material. Other genetic studies in 
this area have concentrated on the linked expression of V markers of 
immunoglobulin expressing latent allotypes. 



21-3 



Honors and Awards 

Dr. Kindt serves on the editorial boards of the Journal of 
Immunology , Proceedings of the Society for Experimental Biology and 
Medicine the Zeitschrift fur Immunotatsforschung , the Journal of 
Experimental Medicine and Molecular Immunology . He serves as a member of 
the program committee of the American Association of Immunologists and was 
elected this year to the American Society for Biological Chemists. Dr. 
Kindt was invited to speak at the Josiah Macy Foundation Conference on 
membranes and human disease which was held in New Orleans and the 
Institute Pasteur Conference on allotypes and idiotypes which was held in 
Paris. Seminars of laboratory work were presented by Dr. Kindt at the 
University of North Carolina at Chapel Hill, at New York University 
Medical School in New York, at Scripps Clinic and Research Foundation in 
La Jolla, at the Molecular Immunology Institute at the University of 
Marseille, at the Institute for Genetics at the University of Koln and at 
the German Cancer Research Institute in Heidelberg. 

Dr. Coligan presented his recent data on the structure of mouse 
histocompatibility antigens in seminars at the Mayo Clinic in Rochester, 
Minnesota and at Harvard University. He also presented data at a 
symposium on T and B lymphocytes held at Keystone, Colorado and at the 
International Congress of Biochemistry in Toronto, Canada. 

Martin Yarmush received his Ph.D. degree from Rockefeller University 
this Spring and was accepted at a number of medical schools. He will 
attend Yale University Medical School in the Fall. 



21-4 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00166-02 LIG 



PH bM V e E r RH l, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Chemical Characterization of Rabbit Histocompatibility Antigens 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Dr. Thomas J Kindt 

Others: Dr. Edward S. Kimball 

Dr. John E. Coligan 

Dr. Frederick. T. Gates 



Chief, LIG LIG NIAID 

Staff Fellow LIG NIAID 

Research Microbiologist LIG NIAID 

Staff Fellow LIG NIAID 



COOPERATING UNITS (if any) 

Dept. of Genetics, University of Illinois at Chicago (Dr. R. Tissot) 



lab/branch 

Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 
1.5 



PROFESSIONAL: 

0.9 



0.6 



CHECK APPROPRIATE BOX(ES) 
Q (a) HUMAN SUBJECTS 

□ (a1 ) MINORS Q (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



XX(=) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

This laboratory has been engaged in the isolation and purification of 
histocompatibility antigens of the rabbit . There are approximately 12 
serologically defined haplotypes in the domestic rabbit and our study has 
concentrated on the RLA 11 haplotype. The major source of antigen has 
been a tumor cell line derived from the Bar Harbor B strain rabbit. 
Antigen isolation was carried out by detergent solubilization of cells 
labelled with tritiated amino acids . Partial amino acids sequence 
analysis was carried out on the histocompatibility antigen isolated and it 
revealed a high degree of homology especially to histocompatiblity 
antigens isolated from humans. Beta-2 microglobulin , a low molecular 
weight protein that is associated with histocompatibility antigens, was 
subjected to total amino acid sequence analysis. Although this protein is 
homologous to analogs from other species some significant differences were 
observed in its structure. 



21-5 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00166-02 LIG 

Chemical Characterization of Rabbit Histocompatibility Antigens 

Graft rejection and control of the ability to respond to specific 
antigens are directed by products of genes residing in the major 
histocompatibility complex. Little is known about the structure and 
function of the major histocompatibility gene products although 
preliminary work, has been reported for proteins from the mouse and human. 
It is our intention to study the rabbit as a third species in order to 
compare MHC gene products structures. There are about twelve 
serologically defined MHC haplotypes in the domestic rabbit. 

Current work has concentrated on the RLA-11 haplotype. The major 
source of antigen has been a tumor cell line derived from the Bar Harbor B 
strain of rabbits. Antigen isolation has been carried out by detergent 
solubilization of cell membranes that were intrinsically labeled with 
tritiated amino acids. A glycoprotein fraction was isolated by lentil 
lectin chromatography and the histocompatibility antigen isolated from 
this by affinity chromatograhy on a column of highly purified antibodies 
directed against beta-2 microglobulins. Amino terminal amino acid 
sequence analysis using radiochemical methods allowed the assignment of 28 
of the amino terminal 35 residues. The data obtained revealed 89% 
homology of the RLA-11 protein with the human HLA B7 and 82% with the 
mouse H-2K . Comparison of the RLA-11 to sequence data obtained from 
major histocompatibility antigens of other species revealed no 
substitutions unique to the rabbit antigen. 

The complete amino acid sequence of rabbit beta-2 microglobulin has 
been determined and compared to the sequence reported for human 
beta-2 microglobulin. This comparison showed a homology of 71% with a 
minimum of 13% difference in nucleotide sequences of genes encoding the 
two proteins. However, a single insertion must be introduced before 
position 68 of the rabbit protein to maintain this maximum homology. 
Amino acid substitutions are distributed throughout the molecule. 
Although the majority of those requiring multiple base changes are 
restricted to the carboxy-terminal third of the molecule. Although the 
rabbit protein analyzed in this study was isolated from the pooled urine 
of 15 rabbits no heterogeneity in amino acid sequence was observed. 

Publications 

Kimball, E.S., Coligan, J.E., and Kindt, T.J.: Structural 
characterization of antigens encoded by rabbit RLA-11 histocompatibility 
genes. Immunogenetics 8: 201-211, 1979. 

Kindt, T.J., Coligan, J.E., Kimball, E.S., Ewenstein, B., Uehara, H. , 
Martinko, J., and Nathenson, S.G. : Use of radiochemical techniques for 
primary structural analysis of mouse and rabbit histocompatibility 
antigens. Proceedings of the Josiah Macy Foundation , in press, 1979. 



21-6 



Z01 AI 00166-02 LIG 

Gates, III, F.T., Coligan, J.E., and Kindt, T.J.: The complete amino acid 
sequence of rabbit beta-2-microglobulin. Biochemi stry 18: 2267-2272 
1979. 



21-7 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION. AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00167-02 LIG 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Mass Spectrometry and Sequence Analysis of Polypeptides 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 

Others: 



Dr. Henry C. Krutzsch 
Dr. Thomas J. Kindt 



Sr. Staff Fellow 
Chief, LIG 



LIG NIAID 
LIG NIAID 



COOPERATING UNITE 

None 



lab/branch 
Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

1.9 



PROFESSIONAL: 

0.9 



1.0 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS 



□ (b) HUMAN TISSUES 



□ 



MINORS Q (a2) INTERVIEWS 



SUMMARY OF WORK (200 words or less - underline keywords) 

A major need in immunogenetic studies are fast, reproducible and 
sensitive methods for amino acid sequence analysis of proteins. 
Mass spectrometric (MS) techniques provide a promising means of meeting 
tnese goals. Methods under study involve enzymatic digestion of 
polypeptides into dipeptides using either dipeptidyl aminopeptidase or 
dipeptidyl carboxypeptidase (DAP/DCP method) and use of MS for rapid 
identification of dipeptides produced. Another employs direct introduction 
of larger derivatized peptides into the MS for analysis (DI method) . 
Computer assisted data analysis programs for both MS methods are under 
development . 

The DAP/DCP and DI methods are being tested using rabbit and mouse 
heavy chains as model proteins because these proteins are not readily 
amenable to routine sequence analysis techniques. The structure of 
rat g-2 microglobulin is also under study using this method. 

21=3 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00167-02 LIG 

Mass Spectrometry and Sequence Analysis of Polypeptides 

It is the aim of this program to develop rapid and sensitive methods 
for polypeptide sequence analysis and to apply these methods to molecules 
of immunogenetic interest. Development of this method will include 
devising strategies for isolaton and purification of peptides in amounts 
sufficient for characterization. These techniques will facilitate 
polyeptide sequence analysis, helping to determine more rapidly the 
primary structures of immunogenetically important molecules such as 
antibodies and histocompatibility antigens. 

An LKB Model 2091 EI-CI gas chromatograph-mass spectrometer (GC-MS) 
was used to identify trimethylsilylated (TMS) dipeptides from dipeptidyl 
peptidase (DP) digestion of polypeptides. Two MS ions are necessary for 
identification; the sequence-determining ion resulting from loss of the 
C-terminal residue plus the N-terminal residue's carbonyl group, and the 
molecular weight-determining ion. Alignment of the dipeptides to give the 
polypeptide sequence is done by using the dipeptides from a second DP 
digestion of the polypeptide after addition of another amino acid residue, 
or by homology to a similar peptide of known structure. 

Some more areas of the DP/GC-MS method have been investigated. The 
utilization of dipeptidyl carbonsypeptidase (DCP) , in this case 
Angiotensin-I converting enzyme, in place of DAP for sequence analysis 
from the C-terminus (instead of the N-terminus) of the polypeptide has 
been one of these. Use of this type of enzyme system will provide 
sequence information that may not be available from Edman chemistry (or 
DAP digestion) due to a blocked N-terminus, or to inability to go 
completely to the C-terminus in peptide with an unblocked N-terminus. At 
the present time, no totally reliable technology exists for such an 
operation. 

In this area, three sections were investigated; enzymology, scope of 
digestion, including available sensitivity and associated chemistry. The 
enzymology was studied to determine what enzyme concentration, buffer 
conditions and associated miscellaneous conditions, such as digestion 
times, which gave maximum dipeptide yields. The enzyme system was also 
checked for the presence of materials that would interfere with the 
subsequent GC MS analysis, such as contaminating proteases or components 
that would create spurious GC peaks. The scope of the digestion was 
studied to determine what types and lengths of polypeptides could be 
digested, as well as the amount, in nmoles, of polypeptide that could be 
determined. The chemical studies were mainly concerned with determining 
and optimizing (and carrying out) the best method for obtaining the 
"overlapping" peptide to provide the necessary alignment information for 
the dipeptides from DCP digestion of the native polypeptide. 

The results obtained up to the present indicate that the method has 
broad applicability. Thus, this system has been applied to peptides 
containing all types of amino acids, with chain lengths of up to greater 
than 50 residues, and at levels less than 5 nmoles. 

21-9 



Z01 AI 00167-02 LIG 

Four computer programs, written in Fortran- IV, have been almost 
completely developed to allow rapid manipulation of the GC-MS DP digestion 
data. Two of these programs are for dipeptide identification, one a 
manual system, one an automatic system. The other two are for dipeptide 
alignment, either from the N-terminus (DAP digestion) or from the 
C-terminus (DCP digestion) . 

Two proteins are currently under study with these enzyme systems; the 
N-terminal 225 residue fragment resulting from CNBr cleavage of a rabbit 
(??4135, a3 allotype) homogeneous antibody heavy chain, and rat B-2 
microglobulin. All of the work, up to the present time has been with the 
DAP system, using as substrates peptides from both restricted (Arg 
cleavage only) and total tryptic cleavage of the rabbit H-chain CNBr 
fragment. Several partial or total sequences have been obtained for these 
peptides. Because several of these tryptic fragments are blocked at the 
N-terminus due to glutamine-pyroglutamate coversion, the DCP enzyme will 
be a useful addition to the present technology. 

Publications 

Krutzsch, H.C. and Pisano, J. Preparation of dipeptidyl aminopeptidase IV 
for polypeptide sequencing. Biochem. Biophys. Acta. 576: 280-289, 1979. 

Krutzsch, H.C. and Kindt, T.J. The identification of trimethylsilylated 
dipeptides with chemical ionization mass spectrometry. Analytical 
Biochemistry 92: 525-531, 1979. 

Rao, D.N., Rudikoff, S. , Krutzsch, H. , and Potter, M. Structural evidence 
for independent joining region gene in immunoglobulin heavy chains from 
anti-galactan myeloma proteins and its potential role in generating 
diversity in complementary-determining regions. Proc. Nat. Acad. Sci. 76: 
2890-2894, 1979. 

Keeton, T.K., Krutzsch, H.C, and Lovenberg, W. A specific 
radioimmuneassay for 3-methoxy-4-hydroxyphenyl-ethylene glycol (mopeg) . 
Proc. 4th International Catecholamine Symposium., Pergamon Press, 1978. 

Yarmush, M.L., Krutzsch, H.C, and Kindt, T.J. Amino acid sequence 
analysis of immunoglobulin light cains by gas chromatographic-mass 
spectrometric techniques: structural identity of latent b9 and nominal b9 
molecules. Molecular Immunology , in press, 1979. 



11-10 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Oo NOT use this sDace) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00168-02 LIG 



P TOoTO EC L, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Structure of Rabbit Immunoglobulin and Antibody Heavy and Light Chains 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, ANO TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Dr. Thomas J. Kindt 
Others: Dr. John A. Sogn 

Dr. Henry Krutzsch 



Chief 

Chemist 

Sr. Staff Fellow 



LIG NIAID 
LIG NIAID 
LIG NIAID 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 

Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

1.0 



PROFESSIONAL: 

0.3 



0.7 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



XX(c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Homogeneous rabbit antibodies to streptococcal carbohydrates , are 
used to investigate structural aspects of the allotypic and 
idiotypic markers of immunoglobulins . The partial amino acid sequence was 
determined for a rabbit light chain bearing latent b9 allotype . In 
addition, structural work on a rabbit heavy chain bearing the a3 allotype 
is presently under way. 

Studies such as these that combine serological and structural 
investigations of antibodies have the potential to define genetic 
mechanisms involved in antibody synthesis . 



21-11 



PHS-6040 
(Rev. IO-76) 



Z01 AI 0U168-02 LIG 

Structure of Rabbit Immunoglobulin and Antibody Heavy and Light Chains 

The structural aspects of serologically detected polymorphic forms of 
rabbit immunoglobulins will aid in understanding the genetic mechanisms 
involved in antibody synthesis. Study of the covalent structure of 
immunoglobulins involves both separation of the protein into component 
peptides and amino acid sequence analysis of these materials. Separation 
of heavy and light chais from rabbit IgG is a standard procedure, but 
structural analysis of the components must be treated in a unique manner 
depending on the chain and its allotypic specificities. During the past 
year we have been determining structural features of light and heavy 
chains representing various allotypes. 

In the area of light chain structure, the amino acid sequence for a 
latent b9 allotype light chain was determined, using the DAP/GC-MS method 
for residues 110-118, following mild acid cleavage and full reduction and 
alkylation of the whole isolated light chain. Comparison with similarly 
treated and DAP/GC-MS analyzed nominal b9 allotype isolated light chain 
showed the same sets of dipeptides, indicating that they are structurally, 
(latent and nominal b9 allotype) as well as serologically, identical. 

In the area of heavy chain structure, work, has been concentrated on 
determining the amino acid sequence for the N-terminal 225 residue piece 
from CNBr cleavage of heavy chain of allotype a3, isolated from 
homogeneous rabbit anti-streptococcal antibody. This fragment has been 
fully reduced and alkylated and subjected to partial and full tryptic 
cleavage. The amino acid sequence determination of these tryptic peptides 
is currently under way; both the conventinal Edman method and the 
DAP-DCP/GC-MS method are being employed. The structural studies on this 
CNBr fragment will serve as a prototype for future structural work on 
otner proteins and peptides. New methods of peptide isolation and 
detection have been developed and are currently being applied to obtain 
peptides from chains such as this to allow complete structural analysis. 

Publications 

Yarmush, M.L. , Krutzsch, H.C. , and Kindt, T.J.: Amino acid sequence 
analysis of immunoglobulin light chains by gas chromatographic-mass 
spectrometric techniques: Structural identity of nominal and latent b9 
molecules. Molecular Immunology , in press, 1979. 

Fraser, B.A. , Thunberg, A.L. , and Kindt, T.J.: Variable region correlates 
of group b allotypes: amino acid sequence studies of b9 L chains from 
homogeneous antibodies. Eur. J. Immunol. 8: 380-385, 1978. 

Kindt, T.J. Antibody (Immunoglobulin) Structure. In Baron, S. (Ed.): 
Medical Microbology: Principles and Concepts , Addison-Wesley Publishing 
Company, Inc., California, 1979, in press. 



21-12 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00169-02 LIG 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Primary Structural Analysis of Murine Transplantation Antigens 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



Research Microbiologist LIG NIAID 

Chief, LIG LIG NIAID 

Staff Fellow LIG NIAID 

Staff Fellow LIG NIAID 

Staff Fellow LIG NIAID 



PI: 


Dr. 


John E. Coligan 


Others: 


Dr. 


Thomas J. Kindt 




Dr. 


Frederick. T. Gates 




Dr. 


W. Lee Maloy 




Dr. 


Edward S. Kimball 



COOPERATING UNITS (if any) 

Dept. of Microbiology, Albert Einstein School of Medicine (Dr. S. Nathenson) 
National Cancer Institute, National Institutes of Health (Dr. David Sachs 
and Dr . Ted Hansen) 



lab/branch 
Laboratory of Immunogenetics , NIAID 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 
4.5 



PROFESSIONAL: 

2.7 



1.8 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS j_ ( a 2) INTERVIEWS 



□ (b) HUMAN TISSUES 



□__?_) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The genes in the mouse H-2 major histocompatibility complex determine 
a diversity of functions associated with immune recognition and reaction . 
Three of these genes, H-2K , H-2D and H-2L encode the major antigens 
involved in allograft rejection ; a feature undoubtedly related to the 
genetic polymorphism of these H-2 molecules. In addition, these molecules 
play a role in T-lymphocyte responses to altered cell-surface antigens. 
These H-2 molecules are integral membrane glycoproteins and are associated 
on the cell surface with B n -microglobulin . The purpose of this work is 
to determine the primary structure of these molecules in order that we 
may better understand their function and mechanism of action . Toward 
this goal, we are determining the primary structure of each of these 
molecules from mice of the b and d haplotypes (i-£. H-2K , H-2D , H-2D , 
H-2L ) . The amino acid sequence analysis of H-2K is near completion and 
limited amounts of sequence data are available for the other molecules. 
In addition, the determination of tne primary structure of murine 
g 9 -microglobulin is near completion. 21-13 

PHS-6040 
(Rev. 10-76) 



Z01 AI 00169-02 LIG 

Primary Structural Analysis of Murine Transplantation Antigens 

In order to relate structural differences among mouse H-2 
glycoproteins to changes in their biological activity and to examine 
various genetic and evolutionary relationships among the genes of the 
major histocompatibility complex, the complete amino acid sequences of 
the H-2K and H-2D glycoproteins are being determined. 

Because of the limited amounts of these materials available, 
radio-chemical methodology has been developed in order to pursue these 
studies. H-2 molecules are intrinsically radiolabeled by growing 
appropriate tumor cell lines in the presence of radioactive amino acids. 
Initial studies have utilized the EL4.BU (H-2 ) and C14 (H-2 ) 
lymphoblastoid cell lines. Following detergent extraction, the H-2 
glycoproteins are isolated by immunoprecipitation with alloantiserum. 
Peptide fragments are produced by CNBr cleavage at methionine residues 
and are isolated by gel filtration and ion exchange chromatographic 
procedures. 

Microsequencing analyses utilizing radiochemical methodology have 
made possible the complete primary structural determination of the H-2K 
(H-2. 33) molecule. Approximately 95% of the amino acid sequence of the 
NH-terminal 270 residues have been assigned. This portion of the 
molecule is roughly equivalent to the papain fragment and accounts for 
the extra cellular portion of the molecule. Amino acid sequence 
determinations of the transmembrane (30 residues) and the intracellular 
(40 residues) portions of the molecule are approximately 50% complete. 

Comparison of the amino acid sequences for the H-2K molecule and 
the human HLA-B7 molecule in regions currently available for comparision 
indicate an overall homology of 70%. Greater than 90% of the amino acid 
interchanges require only one nucleotide base change at the DNA level. 
To date, comparision of the molecules has shown two regions 
(positions 61-82 and 87-105J of less than 50% homology and in which many 
of the amino acids require multiple base changes for interconversion. 
These regions may be involved in alloantigenic specificity. 

Comparison of the H-2K amino acid sequence to the primary structure 
of other proteins reveals that an internal region of the molecule 
encompassing the second disulfide is homologous to the constant region 
domains of immunoglobulin heavy and light chains as well as 
3 -microglobulin. This indicates that all these molecules of the immune 
system evolve from the same ancestral gene. 

Studies on the H-2D b (H-2. 2) and H-2D (H-2. 4) molecules have 
progressed to the point where the CNBr peptides of the H-2 molecules have 
been isolated and aligned by homology to H-2K . NH -terminal sequence 
analyses of each of these peptides has allowed the assignment of residues 



21-1L 



Z01 AI 00169-02 LIG 

which account for approximately 40% of the total sequence. The homology 
of these molecules to the H-2K molecule is approximately 85% for the 
positions available for comparision. 

Similar studies on the H-2K and H-2L molecules have been 
initiated. Because of their proximity in the genome, it has been 
difficult to differentiate the H-2D and H-2L molecules. NH -terminal 
sequence analysis of the H-2L indicated that this molecule can be 
distinguished from H-2D by its lack of a valine and phenylalanine at 
positions 9 and 17, respectively, and by the substitution of an 
isoleucine for a methionine at position 23. 

The determination of the amino acid sequence of murine g - 
microglobulin is 95% complete and, as expected, it's primary structure is 
highly homologous (~80%) to that of 6 ? M from other species. 

A major goal of our structural studies on the H-2 glycoproteins is 
to locate positions of structural changes in the H-2 molecules isolated 
from the series of H-2K and H-2D utants of the b and d haplotypes. 
Strains of each K and D mutant series show strong histogenic reactivity 
in vivo and MLR and CML in vitro with the parents. In five of the H-2K 
mutants that have been examined, there appear to be only one or two amino 
acid interchanges between the H-2K molecules, produced by the parent and 
mutant strain. Further studies on the precise nature of the amino acid 
interchanges in these and other H-2K molecules of this mutant series will 
serve to localize the region of the molecule in which polymorphism can 
serve as a specific and effective signal for recognition by the immune 
system. 

An added impetus to the study of these molecules is given by the 
relationships in humans between the homologous loci (HLA A and B) and 
predisposition to certain diseases. Furthermore, molecules coded for by 
the K and D loci of the major histocompatibility complex appear to play a 
major role in the stimulation and specificity of T-lymphocyte response to 
virally induced and other cell surface antigens. 

Publications 



Coligan, J.E., Kindt, T.J., Ewenstein, B.M. , Uehara, H. , Nisizawa, T. , 
and Nathenson, S.G. Primary structure of murine MHC alloantigens: II. 
Aminp acid sequence studies of the cyanogen bromide fragments of the 
H-2K glycoprotein. Proc. Natl. Acad, of Sciences, USA 75: 3390-3394, 
1978. 

Coligan, J.E., Kindt, T.J., Ewenstein, B.M. , Uehara, H. , Martinko, J.M. 
and Nathenson, S.G. Further analysis of the murine H-2K glycoprotein 
using radiochemical methodology. Mol. Immunol. 16: 3-8, 1979. 



21-15 



Z01 AI 00169-02 LIG 

Kindt, T.J., Coligan, J.E., Kimball, E.S, Ewenstein, B. , Uehara, H. , 
Martinko, J., and Nathenson, S.G. Use of radiochemical techniques for 
primary structural analysis of mouse and rabbit histocompatibility 
antigens. Proceedings of the Josiah Macy Foundation , in press, 1979. 

Nathenson, S.G., Ewenstein, B.M., Uehara, H. , Martinko, J.M., Coligan, 
J.E., and Kindt, T.J. : Recent Studies on the Structure of the H-2K 
Glycoprotein and on the H-2K MHC Mutants. In Ferrone, S. and Resfeld, 
R.A. (Eds.): Current Trends in Histocompatibility . Plenum Press, 1979, 
in press. 

Uehara, H. , Ewenstein, B., Martinko, J.M. , Nathenson, S.G. , Coligan, 
J.E., and Kindt, T.J. Primary structure of murine MHC alloantigens: 
amino acid sequence of the amino terminal 173 residues of the K 
glycoprotein. Biochemistry , in press, 1979. 



il-16 



SMITHSONIAN SCIENCE INFORMATION cXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00170-02 LIG 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Structural Studies on Precursors to Immunoglobulin Molecules 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

PI: Dr. Thomas J. Kindt Chief, LIG LIG NIAID 

Others: Dr. Frederick T. Gates Staff Fellow LIG NIAID 



COOPERATING UNITS (if any) 

Institute of Biochemistry, University of Glasgow, Scotland (Dr. A. Williamson, 
H. Singer) 



lab/branch 
Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



CHECK APPROPRIATE BOX(ES) 
□ (a) HUMAN SUBJECTS □ (b) HUMAN TISSUES &(<=) NE!THER 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

0.8 



PROFESSIONAL: OTHER: 

0.5 I 0.3 



□ (al) MINORS C (a2) INTERVIEWS 



SUMMARY OF ilORK (200 words or less - underline keywords) 

The in vitro translation of the mRNA for some proteins yields 
products which are larger than those found in vivo . The amino termini of 
these precursor proteins are extended by up to thirty amino acids when 
compared to their mature counterparts. Precursor sequences have been 
reported for murine (plasmacytoma) immunoglobulin light and heavy chains, 
with the conclusion that these sequences represent extensions of the 
variable region genes. In order to better understand the multi-gene 
nature of immunoglobulin genes , the purpose of this work, is to sequence 
other mouse immunoglobulin precursor molecules for comparison and to 
establish the nature of such sequences in the rabbit. The latter goal 
requires the construction of hybrid cell lines capable of secreting rabbit 
immunoglobulin chains as sources for the isolation of rabbit 
immunoglobulin mRNA. This mRNA must then be isolated and translated 
in vitro , and the translation products must be analyzed using 
radioseque nce techniques. 

21-17 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00170-02 LIG 

Structural Studies on Precursors to Immunoglobulin Molecules 

Peptides of as many as thirty amino acids may be present at the amino 
terminus of a protein when it is synthesized and these peptides may then 
be cleaved from the rotein, presumably durng the secretion process. These 
precursor peptides, which are hydrophobic, have been identified for 
hormones, digestive track enzymes, and serum proteins. It is postulated 
that these peptides direct the passage of the nascent polypeptide chain 
through the membrane of the endoplasmic reticulum. 

Precursor peptides for several BALB/c mouse immunoglobulin light 
chains and a single heavy chain have been previously reported. The amino 
acid sequence data have indicated that light chan precursors appear to be 
somewhat homologous within and between light chain subgroups, while much 
less homology exists between precursor peptides of Kappa and lambda light 
chains. In collaboration with A. Williamson and H. Singer (University of 
Glasgow) we have determined a partial amino acid sequence for the 
precursor peptides of a gamma heavy chain and a Kappa light chain from a 
C3H mouse myeloma. These sequences are not homologous to those reported 
for BALB/c mice, indicating a broader diversity of precursor peptide 
sequence than was previously assumed. 

In order to study the structure of precursor peptides of 
immunoglobulins from another species, we are constructing mouse-rabbit 
hybrid cell lines (hybridomas) which secrete rabbit immunoglobulin chans. 
With these cell lines as sources of mRNA for in vitro protein synthesis, 
we are immunoprecipitating the subsequent rabbit immunoglobulin products 
prior to radiosequence analysis. The multiplicity of kappa constant 
region genes in the rabbit allows a study of gene rearrangement and 
regulation of gene expression which is not possible in the mouse system. 
This analysis will help to define the structure-function relationship of 
immunoglobulin precursor peptides and the genes which encode them. 

Publications 

None 



21-18 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00171-02 LIG 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Genetic Studies on Rabbit Immunoglobulins and Other Serum Proteins 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Dr. John A. Sogn 

Others: Dr. Thomas J. Kindt 

Dr. Martin Yarmush 

Mr. Mark Simpson 



Chemist 
Chief, LIG 
Chemist 
Veterinary-Costep 



LIG NIAID 

LIG NIAID 

LIG NIAID 

LIG NIAID 



COOPERATING UNITS (if any) 

Dr. Ben Wolf, Univ. of Penn. , School of Vet. Medicine, Philadelphia, Pa. 
Dr. John Coe, Rocky Mountain Laboratory, NIAID 



LAB/BRANCH 

Laboratory of Immunoaenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID. NIM. Bethesda. 



Maryland 20205 



TOTAL MANYEARS: 

1.35 



PROFESSIONAL: 

0.5 



0.85 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS 

□ (al) MINORS 3 (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



D^c) NE 



SUMMARY OF WORK (200 words or less - underline keywords) 

The serologic markers of rabbit immunoglobulins-allotypes and 
idiotypes , are under study because they can provide information concerning 
the number and arrangement of genes encoding the immunoglobulins. A major 
effort has been made to characterize allotype b4 , a new allele of the 
group b allotypes of the C region. This allotype, first discovered in 
this laboratory during amino acid sequence studies, has now been shown by 
genetic analysis to be an allele of the other group b allotypes and has 
been characterized serologically. The utility of chain specific idiotypes 
for genetic studies in the rabbit has been confirmed, and the initial 
study of an L chain specific idiotype has been extended with an H 
chain-specific idiotype. It was possible to show linkage of this idiotype 
to V and C allotypes within a single allogroup, and to demonstrate 
latent expression of an allogroup. Attempts to improve breeding 
efficiency of inbred rabbits are underway at present. Idiotypy studies 
will be extended to these animals when sufficient numbers are available. 

21-19 

' PHS-6040 
(Rev. 10-76) 



Z01 AI 00171-02 LIG 

Genetic Studies on Rabbit Immunoglobulins and Other Serum Proteins 

The various serologic markers of rabbit immunoglobulins have been 
valuable probes for the study of genetic interactions involved in the 
humoral immune response. The rabbit has a uniquely diverse repertoire of 
allotypes (intra-species antigenic variants), encompassing most regions of 
the antibody molecule. The structural correlates for some allotypic 
markers are simple, while for others they are either complex or unknown. 
Studies within this project are designed to expand the allotypic 
repertoire, to increase its structural definition, and to use these 
serologic markers to examine questions about the number of genes involved 
in immunoglobulin synthesis and the mechanisms controlling interactions 
among these genes. The studies based on allotypes have been complemented 
and extended by use of idiotypes as markers for the antibody combining 
site. The obvious complexity of idiotypes as genetic markers has been 
lessened somewhat by increasing the specificity of idiotypic antisera, 
either by fractionation based on antigen competition or by limiting 
idiotypic recognition to determinants on one of the antibody chains. 

Serologic characterization is now complete for a new C, allotype, 
b4 V , which has been under study for two years. While conventional 
antiallotypic reagents do not distinguish b4 from b4 , three methods of 
serologic distinction have been found. First, as reported previously, one 
anti-b4 serum, upon adsorption on a column containing bound cottontail 
rabbit IgG, yielded an antibody fraction which reacted well with b4 IgG 
but poorly with b4 v IgG. An inhibition of binding radioimmunoassay could 
then be used to differentiate b4 from b4. Second, in collaborative 
studies with Dr. John Coe, several bull-frog antisera raised against b4 
IgG or crosslinked b4 v L chains reacted specifically with b4 IgG after 
adsorption with b4 IgG. The reaction could be detected either by 
radiobinding or precipitin assays. Although only low levels of activity 
were found, the result established that b4 IgG does possess antigenic 
determinants not found on b4 IgG. Third, collaborative studies with Dr. 
Jan Naessens have proven that b4 and an allotpe described in Belgium, 
b4.2, are serologically and structurally identical. This finding has made 
tne serologic detection of b4 in serum much easier because an antiserum 
exists (anti-MS7) which reacts only with IgM containing L chains of 
allotype b4.2 (or b4 v ) . The reaction can be seen readily by precipitin 
reaction. Another serum of the MS series, anti-MS3, reacts in a similarly 
specific way with IgM containing b4.1 (b4) L chains. Structural identity 
of b4 V with b4.2 was shown by sequence analysis of succinylated, acid 
cleaved L chains. 

Further genetic analysis of the rabbit immune response will be 
difficult without rabbit of more defined genetic composition. To take 
advantage of available in vitro techniques for dissecting the cellular 
components of the unimmune response fully inbred rabbits would be 
particularly valuable. Some attempts to incorporate existing inbred 
rabbits into studies in ths laboratory have been made in the past year. 
The B/J strain has been examined to determine optimal parameters for 



il-20 



Z01 AI 00171-02 LIG 

immunization with antigens inducing monoclonal responses. Idiotypic 
analysis will be done to examine idiotypic diversity in an inbred 
population. Large scale studies will require numbers of B/J rabbits not 
available with current breeding techniques. Methods for enhancing 
fertility and reproductive rate are being examined in cooperation with the 
Veterinary Resources Branch and Dr. Ben Wolf. 

Limited structural studies are underway in collaboration with Dr. 
John Goe on a protein isolated by Dr. Coe from the serum of female 
hamsters. The protein is present only in trace amounts in nude hamsters. 
Amino acid composition and sequence analysis are being used to determine 
whether the female protein exhibits structural homology to biological 
active proteins of several types already sequenced in other species. 

Publication 



Sogn, J. A. and Kindt, T.J.. Genetic characterization of a new allele of 

the rabbit group b C allotypes. Immunogenetics 7: 141-147, 1978. 
K 

Kindt, T.J. and Capra, D. Gene-insertion theories of antibody diversity: 
A re-evaluation. Immunogenetics 6: 309-321, 1978. 

Smith, L.J., Sogn, J. A., Kindt, T.J., and Mandy, W.J. Serologic 
distinction between the rabbit kappa L chain allotype b4 and an allele 
b4 . European Journal of Immunology 9: 27-31, 1979. 

Yarmush, M.L. and Kindt, T.J. Idiotypes of Rabbit Antistreptococcal 
Antibodies: Probes for Inheritance and Immune Regulation. In Rudbach and 
Baker (Eds.): Immunology of Bacterial Polysaccharides , Elsevier North 
Holland, Inc., 1979, pp. 41-65. 

Capra, J.D. and Kindt, T.J. One From Many: Immunoglobulin V Regions are 
the Products of Interacting Genes. In Cunningham and Sercarz (Eds.): 
The Strategy of Immune Regulation , Academic Press, New York, 1979, in 
press. 



21-21 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl Al 00172-02 LIG 



PERIOO COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Carbohydrate and Glycoprotein Antigens of Microbial Cell Walls 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: 



Dr. John Coligan 



Research Microbiologist 



LIG NIAID 



COOPERATING UNITS (if any) 

Dept. of Micro. Univ. of Alabama at Birmingham (Dr. David Pritchard) 



LAB/BRANCH 

Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NIAID. NIH. Bethesda. Maryland 202U5 



TOTAL MANYEARS: 

0,2 



PROFESSIONAL: 



.1 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (a!) MINORS C (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



sxf 



c NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Previous compositional and immunological studies have defined the 
chemical and antigenic structure of the group-specific carbohydrates for 
Groups A, A variant and C streptococci . These polysaccharides were shown 
to have a common core structure . Work is in progress to determine the 
structure of the group specific carbohydrate of the Group B streptococcus 
in order to chemically define its antigenic determinants and to determine 
its chemical relationship to the other streptococcal group specific 
carbohydrates . 



21-22 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00172-02 LIG 

Publications 

Coligan, J.E. and Slayter, H. Physical, chemical and immunological 
characterization of saline-extracted, concanavalin A purified 
carcinoembryonic antigen. Mol. Immunol. 16: 129-135, 1979. 

Coligan, J.E. and Kindt, T.J. Use of Structurally Defined Streptococcal 
Carbohydrate Antigens in Studies of Rabbit Antibody Idiotypes. In Read, 
S. and Zabriskie, J. (Eds.): Streptococcal Disease and the Immune 
Response . Academic Press, New York, in press, 1979. 

Coligan, J.E., Kindt, T.J., and Krause, R.M. Structure of the 
streptococcal groups A, A-variant and C carbohydrates. 
Immunochemis try 15: 755-760, 1978. 

Coligan, J.E. and Krause, R.M. Antibodies to streptococcal carbohydrate, 
substitutes for the myeloma proteins. J. Infect. Pis. , in press, 1979. 



21-23 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF PROJECT NUMBER 

HEALTH, EDUCATION, AND WELFARE ! 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT ZQ] _ ^ q Q173 _ q2 l1q 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (30 characters or less) 



Nonallelic Expression of Genes Encoding Rabbit Immunoglobulins 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Dr. T.J. Kindt Chief, LIG 

Others: Dr. Martin Yarmush Chemist 

Dr. John A. Sogn Chemist 

Dr. F.T. Gates, III Staff Fellow 



LIG NIAID 

LIG NIAID 

LIG NIAID 

LIG NIAID 



COOPERATING UNITS (if any) 

Dr. William J. Mandy, University of Texas, Austin, Texas 

Dr. Ben Wolf, University of Pennsylvania School of Veterinary Med. 

Philadelphia, Pa. 



lab/branch 

Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



INSTITUTE AND LOCATION 

NTATD, NTH, RpfhPsHa,, Maryland 2Q205 



TOTAL MANYEARS: 

2.3 



PROFESSIONAL: 

1.3 



CHECK APPROPRIATE BOX(ES) 
Z (a) HUMAN SUBJECTS 

G (al) MINORS Q (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



GX&) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Rabbit immunoglobulin allotypes are antigenic determinants thought to 
be inherited as autosomal co-dominant alleles . This notion has been 
challenged by observations of low concentrations of allotypes not detected 
by qualitative tests or predicted by parental genotypes. In the past 
year, L chains with "latent" allotypes were isolated from the sera of 
pedigreed rabbits ana shown by amino acid sequence analysis to be 
indistinguishable from the normal allotypes. Current research focus on 
hybridoma cells maintained in culture that synthesized rabbit 
immunoglobulin RNA will be isolated from these cell lines and cDNA probes 
will be prepared to carry out molecular biological studies in order to 
determine the basis for inheritance of latent allotypes. Other studies in 
this area have included clearance of normal versus latent allotypes in 
animals using doubly labelled material and studies on the linked 
expression of V^ markers on immunoglobulins expressing latent allotypes. 



21-; 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00173-02 LIG 

Nonallelic Expression of Genes Encoding Rabbit Immunoglobulins 

In the past year, studies on latent allotypes of rabbit 
immunoglobulins have included complete serologic characterization of 
allotypic markers of both the constant and variable regions of the rabbit 
molecules. It has been shown by structural studies that the molecules 
expressing latent allotypes have markers that are indistinguishable from 
those of the allotypes normally expressed. Latent Group b markers of the 
constant region of the rabbit antibody light chain were previously 
detected in sera, in IgG preparatins, and on isolated L chains from 
rabbits bred for homozygosity at the b locus. Serologic analysis of sera 
from an extended family of homozygous b4 rabbits revealed the presence of 
latent b allotypes in 5 of 37 sera tested. Latent b5 and b9 markers were 
identified. None of the sera tested contained latent b6. In two 
instances, the level of latent b9 allotype was sufficiently high to permit 
isolation and detailed characterization of the immunoglobulin population 
bearing this latent allotype. These studies which are completely 
described in another section indicated that the nominal and latent 
allotype light chains were identical in sequence. 

Studies concerning the linked expression of C and V allotype genes 
have involved isolation of molecules on the basis of a latent group a 
allotype. This is carried out by use of immunoadsorbent chromatography 
employing specific antibodies directed against the allotype specificities. 
The isolated molecules were then typed for their group d markers (dll, dl2) 
by serologic and by chemical means. Approximately half of the molecules 
isolated were shown to carry latent group d markers in addition to the 
latent group a allotype. However, in no case was found a molecule with 
combinations of group a and group d markers that are not normally 
expressed in the rabbit populations studied. This finding indicates that 
the mechanism for synthesis of latent allotypes are similar to those for 
nominal allotypes and do not represent products of events such as 
misspairing of multiple genes involved in H chain synthesis. 

A promising new tool for the study of latent allotypes has been 
introduced by the use of interspecies (mouse-rabbit) hybridoma cells. 
Cells have been prepared that will secrete rabbit heavy or light chains in 
culture and in ascites fluid of nude mice. These cells are being prepared 
in bulk culture and RNA encoding the rabbit immunoglobulin chains will be 
isolated and used to prepare cDNA which can be used as a probe to study 
the occurrence of latent allotype genes in various rabbits. 

IgG clearance rates were measured by intravenous injection of rabbits 
with differentially labelled ( I and I) allotype matched and 
nonmatched IgG samples. In no instance was the allotype matched IgG 
cleared faster than nonmatched although the converse was true in several 
instances. These data suggest that there is an in vivo recognition of 
allotypes which is a possible regulatory mechanism. Many questions remain 
to be answered concerning mechanisms for the regulation of synthesis and 
the selection of genes involved in the preparation of normal and latent 
allotypes. 

21-25 



Z01 AI 00173-02 LIG 

Publications 

Yarmush, M.L. and Kindt, T.J. Isolation and characterization of IgG 
molecules expressing latent group b allotypes from pedigreed b b rabbits. 
J. Exp. Med. 148: 522-533, 1978. 

Mudgett-Hunter, M. , Yarmush, M.L. , Fraser, B.A. , and Kindt, T.J. Rabbit 
latent group a allotypes: Characterization and relationship to nominal 
group a allotypic specificities. J. Immunology 121: 1132-1138, 1978. 

Yarmush, M.L., Sogn, J. A., and Kindt, T.J.: Latent allotypes: A window 
to a genetic enigma. Ann. Immunol. (Inst. Pasteur) 130C: 143-156, 1979. 

Yarmush, M.L., Krutzsch, H. , and Kindt, T.J.: Amino acid sequence 
analysis of immunoglobulin light chains by gas chromatographic - mass 
spectrometric techniques: structural identity of latent b9 and nominal b9 
molecules. Molecular Immunol., in press, 1979. 



Wolf, B., Urban, R. , Miller, A.B., Kimball, E.S., Mudgett, M. , Catty, D. 
and Danemann, J.: Nonallelii 
Cell surface and serum studit 
J . Immunol . , in press, 1979. 



and Danemann, J.: Nonallelic inheritance of V region a group allotypes. 
Cell surface and serum studies in double and triple expressing rabbits. 



21-26 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00180-01 LIG 



PERIOD COVERED 

October 1, 1978 to September 30, 1979 



TITLE OF PROJECT (80 characters or less) 



Monoclonal Antibodies Directed Against Cell Surface Proteins 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



LIG NIAID 

LIG NIAID 

LIG NIAID 

LIG NIAID 



PI: 


Dr. 


John A 


Sogn 


Chemist 


Others: 


Dr. 


Thomas 


J. Kindt 


Chief, LIG 




Dr. 


Edward 


S. Kimball 


Staff Fellow 




Dr. 


Thomas 


Folks 


Staff Fellow 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 

Laboratory of Immunogenetics 



SECTION 

Immunogenetics Research Section 



NSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 



20205 



TOTAL MANYEARS: 

2^2 



PROFESSIONAL: 
i 1.2 



±^L 



CHECK APPROPRIATE SOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



&f C 



SUMMARY OF WORK (200 words or less - underline keywords) 

Preliminary studies are under way on antibodies to cell 
surface proteins produced by hybridoma techniques. The initial targets 
for this investigation is cell line RL-5, a rabbit T-cell tumor . The cell 
line has been characterized by conventional techniques and several 
mouse-mouse hybridomas with activity against this cell have been produced, 
cloned and propagated _in vivo and _in vitro . Biochemical characterization 
of the molecular specificity of these antibodies is under investigation. 



21-27 



PHS-6040 
(Rev. IO-76) 



Z01 AI 00180-01 LIG 

The ability to produce monoclonal hybridoma antibodies, in unlimited 
amounts, directed against any antigenic determinant of interest, no matter 
how impure, has revolutionized the study of the biologically important 
proteins of the cell surface. These proteins are difficult or impossible 
to purify by conventional techniques because they are present in 
vanishingly small amounts and because their isolation is complicated by 
the hydrophobic nature of these integral membrane constituents. 
Characterization of such proteins has been feasible only by indirect 
methods using genetically defined organisms, where specific antibodies may 
be produced by cross-immunization of antimals differing at a limited 
number of genetic loci. Structural studies in outbred species such as 
rabbit and man have, therefore, been especially limited. Hybridoma 
technology removes these restrictions. The research interests of several 
groups in the Laboratory of Immunogenetics currently involve molecules of 
immunologic importance found on cell surfaces. The goal of this project 
is to raise hybridoma antisera against these molecules so that structural 
studies can proceed rapidly. 

The principal current interest is the rabbit cell line RL-5 . This 
virus- induced lymphoma has been characterized as a member of the T-cell 
lineage on the basis of reactivity with two specific anti-rabbit thymocyte 
sera, the absence of cytoplasmic and surface immunoglobulin, and the 
absence of receptors for Fc or C3. Mice have been immunized with RL-5 
and spleen cells from the immunized mice have been fused with appropriate 
myeloma cells to obtain hybrid antibodies against RL-5. Antibody activity 
is measured in an ELISA assay. The membrane components against which the 
antibodies react are currently being identified by immunoprecipitation and 
electrophoretic techniques. 

Publication 

None 



21-21 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

Z01-A1-00191-01 LIG 



PERIOD COVERED 

New - October 1 , 1979 



TITLE OF PROJECT (80 characters or less) 

Immunoregulation of T Lymphocytes - The Role of Anti-idiotype and 
Immune Complexes 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



PI: Kenneth W. Sell 
Other: Tom Folks 



Head, Immunobiology Section LIG, NIAID 
Staff Fellow LIG, NIAID 



COOPERATING UNITS 



Dr. Aftab Ahmed (Merck Corporation, Rahway, N.J.), Dr. James Woody (NMRI) 



LAB/BRANCH 

Laboratory of Immunogenetics 



SECTION 

Immunobiology Section 



INSTITUTE AND LOCATION 

NIAID, NIH, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

3- 1/2 



PROFESSIONAL: 

1- 1/2 



CHECK APPROPRIATE BOX(ES) 

□ (a) HUMAN SUBJECTS 

□ (al) MINORS □ (a2) INTERVIEWS 



$ (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

Investigation of mechanisms by which immunoregulatory signals are transmitted to 
T-lymphocytes to cause activation or suppression of their activity. Project 
attempts to examine both antigen and allogeneic receptors on T-lymphocytes and 
the means by which they may be activated, blocked or stimulated to active sup- 
pression of T-cell functions In particular, the role of immune complexes and 
the related role of anti-idiotype antibodies in the regulatory process will be 
examined. The interaction between antigen, antibody, complement, anti-idiotype 
immunoconglutinjn and rheumatoid factor will be examined in the make-up of immune 
complexes to determine the role of each, individually or acting in concert, to 
produce effects on T-cell activation or effector function. In addition, attempts 
will be made to identify immune complexes in disease states such as juvenile 
diabetes and hepatitis to determine what role they may play i_n vivo in the 
suppression of cell-mediated immunity in these diseases which are related to 
latent or persistent viral infections. 



'21-29' 



PHS-6040 
(Rev. IO-76) 



Z01-A1-00191-01 LIG 
Project Description 

Objectives 

The specific suppression or inactivation of those clones of antigen- 
reactive cells directed against foreign antigens or donor histocompatibility 
antigens has been the object of intensive research. A recent finding has 
been the disclosure that F, hybrids between two inbred strains of mice will 
synthesize anti-allo-anti bodies if injected with either parental lymphoid 
cells or in al lo-antiserum produced in the same parental strain against the 
allo-antigens of the other parental strain. These observations have led to 
the concept that the recognition of allo-antigen by the T-lymphocyte is the 
primary step towards allograft rejection and initiation of antigen responsive- 
ness. The exact nature of the T-lymphocyte recognition structure is still an 
unresolved question. Binz and Wigzell have obtained evidence that suggest 
that the recognition structure is similar, if not identical, to the combining 
site of allo-antibody (idio-type). Isolated T-cell receptors have been shown 
to have a single chain structure with a molecular weight of 70,000 daltons. 
It has been suggested that both T and B cells employ a common pool of heavy 
chain V genes in synthesis of the antigen binding site on the respective an- 
tigen receptors. The use of the term "idiotype" for the recognition structure 
on T-cells is based on the evidence for shared idiotypic determi nance on both 
T and B cells. Both normal T-cells as well as T-lymphoblasts which have been 
specifically activated by antigen or the mixed lymphocyte reaction both 
express idiotypic determinants against the relevant histocompatibility or 
foreign antigen. Although these i diotype-posi ti ve cells specific for a 
given set of major histocompatibility complex antigens are found with very 
low frequency, less than 3 percent, in the normal resting population of 
peripheral blood lymphocytes, they are greatly enriched during the in vitro 
proliferation in an MLC response against the relative MHC. 

Auto and anti-idiotype (anti-receptor) antibodies can be produced by immuni- 
zation with lymphoblasts. These can deplete the responder T-lymphocyte popu- 
lation apparently through cytotoxic killing in the presence of complement. 
They are not capable of blocking MLC reactions in the absence of complement 
as they apparently compete ineffectively for binding with the receptor site 
on T-cells responsible for the MLC reaction. 

Inhibitors of T-cell mediated immunity have been identified in certain clini- 
cal diseases. In subacute sclerosing panencephalitis (SSPE) and in CMV virus 
disease, immune complexes have been identified which apparently are capable 
of specifically inhibiting T-cell responses to the virus and specifically 
blocking effector functions such as the release of macrophage inhibition 
factor. We hypothesize that these immune complexes may exert their immuno- 
suppressive activity on T-lymphocytes because of the presence of antiidio- 
types. It is possible that the immune complex serves as a focusing mechanism 
or perhaps a stabilizing mechanism allowing the more effective interaction of 
anti-idio-type with T-cell receptors, thus providing for blocking or initia- 
tion of inactivation events in the T lymphocyte. 

We, therefore, propose to study the role of anti-idiotype and immune complexes 
as they interact with receptors on T-lymphocytes in order to elucidate the 

21-30 



Z01-A1-00191-01 LIG 
mechanisms of immunoregulation of cell -mediated immune events. 

Methods Employed: 

1. Attempts will be made to construct model immune complexes in order to 
evaluate their interaction with T-cell receptors and subsequent T-cell acti- 
vity. Specific antibody and anti-idiotype as well as immunoconglutinin and 
rheumatoid factor will be prepared using hybridomas. Balb/C mice will be 
immunized with specific carrier proteins such as BSA, OVA, BGG, KLH, etc., 
and their spleen cells fused with appropriate mouse myeloma cells in order to 
establish specific clones of antibody producing cells. The Elisa technique 
will be utilized to assay for the presence of specific antibodies. It is an- 
ticipated that spleens of immunized animals will provide cells producing, not 
only specific antibody against the relevant antigen but also cells expressing 
anti-idiotype against antibody. In addition, autoantibodies against C-3 
(immunoconcluti nin) and an auto-antibody against IgG (rheumatoid factor) will 
be anticipated to occur in the immunized mice. Each of these antibodies then 
can be used along with purified complement (C3) to construct immune complexes 
which can be tested in cell-mediated immune tests both for recognition (trans- 
formation) or for effector function (MIF assay) or killer cell assays. 

2. Lymphocytes and serum will be collected from chimpanzees which have been 
infected with hepatitis virus. The lymphocytes will be assayed for cell- 
mediated immune activity including transformation and MIF production. Serum 
will be assayed for the presence of inhibitors of cell-mediated immunity. If 
inhibitors are discovered, specificity will be determined and nature of the 
inhibitor will be examined with particular emphasis on techniques to identify 
immune complexes. Assays will be made regularly during the infective stage 
of the disease in the chimpanzee to determine the sequential appearance of 
cell-mediated immunity and inhibition. 

3. Patients with juvenile diabetes as well as control patients will be tested 
for the presence of humoral and cell-mediated immunity against viruses which 
have been suspected in the etiology of diabetes. In particular, coxsachie B4 
will be evaluated and cell -mediated immunity (transformation and MIF) will be 
studied and compared with a control group of sex and age matched normal child- 
ren. Serum of diabetic patients will be examined for inhibitors of eel 1 - 
mediated immunity against candidate viruses with immunochemical studies to 
determine the presence of immune complexes. 



21-31 



LABORATORY OF IMMUNOLOGY 

1979 Annual Report 

Table of Contents 



Z01-AI 
Project Number 

Summary 

00030-11 

00148-04 

00035-0.6 



Antigen Recognition and Activation 
of Immunocompetent Cells. — Paul 

Lymphocyte Interactions, Receptors 
and Functions. — Green 

Specificity in Immune Responses. — 
Inman 



Page 
22-1 
22-13 

22-19 

22-25 



00036-14 



00037-12 



00038-07 



00040-05 



Immunoglobulin Genetics: Regulation 22-30 
of Gene Expression and Lymphoid 
Differentiation. — Mage 

Immuno gene tics of Mouse Immunoglobulin 22-38 
and Genetic Control of Antibody 
Response . — Lieberman 

Structure and Activity Studies on 22-43 
Immunologically Important Cells and 
Proteins . — Waxdal 



Genetic Control of Immunocompetent 
Cell Interactions. — Shevach 



22-48 



00147-04 



The Mechanism of Activation of 
Thymus -Derived Lymphocytes . — 
Schwartz 



22-56 



PHS-NIH 
Summary Statement 
Office of the Chief 
Laboratory of Immunology 
October 1, 1978 through September 30, 1979 

Recombination between Genes for u and <S Immunoglobulin Heavy Chains 

The genes controlling the constant regions of heavy (H) immunoglobulin 
(Ig) chains of various classes exist as a genetic linkage group termed the 
IgC complex in the mouse. The genes specifying IgA and the various IgG 
Hcnains are very tightly linked. Indeed, in the examination of several 
thousand individual mice and large numbers of humans, no instance of a 
genetic recombination separating these genes had been observed. It is known 
that genes for the \i and 6 H chains are also found in the IgC complex but 
the degree of linkage has not been extensively studied because allotypic 
markers for the p and 6 genes have only recently been described. In the 
course of experiments aimed at studying the linkage of Lyb7 and IgD , 
scientists in the Laboratory of Immunology and at the Naval Medical Research 
Institute, have observed two instances of recombination between the IgG 
and IgD genes. The initial observation was that two progeny (numbers 72 
and 74) of a cross between (C57BL/6 x DBA/2) and C57BL/6 possessed the 
"DBA/ 2" allele of IgD [IgD a ] but lacked the "DBA/ 2" allele of IgG [ IgG C ]. 
This phenotype was transmitted to progeny on subsequent crossings of 
individuals 72 and 74 to C57BL/6 to create families 72 and 74. Proof that 

Q 

the failure of these mice to express IgG was due to its replacement by 
_Ig_G„ [the allele possessed by C57BL/6] was obtained by crossing members of 
families 72 and 74 to A mice and demonstrating that progeny that inherited 
IgD , which is distinguishable from the IgD of A, also inherited IgG ? . It 
has also been possible to show that in both recombinants the genes for IgG.. , 
IgA, IgM and prealbumin segregated with the IgG ? gene. These recombinant 
families represent the first documented examples of intra- IgC genetic 
recombination. Studies of segregation of IgV markers in these mice, which 
are now in progress, will allow construction of more detailed genetic maps 
of this genetic region, which is of key importance in the specification of 
antibody molecules. In turn, this will prove of considerable utility in 
understanding the genetic basis of the immune response (Subbarao, Lieberman 
and Paul, LI/NIAID; Ahmed and Scher, Naval Medical Research Institute). 

Genetic Regulation of the Anti-f ructosan Antibody Response 

Bacterial levan (BL) is a g (2-»-6) linked polyfructosan with g (2-KL) branch 
points. Anti-BL antibodies produced in BALB/c mice consist of two families 
of molecules. One of these bind BL but not inulin, a g (2-K1) polyfructosan, 
and thus appear to be specific for g (2-*-6) fructosans. The other set of anti- 
bodies bind inulin and BL, are principally specific for g (2-KL) linkages and 
express one or more of a family of cross reactive idiotypes (IdX) found on 
inulin-binding myeloma proteins of BALB/c mice. It has previously been shown 
that the capacity to produce both anti-inulin antibody and IdX-bearing mole- 
cules in response to immunization with BL requires the presence of genes 
linked to the IgC gene complex. During the past year, Laboratory of Immunol- 
ogy scientists have demonstrated that non-IgC genes exert major effects on the 

22-1 



amount and clonal diversity of the anti-inulin antibodies produced in response 
to BL immunization. Thus, BALB/c mice immunized with BL produce inulin- 
binding antibodies which, when analyzed by isoelectric focusing (IEF) , consist 
of characteristic "triplet" of bands. C57BL/6 mice, or C.B20, which are 
BALB/c congenic mice expressing IgC genes of C57BL/6, make no anti-inulin 
antibodies after BL immunization"! Nonetheless, F- hybrids between C57BL/6 
and BALB/c make a more vigorous and more diverse anti-inulin response to 
BL, as judged by IEF, than do BALB/c. The genetic influence contributed by 
the C57BL/6 depends on genes outside the Ig H chain genetic region since 
B.C8 mice, which are congenic to C57BL put possess BALB/c IgC genes, make 
a response similar to the F.. in its amount and diversity. Through the analysis 
of amount and diversity of the anti-inulin response to BL of recombinant 
inbred lines between C57BL/6 and BALB/c, it has been tentatively concluded 
that two non-IgC genes regulate the clonal diversity and amount of the anti- 
inulin response. These studies should shed considerable light on the 
genetic factors which regulate immune responses to simple antigens, parti- 
cularly those which are excellent models for polysaccharide antigens used 
in bacterial vaccines (Stein, Bona, Lieberman and Paul, LI/NIAID) . 

A Specialized Subset of B Lymphocytes is Stimulated By Anti-Immunoglobulin 
Antibodies 

One of the most direct and potentially informative approaches to the 
study of the requirements for B lymphocyte activation is the use of anti- 
bodies to B lymphocyte membrane receptors to stimulate these cells. 
Laboratory of Immunology scientists have established that specifically 
purified antibodies to u H chains and to k L chains cause marked stimula- 
tion of proliferative responses by B lymphocytes. The response is 
independent of any requirement for T lymphocytes or macrophages. However, 
anti-Ig antibodies do not stimulate the synthesis of Ig by B lymphocytes; 
indeed, these antibodies are powerful inhibitors of stimulation of Ig 
synthesis by both antigens and polyclonal B lymphocyte activators. 
During the past year, scientists in the Laboratory of Immunology and the 
Naval Medical Research Institute have shown that the capacity of lympho- 
cytes to respond to anti-u and anti-K is a property of a specialized 
subset of B lymphocytes which has a characteristic density of membrane (m) 
Ig and expression of differentiation antigens. To show this, B lymphocytes 
were incubated with fluorescein (Fl) conjugated anti-Ig, anti-u or anti-6 
reagents and separated into populations with characteristic densities of 
mlg using the fluorescence activated cell sorter (FACS) . Cells with a high 
density of total Ig, a relatively low density of IgM, and a moderate to 
high density of IgD were most responsive to anti-Ig. By contrast, cells 
with a low density of total Ig , a high density of IgM, and a low density 
of IgD were poorly responsive. The results also suggest that the bulk 
of cells responsive to anti-Ig are Lyb5 . These results establish that 
responsiveness even to simple stimulants such as anti-u is a property of 
a discrete B lymphocyte subpopulation. Further progress in delineating the 
biochemical events in B lymphocyte activation by specific stimuli will require 
techniques to purify and/or clone specific populations of B lymphocytes. 
Sieckmann and Paul, LI; Scher, Naval Medical Research Institute). 



22-2 



Failure to Detect V Framework Determinants on T Lymphocytes 

The delineation of the chemical nature of the antigen-binding re- 
ceptors of T lymphocytes has been one of the most challenging and critical 
problems of modern immunology. There is an increasing body of functional 
and genetic data which indicates that some idiotypic determinants, pre- 
sumably markers of hypervariable and J segments of immunoglobulin H chains, 
are expressed on T lymphocyte receptors as well as on antibodies and B 
lymphocyte receptors. These findings provide strong evidence that 
structural genes in the IgV genetic region code for at least part of the 
T lymphocyte receptor. Laboratory of Immunology scientists have sought to 
determine whether antigenic determinants associated with framework areas of 
Ig H chain variable (V ) regions were also expressed on T lymphocytes. In 
order to examine this, anti V framework antibodies against allotypic and 
species-specific determinants have been prepared. The former are the well 
known antibodies to ^ locus allotypic determinants; the latter are anti- 
sera to rabbit V determinants prepared in goats. Such reagents have been 
fluoresceinated and their ability to stain B and T lymphocytes determined 
by direct examination and by use of the fluorescence activated cell sorter 
(FACS). Thus far, no evidence for T lymphocytes expressing V determinants 
has yet been obtained. Since T lymphocytes have been shown to lack 
classical light (L) chains, efforts have been made to chemically identify 
V bearing molecules which lack L chains. Again, no such molecules have 
yet been found. This data is consistent with the failure of V framework 
determinants to be expressed on T lymphocytes at all, even on T lymphocytes 
which appear to express idiotypic determinants. It is also consistent with 
the possibilities that only a very small fraction of T cells possess V - 
bearing receptors or that each T cell expresses very small amounts of such 
receptors. These studies are being pursued both with an effort to in- 
crease their sensitivity and to obtain data on the effects of anti-V 
sera on T lymphocyte functions. They promise to offer important insights 
into the nature of antigen-recognition by T lymphocytes (Wilder and 
Mage, LI/NIAID; Scher, Naval Medical Research Inst.). 

Phospholipid Methylation is an Early Event in T Lymphocyte Activation 

T lymphocytes can be stimulated to proliferate by exposure to a wide 
variety of agents of which the plant lectin concanavalin A (Con A) is a 
prototype. It has long been known that many intracellular events are 
associated with stimulation of DNA synthesis by Con A but no clear under- 
standing of the critical initial events which lead to the proliferative 
response has yet been developed. Recently, scientists in the Laboratory of 
Immunology and in the National Institute of Mental Health have shown very 
rapid changes in membrane phospholipid methylation following exposure of T 
lymphocytes to Con A. Thus, there is a maximum incorporation of H-methyl 
groups into membrane phospholipids within 10 minutes after the addition of 
Con A followed by a somewhat slower calcium dependent degradation so that 
normal levels of methylation are found at 40 minutes. Increases in phos- 
pholipid methylation are closely correlated with subsequent proliferative 
events. Thus, the dose-response curve for increased phospholipid methylation 
and for stimulation of DNA synthesis are similar; only cell types which 
' proliferate in response to Con A exhibit increased methylation; and, only 

2 2-3 



those lectins which are mitogenic induced methylation. Furthermore, 
inhibition of methylation by addition of a methylation- inhibitor prevents 
subsequent DNA synthesis but only if the inhibitor is added before the 
increase in methylation caused by Con A. These results suggest that 
membrane transmethylases are activated by binding of Con A to the cell, 
that this leads to an increase in several methylated phospholipids in- 
cluding monomethylethanolomine. Such an increase may lead to decreased 
membrane viscosity and the entry of calcium into the cell. Calcium is 
apparently associated with the accelerated degradation of methylated phos- 
pholipids and release of a series of compounds, such as thromboxane, 
prostaglandins, and lysolecithin which may be of considerable importance 
in subsequent events of lymphocyte activation. These early biochemical 
events appear to be excellent candidates to be critical steps in the 
cellular processes which determine the capacity of physiologic stimulants, 
such as antigens, to activate lymphocytes (Toyoshima and Waxdal, LI/NIAID; 
Hirata and Axelrod, NIMH). 

Antigen Presentation by Macrophages: both Antigen and la Gene Products must 
be Expressed on the Surface of the Antigen-presenting Cell 

The activation of proliferative responses and of lymphokine production 
by specific T lymphocytes depends on presentation of antigen by a special- 
ized macrophage- like cell. Antigen-recognition by T lymphocytes has been 
shown by Laboratory of Immunology and Laboratory of Clinical Investigation 
scientists to involve recogntion of both I-region associated (la) major 
histocompatibility complex antigens and the conventional antigen to which 
the donor of the T lymphocytes had been primed. The role of la antigen 
recognition in this process has been strongly supported by the finding that 
anti-la antisera are potent inhibitors of T cell activation. Paradoxically, 
antisera to the "conventional" antigen have been uniformly ineffective in 
the blocking of T lymphocyte activation by antigen-pulsed macrophages. 
Although this failure of antibody to cause inhibition has certain physiologic 
advantages, it also raises the possibility that the stimulatory antigen 
may not be on the surface of the antigen-presenting cell. To further study 
this critical point, Laboratory of Immunology scientists have developed 
a T lymphocyte activation system in which the antigen-presenting cell is 
covalently derivatized with trinitrophenyl (TNP) groups and responding T 
cells are primed to TNP-macrophages _in vivo or in vitro . The antigen- 
presenting retains its capacity to stimulate responses by T lymphocytes 
even if it is incubated for 24 hours after derivatization before addition 
to T cells. Despite the capacity of these "aged" TNP-macrophages to 
stimulate a TNP-specific response, anti-TNP antibody has no inhibitory, 
effect on the activation process. However, if the macrophage is subject 
to a second round of derivatization with either TNP or DNP , just prior to 
being added to the T cells, its stimulatory activity is now sensitive 
to inhibition by anti-TNP antibody. This is particularly striking for the 
case in which DNP derivatization is used because DNP-derivatized macro- 
phages do not stimulate responses by T cells primed to TNP-macrophages, 
although, anti-TNP antibody does bind DNP. This result provides strong 
evidence that the stimulatory antigen is on the surface of the antigen- 
presenting cell. it appears the antigen is present at a density too low 



22-4 



to be cross-linked and cleared from the surface by anti-TNP antibody. The 
second round of derivatization increases the antigen density and thus leads to 
the result that the cell is now susceptible to blockade. However, these 
results still lead to the conclusion that the antigen on the macrophage, 
surface can stimulate T cell responses even in the presence of saturating 
amounts of antibody. These results have considerable significance in 
the understanding of the regulation of T lymphocyte immunity by antibody 
and in the elucidation of the molecular events in antigen recognition. 
(Thomas and Shevach; LI/NIAID) . 

Cellular Expression of Responder Phenotype in Complementing Immune Response 
Gene Systems 

Specific immune response ( Ir ) genes of the major histocompatibility 
complex control the ability of T lymphocytes to be activated by specific 
antigens. In certain systems, such as responses to poly (Glu,Lys,Phe) 
[GL$] and to pigeon cytochrome C, responsiveness depends upon the possession 
of responder alleles of Tr genes in both the I -A and I-E/C subregions of 
the mouse MHC. It had previously been shown in these and related systems 
that antigen-specific T lymphocyte activation by antigen-presenting cells 
(APC) required that the donor of the APC be a responder to the antigen 
under study. However, whether the responding T cell had to be derived 
from a responder donor was uncertain. Laboratory of Immunology scientists 
have studied this problem through the creation of bone marrow chimeras. 
Thus, F.. hybrids prepared by crossing complementing non-responder parents 
(i.e. Ir-GL$a 3 and Ir-GL$oi 8 mice) are capable of responding to GL$. 
If these mice are lethally irradiated and reconstituted with bone marrow 
from both parents, the resulting mice are unresponsive to immunization 
with GL$, indicated that some cell must possess both complementing Ir 
genes in order for a response to ensue. However, if T lymphocytes are 
transferred from such a reconstituted mouse to a lethally irradiated F.. 
recepient together with a source of F, APC and immunized immediately 
with GL$, they respond to GL$. This indicates that the inability of 
non-responder T lymphocytes to respond is not a genotypic character of 
the T cells, but depends, at least in part, upon cells in their sensitizing 
microenvironment. On the other hand, when bone marrow cells from F 
(responder) donors were used to reconstitute lethally irradiated mice of 
either of the non-responder parental types or of the responder F, type, 
only the F-i+F-, chimera could subsequently respond to antigen. This 
indicates that the developmental, as well as the sensitizing, microenviron- 
ment is critical for the acquisition of the responder phenotype by the 
T lymphocytes. These results provide further insight into the nature of 
the reagulation of immune responses by MHC I_r genes and should allow a 
better understanding of the linkage of disease susceptibility and resistance 
to MHC genes. (Longo and Schwartz, LI/NIAID). 

Three Cells Participate in the In Vitro T Lymphocyte Proliferative Response 
to Antigen 

It is now well recognized that antigen-specific activation of T lympho- 
cytes for proliferative, lymphokine producing, or helper responses can be 



22-5 



effeciently achieved by the presentation of antigen to the T cells in 
association with an la macrophage-like cell. The analysis of this antigen- 
presentation system has led to the demonstration that T lymphocytes recog- 
nize both antigen and I_-region gene products expressed on the surface of 
the antigen-presenting cell. Nonetheless, this analysis only demonstrates 
that cellular interactions are important in the artificial condition of 
addition of antigen-pulsed cells to a primed T lymphocyte population. It 
does not give a picture of the relative importance of such interactions in 
the normal in vitro response of unseparated lymphoid cells. 

In order to examine cellular interactions in unseparated cell popula- 
tions, Laboratory of Immunology scientists have studied the relation between 
numbers of cells cultured and response to added antigens. In an ideal 
situation, the diminution of response upon diluting a cell population by a 
factor of two, should be two-fold if a single cell type limits the response, 
four-fold if there are two interacting cell types both of which are limiting, 
and eight-fold, if there are three such limiting cell types. A plot of the 
log of number of added cells against log of the response will yield a 
slope of 1 for one limiting cell type, 2 for two limiting cell types, 3 for 
three limiting cell types, etc. In a large series of experiments, we found 
that the relationship between the log of number of primed nylon wool passed 
lymph node cells and the log of thymidine uptake in response to the immuniz- 
ing antigen was 3. This suggests that the _in vitro response normally in- 
volves the participation of three cell types. The nature of these inter- 
acting cells can be determined by adding a constant (and excess) number of 
a given cell type to all dilutions of the primed lymph node cells and 
measuring its effect on the slope of the resultant response. Using this 
analysis, it was shown that one of the cells was a radio- resistant cell 
present in spleens of non-primed mice; this cell is very likely an antigen- 
presenting cell. A second cell was a radio-sensitive T lymphocyte found 
in lymph nodes of primed and unprimed donors. This cell appears to 
represent a cell which is non-specif ically "recruited" to proliferate. The 
third cell type appears to be the antigen-specific T lymphocyte. Analysis 
of histocompatibility restrictions and expression of immune response gene 
function in these systems are now in progress. These experiments provide 
an approach to determine the extent to which rules derived from "synthetic" 
systems apply to cellular interactions in unseparated lymphoid cell popula- 
tions and will thus give a more complete understanding of the relative role 
of various cellular interactions in intact systems. (Tse, Schwartz and 
Paul; LI/NIAID). 

Patients with Systemic Lupus Erythematosus have a Defect in Suppressor 
Cell Function Associated with a Serum Autoantibody 

Systemic lupus erythematosus (SLE) is complex disorder associated 
with derangements in the regulation of immune responses and with the 
presence of antibodies to various autoantigens. Scientists in the 
Laboratory of Immunology and in the Arthritis and Rheumatism Branch of 
NIAMDD have shown that blood lymphocytes of SLE patients have a markedly 
diminished capacity to develop into suppressor cells as a result of 
stimulation with concanavalin a (Con A) . Con A induced suppressor cells 



22-6 



from normal individuals will suppress in vitro proliferative responses to 
Both pokeweed mitogen and autologous lymphoid cells (i.e. mixed lymphocyte 
responses [MLR]). In many patients with SLE, Con A fails to induce cells 
capable of suppressing either the pokeweed mitogen response or the MLR, 
whereas as other patients are defecient in only one of these suppressor 
functions . Sera from SLE patients with defects in capacity to develop 
suppressor cells contain cytotoxic antisera capable of causing complement 
mediated lysis of suppressor cell precursors in normal lymphocyte populations. 
Moreover, serum from patients with selective defects caused the same 
selective defect in normal lymphocyte populations treated with that serum and 
complement. These results suggest that separate T lymphocyte populations 
suppress the pokeweed mitogen response and the MLR. Fractionation of T lympho- 
cytes into those possessing Fc receptors for IgG (T cells) and those lacking 
such receptors indicates that T cells preferentially suppress the MLR. 
These studies provide important insights into the pathophysiology of the 
immune response in SLE patients and help to expand our understanding of 
the normal regulation of the human immune response. (Sakane and Green, 
LI/NIAID; Steinberg, Arthritis and Rheumatism Branch, NIAMDD) . 

Lack of Role for the Fourth Component of Complement (C4) in In Vitro T 
Lymphocyte Activation 

Other investigators have reported that antisera to human C4 inhibit the 
in vitro mixed lymphocyte response (MLR) . This implies that C4, perhaps as a 
membrane molecule, may have a critical role in the response of T lymphocytes, 
either by acting on the stimulatory or responding cell. Because of the 
potential importance of this finding, Laboratory of Immunology scientists 
have carefully examined this issue in guinea pig systems. Antisera to C4, 
raised in guinea pigs, rabbits and goats, were found to have no effect on 
in vitro proliferative responses to antigens and mitogens and did not 
block the MLR. Furthermore, T lymphocytes from guinea pigs with a genetic 
defeciency in C4 (C4D) responded normally to each of these stimulants and 
cells of C4D guinea pigs had normal capacity to present antigens and to 
stimulate mixed lymphocyte responses. These results suggest that if C4 
has an important role in the regulation of T lymphocyte activation, that 
role cannot be a general one involving all (or even most) types of such 
activation (Burger and Shevach, LI/NIAID) . 



22-7 



22-8 



Administrative, Organization and Other Changes 

The Laboratory of Immunology plays an important role in the training of 
young scientists. During the past year, Drs. Theo Kirland, Virgil Woods, Jr., 
Loran Clement and Ronald Wilder completed their appointments as research 
associates. Dr. Constantin Bona, who was a Visiting Scientist; Drs. Bondada 
Subbarao, Tsuyoshi Sakane and Franc Meloni, who were Visitng Fellows; and 
Drs. Herbert Herscowitz, Ole Werdelin, Kim Bottomly and Harley Tse, who were 
Guest Workers, completed research and training programs in the Laboratory of 
Immunology. Margaret Simons received her Ph.D. in genetics for research 
carried out in the Laboratory under the supervision of Dr. Rose Mage. Each 
of these scientists made substantial research contributions during their ten- 
ure in the Laboratory of Immunology. 

Drs. Laurie Glimcher, Dan Hansburg, John Schmidt and David Cohen entered 
the Laboratory as research associates while Dr. Dan Longo was converted from 
a Guest Worker to a Research Associate. Drs. Louis Matis and Robert Clark, 
clinical associates in the Laboratory of Clinical Investigation, were assigned 
to the Laboratory of Immunology. Drs. John Kung, Joe Chiba, Benjamin Sredni, 
and Shunichi Kumagai were appointed as Visiting Fellows and Dr. Leona 
Fitzmaurice entered the Laboratory as an Expert Consultant. 

Finally, one of the senior staff of the Laboratory, Dr. Donald Mosier, 
resigned his position in order to accept a major appointment at the 
Institute for Cancer Research, Fox Chase, Pennsylvania. Dr. Mosier had 
established an internationally recognized research program in cellular 
immunology. His loss as a contributor to the Laboratory program will be 
deeply felt. 



22-9 



22-10 



Honors, Awards, and Scientific Recognition 

Laboratory scientists play important roles in the national and inter- 
national scientific community. Many serve on editorial boards of important 
journals. Dr. Paul was appointed to the editorial board of the Journal of 
Immunology; Drs. Schwartz and Shevach are associate editors of this journal. 
Dr. Green and Dr. Shevach are referee editors of the Proceedings of the 
Society for Experimental Biology and Medicine. Dr. Mage is chairman of the 
editorial board of Federation Proceedings and is a member of the editorial 
board of the Journal of Immunologic Methods. In addition, Dr. Paul is an 
advisory editor of the Journal of Experimental Medicine and an associate 
editor of Immunologic Reviews, The Scandinavian Journal of Immunology, 
Immunologic Communications, CRC Critical Reviews in Immunology, and 
Human Immunology. Dr. Green is an associate editor of Clinical Immunology 
and Immunopathology, and Dr. Inman is an advisory editor of Molecular 
Immunology. Ms. Lieberman completed a term as an advisory editor of that 
journal. Dr. Shevach is a member of the editorial advisory board of the FASEB 
Handbook on Inbred Strains of Laboratory Animals. 

Dr. Paul presented invited lectures at the Southeastern Immunology 
Workshop, the Armand Hammer Conference on the Regulation of the Immune 
Response, the Midwest Student Medical Research Forum, the Scottsdale 
Conference on B Lymphocytes in the Immune Response, the Jane Coffin Childs 
Symposium, the Fogarty Center Workshop on Mediation of Effector 
Functions by Antibodies and the Symposium on Carcinogeneses of the Given 
Institute. He served as summarizing speaker at the National Institute on 
Aging Conference on Immunology and at the International Symposium on Aging 
and Immunity and was a session chariman at the Brook Lodge Conference on 
Mononuclear Phagocytes and the Scottsdale Conference on B Lymphocytes in the 
Immune Response. In addition Dr. Paul was elected President-elect of the 
American Society for Clinical Investigation and was appointed chairman of 
the American Association of Immunologists Program Committee. He is a member 
of The American Cancer Society Committee on Personnel for Research and the 
Arthritis Foundation Fellowships sub-committee and is co-organizer of the 
Cold Spring Harbor Immunogenetics Course. 

Dr. Green was an invited lecturer at the Intenational Congress on 
Systemic Lupus Erythematosus, held in Kyoto; at the International Congress 
on Vasculitis in Innsbruck; and at the Summer Course in Methods of 
Immunological Research and Diagnosis. He is co-chairmen of the American 
Association of Immunologists Program Committee and is a member of the 
Transplantation Immunology Committee of NIAID. 

Dr. Shevach gave major lectures at the ICN-UCLA meeting on T and B 
Lymphocytes, at the Annual Meeting of the New England Society for Allergy 
and at the Brook Lodge Conference on Mononuclear Phagocytes. He is a 
member of the Allergy and Immunology Study Section of the Division of 
Research Grants, NIH. 



22-11 



Dr. Schwartz gave invited lectures at the. ICN-UCLA Meeting on T and B 
Lymphocytes and at the Brook Lodge Conference on Mononuclear Phagocytes. He 
was co-chairmen of a minisymposium at the annual meeting of the American 
Association of Immunologists. 

Dr. Mage, Ms. Lieberman and Dr. Bona were invited lecturers at the 
International Colloquium in honor of Jacques Oudin at the Institut Pasteur, 
in Paris. Dr. Mage is Vice-President of the D.C. Chapter of the Society of 
the Sigma Xi. Ms. Lieberman is a member of the American Association of 
Immunologists Membership Committee. 

Dr. Inman was an invited speaker at the European Molecular Biology 
Organization Workshop on Accuracy in Biology, at the Netherlands Red Cross 
Transfusion Service and at the Central Laboritorium TNO Ryswegh, Netherlands. 
Dr. Waxdal was a major speaker at the Colloquium on Membrane Glycoconjugates, 
in Seillac, France, and at the Colloquium on Protides of the Biological 
Fluids, in Brussels. He was an invited discussion leader at the Symposium 
on Biomedical Applications of the Horseshoe Crab at Woodshole MA. and was 
chairman of the session on Biochemical Consequences of Mitogen Activation 
at the annual meeting of the American Association of Immunologists. 

In addition, laboratory scientists presented research seminars at major 
universities and research institutes in the United States and abroad. 



22-12 



SMITHSONIAN SCIENCE 

PROJECT NUMBER (Do NOT use th 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT . . . ^ 

Principal Investigator : William E. Paul, Chief, LI/NIAID 

Rose Lieberman, Sr. Investigator, LI/NIAID 

Donald E. Mosier, Inst, for Cancer 



NFORMATION EXCHANGE 
space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl AI 00030-11 LI 



PERIOD COVERED 



ctober 1, 1978 - September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Antigen Recognition and Activation of Immunocompetent Cells 



Other Investigators : 



Research, Phila. , PA. 
Bondada Subbarao , Inst, for Cancer 
Research, Phila., PA. 



Donna G. Sieckmann, Staff Fellow, LI/NIAID 

Kathryn Stein, Staff Fellow, LI/NIAID 

Toby Hecht, Sr. Staff Fellow, LI/NIAID 

Constant in Bona, Visiting Scientist, LI/NIAID 

Patricia Mongini, Guest Worker, LI/NIAID 

John Kung, Visiting Fellow, LI/NIAID 

Ellen Heber-Katz, Staff Fellow, LI/NIAID 

James Mond, Uniformed Services University of the Health Sciences 

Irwin Scher, Naval Med. Res. Inst. 

Aftab Ahmed, Naval Med. Res. Inst. 

Steven Kessler, Naval Med. Research Institute 



COOPERATING UNITS (if any) 

LMI/NIAID; Naval Medical Research Institute; Institute for Cancer Research, 

Philadelphia, PA 



LAB/BRANCH 

Laboratory of Immunology 



institute and locat I ONNat ional Institute of Allergy and Infectious Diseases. 
National Institutes of Health, Bethesda, Maryland 20014 



TOTAL MANYEARS: 
9 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS 

□ (a1 ) MINORS □ (a2) INTERVIEWS 



□ (b) HUMAN TISSUES 



^ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

The general goal of this project is to delineate the mechanisms involved 
in antigen recognition by and activation of thymus-independent (B) and thymus - 
dependent (T) lymphocytes. During the past year our efforts have concentrated 
on: 1) the development of new genetic models of _B lymphocyte deficiency; 
2) the identification and genetic mapping of B lymphocyte differentiation 
antigens ; 3) the study of genetics of IgD and of recombination within the IgC^ 



gene complex; 4) the genetic regulation and chemical characteristics of anti- 
bodies produced in response to polysaccharide haptens ; 5) the regulation of 
antibodies and T_ lymphocytes capable of interacting with membrane immunoglobulin , 



In addition, studies of the regulation of growth of vesicular stomatitis 
yirus in spleen cells, both _in vivo and _in vitro are underway. 



22-13 



PHS-6040 
(Rev. 10-76) 



Z01 AI 00030-11 LI 

Genetic models for the study of B lymphocyte development 

Over the past several years we have investigated the developmental and 
functional heterogeneity of thymus-independent (B) lymphocytes through the 
use of a mutant mouse strain. Mice of this strain, CBA/N, are unresponsive 
to thymus-independent type 2 (TI-2) antigens and lack a subset of B lympho- 
cytes which express the differentiation antigens Lyb 3,5, and 7. Recently, 
we have observed that breeding the mutant CBA/N gene, xid , on to a C3H back- 
ground leads to a much more profound defect due to a genetic synergy between 
the xid gene and one or more C3H genes. The "synergistic defect" (SD) is 
characterized by _in vitro unresponsiveness to TI-1 as well as TI-2 antigens 
and by a failure of B lymphocytes to proliferate in response to a series of 
mitogens including lipopolysaccharide, bacterial lipoprotein, and Nocardia 
water soluble mitogen. Mice with the SD phenotype have diminished numbers of 
B lymphocytes and the B lymphocytes they possess express very low ratios of 
membrane (m) IgD:mIgM. We are currently in the process of preparing inbred 
and congenic mouse strains expressing the SD phenotype in order to make these 
mice available for the detailed study of B lymphocyte function and development. 

A concurrent program is the analysis of B lymphocyte subsets through 
the development of antisera to differentiation antigens expressed by these 
subpopulations. In last year's report, we described the identification and 
mapping of Lyb 5 and Lyb 7. Both markers are found on that subpopulation 
of B lymphocytes which is lacking in mice with the CBA/N immune defect. 
Lyb 7 is particularly interesting because antisera to it block B lymphocyte 
activation by TI-2, but not TI-1, antigens and because Lyb 7 is genetically 
linked to genes specifying immunoglobulin heavy chain constant regions 
(IgC genes) . 

We are attempting to expand our "library" of antibodies to B lymphocyte 
differentiation antigens by the preparation of rat anti-mouse B lymphocyte 
hybridomas. A series of such hybrid tumors have been produced, and pre- 
liminary analysis of their secreted products is in progress. Several of these 
reagents appear to identify B lymphocyte subpopulations and will be of 
considerable interest in our continued study of B lymphocyte function. 

Linkage and recombination of immunoglobulin structural genes . 

Studies of the genetic regulation of mlg expression are also in progress. 
We have available a series of monoclonal, alio- and hetero- anti-6 anti- 
bodies and have recently completed an extensive strain survey allowing us to 
more completely make IgC haplotype assignments among common inbred 
laboratory strains. An interesting result of this is the observation that 
the d and e haplotype groups need to be further subdivided because strains 
previously assigned to each of these groups differ in IgD allotype from other 
strains within the same group. On this basis, we have defined the n and 
o IgC haplotypes. Furthermore, one inbred strain, AL/N, appears to be a 
natural IgC recombinant between the A and AKR strains, possessing the IgD 
alleles of A 1 and the IgG alleles of AKR. This observation is supported by 
our identification of two actual intra- IgC recombinant mice, which arose 

22T-14 



Z01 AI 00030-11 LI 

during backcrossing of (C57BL/6 x DBA/2)F.. hybrids to C57BL/6. These mice, 
individuals 72 and 74, express mlgD and serum IgD of the DBA/2 type but lack 
DBA/ 2 type IgM, IgG , and IgG . This phenotype is stable on subsequent 
breeding of 72 and 74 to C57BE/6. Furthermore, when mice of the 72 and 74 
families are crossed to strain A mice, individuals which inherit DBA/2 IgD 
also inherit C57BL/6 IgG. , proving that these allelic genes are on the same 
chromosome and establishing that a recombination has actually occurred. The 
availability of these IgM-IgD recombinant strains should make possible the 
precise mapping of genes for V region markers with reference to these two 
critical constant region genes. 

Genetics of immune responses 

In association with these studies of genetics of immunoglobulin 
structural genes, studies of the genetic basis of antibody responses to 
highly defined polysaccharide antigens are in progress. Lieberman and her 
colleagues have previously established that the capacity to make antibodies 
to 6(2-^1) fructosan determinants, such as those in inulin, upon immunization 
with bacterial levan, a g (2->6) polyfructosan with (2-*-l) branch points, is 
determined by genes linked to IgC genes. Our current studies indicate that 
non -IgC genes play a critical role in the magnitude and clonal diversity of 
the response. Most strikingly, BALB/c mice immunized with bacterial levan 
make a highly restricted anti-inulin response, dominated by an idiotype- 
positive antibody appearing as a characteristic triplet of bands on iso- 
electric focusing. Although C57BL/6 mice lack the structural genes for the 
triplet and related anti-inulin antibodies, they possess non- immunoglobulin 
genes which increase and diversify the response. This is shown by an 
analysis of responses of (BALB/c x C57BL/6)F.. hybrids, allotype congenic 
B.C8 mice, and recombinant inbred CXB mice. Our current analyses suggests 
that two distinct genes are involved; we are in the process of verifying 
this and mapping those genes. In addition, we are studying the genetic 
regulation of the response to isomaltotriose and isomaltohexose derivatives of 
proteins in normal mice and in mice with the CBA/N immune defect. 

Regulation of B lymphocyte activation through mlg 

Over the last several years, we have developed persuasive evidence 
that purified antibodies to IgM and to L chains can stimulate substantial 
proliferative responses on the part of B lymphocytes while, at the same 
time, inhibiting the secretion of immunoglobulin in response to other 
stimulants. Our data has indicated that this proliferative response does 
not require the presence of either T lymphocytes or macrophages but that it 
is a function of a late appearing subset of B lymphocytes which is absent in 
mice with the CBA/N immune defect. During the past year, a detailed study 
of the phenotype of the responding cells has been carried out. Particular 
emphasis has been placed on the characterization of these cells in terms of 
the density of mlg of various classes expressed on their membranes. To 
carry out these studies, B lymphocytes have been prepared and separated into 
subpopulations with distinct amounts of Ig on their surface through the use 
of the fluorescence activated cell sorter. Through this approach, we have 

22-15 



Z01 AI 00030-11 LI 

shown that cells which proliferate in response to both anti-p and anti-K 
express a high density of total Ig, a low density of IgM, and an intermediate 
to high density of IgD. Preliminary studies indicate these cells also express 
Lyb 5. We are now in the process of preparing hybridoma rat anti-mouse u 
antibodies inorder to more decisively explore the recognition events involved 
in B cell activation. 

In a closely related set of studies, we have shown that T lymphocytes 
with receptors specific for idiotypic determinants found on the TNP-binding 
myeloma protein MOPC460 suppress the production of anti-TNP antibodies which 
express this idiotype. Furthermore, we have evidence that such cells not 
only exist but that they play an important normal role in regulating the 
clonal nature of the anti-TNP response to TNP antigens. These studies are 
particularly interesting because they provide one of the first examples of 
direct action of suppressor T cells on B cells and because they comprise one 
of the first instances of the physiologic activity of the idiotype-anti- 
idiotype network. 

Regulation of infection of spleen cells with vesicular stomatitis virus (VSV) 

VSV is a membrane budding virus which fails to grow in resting lympho- 
cytes and which has been reported to contain host cell membrane proteins, such 
as H-2 antigens, in its envelope. We are in the process of testing the thesis 
that H-2 antigens associated with VSV virions may provide a means of attack of 
the host immune system upon virus and that H-2 antigens associated with virus 
play a strong evolutionary role in promoting H-2 diversity and the high level 
of "spontaneous" immunity to H-2. To study this, we have developed procedures 
for the m. vivo and in vitro infection of mouse spleen cells with VSV obtained 
from various cell lines and have shown that immunity to the cell line in which 
VSV is grown profoundly effects the capacity of the virus to infect spleen 
cells in vivo or in vitro . Studies are in progress to obtain more reproducible 
conditions for inducing in vivo and in vitro infection of lymphocytes by VSV, 
for more precisely evaluating the immunologic mechanisms underlying the 
distinctions made between VSVs obtained from cell lines of distinct H-2 type, 
and to directly determine what host component is, in fact, responsible for the 
capacity of the immune system to determine the "lineage" of the virus. 



22-16 



Z01 AI 00030-11 LI 

PUBLICATIONS 

Paul, W.E., Scher, I., Mond, J.J., Ahmed, A., Subbarao, B. and Mosier, D.E.: 
B lymphocyte development, heterogeneity and function. Arthritis and 
Rheumatism 21: 5175, 1978. 

Mond, J.J., Scher, I., Mosier, D.E., Blaese, M. and Paul, W.E.: T independent 
responses in B cell defective CBA/N mice to Brucella abortus and to TNP 
conjugates of Brucella abortus . Eur . J . Immunol . 8: 459, 1978. 

Sieckmann, D.G., Scher, I., Asofsky, R., Mosier, D.E. and Paul, W.E.: 
Activation of mouse lymphocytes By ant i- immunoglobulin. II. Requirement 
for a mature subset of B lymphocytes. J. Exp. Med . 148: 1628-1643, 1978. 

Mosier, D.E., Zitron, I.M. , Mond, J.J. and Paul, W.E.: Requirements for 
induction of antibody formation in the newborn mouse. Strasburger 
Symposium on Developmental Immunobiology (Eds. G.W. Siskind and M. Weksler) 
Grune and Stratton, New York. In press. 

Paul, W.E., Subbarao, B. , Mond, J.J., Sieckmann, D.G., Zitron, I.M., 
Ahmed, A., Mosier, D.E. and Scher, I. B lymphocyte development and 
activation: Analysis with a mutant mouse strain. Cells of Immunoglobulin 
Synthesis (Eds. B. Pernis and H.J. Vogel) . In press. 

Paul, W.E.: Genetic regulation of the immune response. Immunopa t ho lo gy 
(Eds. J. Mohn and F. Milgrom) S. Karger AG, Basel, Switzerland. In press. 

Ahmed, A., Subbarao, B., Scher, I., Ryan, J.J., Paul, W.E., Mosier, D.E. and 
Sell, K.W. : Preliminary evidence for a new non-H-2 locus which codes for 
lymphocyte activating determinants distinct from Mis. Transplant. Proc . 
In press. 

Paul, W.E.: Lymphocyte biology. Clinical Immunology (Ed. C.W, Parker) 
Saunders, New York. In press. 

Bona, C. and Paul, W.E.: Cellular basis of regulation of expression of 
idiotypes. I. Suppressor cells specific for MOPC460 idiotype regulate the 
expression of cells secreting anti-TNP antibodies bearing 460 idiotype. 
J. Exp. Med . 149: 592-600, 1972. 

Bona, C, Mond, J.J. , Kessler, S., Scher, I. and Paul, W.E.: "Synergistic" 
immune defect in backcross male mice from matings of CBA/N and C3H/HeJ lines. 
In B-Cell Differentiation (Edited by I. Scher). Academic Press. In press. 

Bona, C. , Hooghe, R. , Cazenave, P. A., Leguern, C. and Paul, W.E.: Cellular 
basis of regulation of expression of idiotype. II. Enhancement of MOPC460 
idiotype component of anti-TNP antibodies by antibody against anti-MOPC460 
Id antibodies. J. Exp. Med 149: 815-823, 1979. 



22-17 



Z01 AI 00030-11 LI 

Bona, C. , Cazenave, P. A. and Paul, W.E.: REgulation of anti-TNP response by 
antiidiotypic and anti-(antiidiotypic) antibodies. Ann. Immunol. (Inst . 
Pasteur ) 130C: 303, 1979. 

Paul, W.E.: Genetic control of the immune response. In Textbook of 
Rheumatology . 

Sieckmann, D.G., Scher, I. and Paul, W.E.: B lymphocyte activation by 
anti- immunoglobulin antibodies. In Physical-Chemical Aspects of Cell Surface 
Events in Cellular Regulation . (edited by R. Blumenthal and C. DeLisi) 
Academic Press. In press. 

Subbarao, B. , Hosier, D.E., Ahmed, A., Mond, J.J., Scher, I. and Paul, W.E.: 
Role of a non-Ig cell surface determinant in the activation of B lymphocytes 
by thymus- independent antigens. J . Exp . Med . 149: 495-506, 1979. 

Subbarao, B. , Ahmed, A., Paul, W.E., Scher, I., Lieberman, R. and Mosier, D.E. 
Lyb 7, A B cell alloantigen controlled by genes linked to the IgC,, locus. 
J. Immunol . In press. 

Bona, C. and Paul, W.E.: Cellular basis of the regulation of production of 
anti-TNP antibodies carrying MOPC460 idiotype. 1979 ICN-UCLA Symposia, 
Keystone, Colorado. 

Mond, J.J., Stein, K. and Paul, W.E.: Analysis of B cell activation require- 
ment using the conjugated polyacrylamide beads. J . Immuno 1 . In press. 

Perlmutter, R.M. , Nahm, M. , Stein, K.E. , Slack, J., Zitron, I., Paul, W.E. 
and Davie> J-M..: Immunoglobulin subclass-specif ic immunodeficiency in mice 
with an X-linked B-lymphocyte defect. J. Exp. Med . 149: 993-998, 1979. 

Stein, K.E., Mond, J.J., Brennan, C, MatikelH, 0. and Paul, W.E.: Antibody 
affinity in CBA/N mice. ICN-UCLA Symposia, Keystone, Colorado. 

Mond, J.J., Sehgal, E., Sachs, D.H. and Paul, W.E.: Expression of la antigen 
on adult and neonatal B lymphocytes responsive to thymus independent antigens. 
J. Immunol . In press. 



22-18 



ITHSONIAN SCIENCE INFORMATION EXCHAN 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



Z01-AI-00148-04 LI 



PERIOD COVERED 

October 1, 1979 - September 30, 1980 



TITLE OF PROJECT (80 characters or less) 

Lymphocyte Interactions, Receptors and Functions 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Principal Investigator: Ira Green, M.D., Senior Investigator, LI/NIAID 



Other Investigators: 



Tsuyoshi Sakane, LI/NIAID 

Myron Waxdal, LI/NIAID 

Ethan Shevach, LI/NIAID 

Alfred Steinberg, Arthritis & Rheumatism Branch, 

NIAMDD/NIH 
Herbert Herscowitz, Dept. of Microbiology, Georgetown 

University School of Medicine 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Immunology 



institute and location National Institute of Allergy and Infectious Diseases, National 
nrgs of HPalfh. RpfhPsria, Maryland 2 0? 05 



Tnsti ti 



TOTAL MANYEARS: 



PROFESSIONAL: 
2_ 



CHECK APPROPRIATE BOX(ES) 
Q (a) HUMAN SUBJECTS 

□ (a1 ) MINORS □ (a2) It 



EJj] (b) HUMAN TISSUES 



□ (c) NEITHER 



SUMMARY OF WORK (200 words or less - underline keywords) 

A B cell leukemia (L C) of inbred strain 2 guinea pigs and a new myelogenous 
leukemia (G-13) of inbred strain 13 guinea pigs are being investigated as models 
of human leukemia. The chemical nature of the tumor specific transplantation 
antigen (TSTA) of the L„C leukemia is being investigated by immunization protec- 
tion tests in syngeneic animals. The TSTA of the L C leukemia is unusual in 
that it is of low molecular weight and is extremely resistant to heating. The 
G-13 leukemia is being examined for the presence of a TSTA and the presence of 
cell surface markers. As in the human, the myeloblasts of this leukemia are 
la . The immune function of these cells are also being investigated. 

Patients with systemic lupus erythematosus (SLE) have a defect in the 
development of Con A induced suppressor cells. Moreover, the sera from lupus 
patients (more particularly the IgM fraction) can induce suppressor cell defects 
in populations of normal lymphocytes. T and T cells mediate different type^ 
of suppression; certain lupus sera are specifically cytotoxic for T cells. 
Finally T cells able to proliferate in response to autologous non T cells act as 
precursors of Con A induced supppressor cells. 



PHS-6040 
(Rev. IO-76) 



-±9- 



Z01-AI-00148-04 LI 

I. Studies of the Guinea Pig L C Leukemia and a New Myelogenous Leukemia 

The L C lymphatic leukemia of inbred strain 2 guinea pigs arose about 25 
years ago in an old female strain 2 guinea pig. Studies in our laboratory 
starting 8 years ago have shown that this leukemia cell is of B cell origin 
and has surface IgM and C3 receptors. There are several variants of this 
leukemia; one variant exists which lacks la antigens (an antigen of the major 
histocompatibility complex of the guinea pig) . Our laboratory has demonstra- 
ted that, as measured by immunization protection tests in inbred strain 2 
guinea pigs, the L C cell has a powerful tumor specific transplantation 
antigen (TSTA) . However, in the la negative variant of the L C cell, this 
TSTA is not immunogenic. Our previous studies suggested that the TSTA is 
not related to the surface IgM molecule. 

The main goal of the present study is to identify the chemical nature of 
the TSTA of the L„C leukemia cell. A 3M KC1 extract of the L 9 C cell contains 
the TSTA as measured by immunization protection tests in syngeneic strain 2 
animals. Immunization with as little as 100 ug of this material per animal 
confers protection against a lethal challenge of L 9 C cells. Fractionation 
of the KC1 extract on Sephadex G-200, DEAE cellulose and CM cellulose 
indicate that the immunogenic molecule has a M.W. of less than 20,000 and is 
basic. The material is very resistant to heat and extremes of pH but is 
destroyed by treatment with trypsin, neuramnidase and periodate. These 
properties suggest that the TSTA is a low m.w. glycoprotein. Attempts to 
further fractionate the material by polyacrylamide gel electrophoresis were 
unsuccessful, none of the fractions eluted from these gels were immunogenic. 

Most recent studies have used CM cellulose followed by G-50 chromato- 
graphy. Several immunogenic regions from G-50 chromatography have been 
obtained and are now being currently investigated. Progress in the chemical 
identification of the TSTA of the L C cell has been slow because each 
individual assay takes 7-8 weeks to~complete (in vivo protection being the 
final end point) . 

In addition to the studies of the L„C leukemia, we have been studying 
a new myelogenous leukemia of strain 13 guinea pigs induced with the carcino- 
gen N-nitroso-N-butylurea (Evans et al. , Cancer Research, Vol. 38: 130, 1978). 
This leukemia is serially transplantable in inbred strain 13 guinea pigs 
using intradermal or intraperitoneal injection of whole cells. Two to three 
weeks after injection of the cells there is a rise in polymorphonuclear 
leukocytes; this is soon followed by an increased number of myeloblasts. The 
WBC finally rises to > 100,000 cmm and the animal succumbs. Paralysis of the 
hind legs is often observed as a terminal event. 

We have characterized the surface markers on these myeloblasts; these 
cells lack surface Ig and do not have C3 or Fc receptors. These cells do 
have la antigens on their surface as determined by immunof luorescent 
techniques; biosynthetic studies indicate that the la antigens found on these 
cells are actually synthesized by the cells. 



22-20 



Z01-AI-00148-04 LI 

To determine whether la antigens were also found on more mature cells 
of the myelocytic series the cells were first stained in suspension in the 
living state for the presence of surface la antigens. The cells were then 
smeared on gridded slides (each grid having a number) and the position of the 
la positive (stained) cells was noted. The smear was then stained by Giemsa 
stain, the same cells were again located and their morphology noted. The 
results of such experiments demonstrated that most myeloblasts were la where- 
as most bands and polymorphonuclear leukocytes were negative. 

The la positive myeloblasts were then studied for two immunological 
functions. First the myeloblasts were tested for their ability to act as 
stimulator cells for the MLR. Second the myeloblasts were tested for their 
ability to act as antigen presenting cells for the T cell proliferative 
response to antigen. They failed in both of these functional tests. This 
observation strongly suggests that the presence of surface la antigens is 
necessary but not sufficient to allow a cell to perform such immunological 
functions. 

Preliminary immunization protection studies to determine whether these 
cells possess a tumor specific transplantation antigen (TSTA) indicate that 
the cells do have a TSTA. In immunized animals, large f ungating tumor masses 
were observed to regress. 

Significance to Biomedical Research and the Program of the Institute 

The properties of the TSTA of the L ? C leukemia cell are highly unusual 
and differ from the properties of most other TSTA described by other investi- 
gators. Most other TSTA have a m.w. greater than 40,000 daltons and are 
sensitive to heat. 

The fact that this TSTA is on a malignant B lymphocyte is of particular 
interest; once the L„C TSTA is more completely identified one could look for 
a similar TSTA in several different human B cell leukemias. Another possi- 
bility is that this TSTA could represent a marker for a particular stage of B 
cell differentiation present on only a very small number of normal B cells. 

Studies of this myelogenous leukemia are of potentially great interest 
since in some cases of human myeloid leukemia the myeloblasts are also la pos- 
itive. The availability of large numbers of la myeloblasts may make possible 
further chemical studies of these la antigens and to allow comparison of these 
la antigens with those on lymphoid cells. Further functional studies using 
these la myeloblasts may provide a clue as to why la antigens are present on 
myeloblasts. This leukemia may also serve as a model for chemo- immunotherapy 
of myelogenous leukemia. 

Human Suppressor Cells in Health and Disease 

The second aspect of our studies concerns suppressor cells in patient 
with systemic lupus erythematosus (SLE) . The system we are using is adapted 
'from Shou _et al. , J. Exp. Med. 143, 1100, 1976. The use of this system to 

22-21 



Z01-AI-00148-04 LI 

describe concanavalin induced suppressor cells in normal individuals has 
already been published by us in the J. of Immunology 119 , 1169, 1977. 

A two stage assay system is employed in which T lymphocytes are exposed 
to concanavalin A (Con A) for 3 days to induce suppressor function and then 
these cells, after mitomycin C treatment, are added to an assay system con- 
sisting of lymphoid cells obtained from the same donor 3 days later. As a 
control, another aliquot of cells incubated without Con A is used. These 
assay cultures are then stimulated by either mitogen or allogeneic cells, 
cells from the first culture are added and the degree of proliferation is 
measured by H-thymidine incorporation. 

We have recently demonstrated that aliquots of these same Con A activated 
T cells can also suppress a pokeweed mitogen (PWM) induced polyclonal B cell 
response. 

Our major effort has been to analyze suppressor cell defects in patients 
with SLE. We first demonstrated that patients with SLE have a relative lack 
of T cells capable of being induced to form suppressor cells. Furthermore 
when certain SLE sera are added to cultures of normal lymphocytes during the 
Con A mediated induction of suppressor cells, no suppressor cells develop. 
We had previously shown that the IgM fraction of SLE sera was responsible for 
this effect and that complement is necessary. Our most recent studies have 
revealed that the SLE sera is only effective when added at the beginning of 
the Con A inducing culture but not at the end. We also observed that certain 
patients with SLE had a selective loss of only one type of suppressor cell. 
That is, the patients lymphocytes could suppress the PWM proliferative re- 
sponse but not the MLR. The sera from these same patients could induce in an 
in vitro culture exactly this same pattern of defects in the lymphocytes from 
normal individuals. The fine cytotoxic specificity of the sera from these 
selected patients was then tested on different classes of T cells. T cells 
can be separated into two classes, one class having a surface receptor for the 
Fc portion of IgG. (T^ cells) and another class lacking this receptor (T 
cell). These sera were selectively cytotoxic for T cells. We then investi- 
gated whether different T cell classes could differentially suppress either 
the PWM proliferative response or the mixed lymphocyte response (MLR) . We 
observed that T cells could preferentially suppress the MLR. Thus the ability 
of certain SLE sera to eliminate suppressor cells for the MLR can be ascribed 
to their preferential cytotoxicity for T cells. 

We have also performed two other related studies. First we have serially 
studied patients with SLE during the active and inactive phases of their 
disease for both defects in suppressor cells development for defects in the 
autologous MLR (discussed in last year's annual report). 

In each case these _in vitro tests were abnormal during the active phase 
of disease and returned to normal when the disease activity decreased. These 
results strongly suggest that the abnormalities of suppressor cell generation 
and the autologous MLR observed in lymphocytes from patients with SLE are not 
"intrinsic to the lymphocyte or genetically determined. 

22-22 



Z01-AI-00148-04 LI 

In the second study we wished to determine whether cells proliferating 
in the autologous MLR were particularly able to develop Con A induced supp- 
ressor function as compared to cells proliferating in the usual cells MLR. 
Therefore cells undergoing either auto MLR or allogeneic MLR were treated or 
not treated with BUdR and light. 

The T cells were then harvested and tested for their ability to generate 
suppressor function. The cells undergoing an autologous MLR in the presence 
of BUdR and light failed to subsequently develop suppressor function whereas 
the cells undergoing an allogeneic MLR were able to develop suppressor func- 
tion. 

Significance to Biomedical Research and the Program of the Institute 

The defect in the development of suppressor cells in patients with SLE 
may be one of the causes of increased auto antibody production in this 
disease. Furthermore, sera from patients with SLE can induce suppressor cell 
abnormalities in normal T lymphocyte. Strategies to increase suppressor 
function in patients with SLE might have therapeutic potential. Selective 
removal of antibodies to suppressor cells might be a step in this direction. 

PUBLICATIONS : 



Sakane, T. , Steinberg, A.D. and Green, I.: Failure of suppressor T cell 
activity in patients with systemic lupus erythematosus (SLE) . Arthritis 
and Rheumatism . 21: 657, 1978. 

Gelfand, M.C., Frank, M.M. , Green, I. and Shin, M.L.: Binding sites for 
immune- complexes containing IgG in the renal interstitum of man and 
other species. Clinical Immunology and Immunopathology 13: 19, 1979. 

Stingl, G. , Katz, S.I., Clement, L., Green, I. and Shevach, E.M. ; Immuno- 
logical functions of la-bearing epidermal Langerhans cells. J. Immunol . 
121: 2005, 1978. 

Carter, R. , Gwadz, R.W. and Green, I.: Plasmodium gallinaceum : Transmission 
Blocking Immunity in the Chicken with Aedes algypti as the Mosquito Vector. 
II. The Effect of Anti-Gamete Antibodies _In Vitro and InVivo and their 
Elaboration During Infection. Experimental Parasitology . In press. 

Nilsson, S.F., Edelson, R. , Mann, D. , Green, I. and Waxdal, M.J.: Concanavalin 
A binding proteins on the surface of human malignant and normal lymphocytes. 
Scand. J. Immunol . In press, 1979. 

Bona, C, Lieberman, R., House, S., Green, I. and Paul, W.E. : Immune 
Response to Levan. II. T-Independence of Suppression of cross-Reactive 
Idiotypes by Anti-Idiotype Antibodies. J. Immunol . 122: 1614, 1979. 



22-23 



Z01-AI-00148-04 LI 

Sakane, T., Steinberg, A.D., Reeves, J. P. and Green, I.: Studies of 
Immune Functions of Patients with Systemic Lupus Erythematosus. Complement 
Dependent IgM anti-T Cell Antibodies Preferentially Inactivate Suppressor 
Cells. J. Clin. Investigation . 63: 954, 1979. 

Sakane, T., Steinberg, A.D., Arnett, F.C., reinertsen, J.L. and Green, I.: 
Studies of immune function of patients with systemic lupus erythematosus. 
III. Characterization of the lymphocyte subpopulations responsible for 
the defective autologous MLR. Arthritis and Rheumatism . In press. 

Sakane, T. and Green, I.: Specificity and suppressor function of human 
T cells responsive to autologous non-T cells. J. Immunol . In press. 

Sakane, T., Steinberg, A.D., Reeves, J. P. and Green, I.: Studies of 
immune functions of patients with systemic lupus erythematosus. T cell 
subsets and antibodies to T cell subsets. J. Clin. Investigations . In 
press. 

Sakane, T., Steinberg, A.D. and Green, I.: Studies of immune functions of 
patients with systemic lupus erythematosus. V. T-cell suppressor function 
and autologous MLR during active and inactive phases of disease. Arthritis 
and Rheumatism. In press. 



22-24 



SMITHSONIAN SCIENCE INFORMATION EXCHANGE 
PROJECT NUMBER (Do NOT use this space) 



U.S. DEPARTMENT OF 
HEALTH, EDUCATION, AND WELFARE 
PUBLIC HEALTH SERVICE 
NOTICE OF 
INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl AI 00035-06 LI 



PERIOD COVERED 

October 1, 1978 - September 30, 1979 



TITLE OF PROJECT (80 characters or less) 

Specificity in Immune Responses 



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER 
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



Principal Investigator : 
Other Investigators: 



John K. Inman, Senior Investigator, LI/NIAID 

Dr. Ettore Appella, LCB/NCI 
Dr. Garrett C. DuBois, LCB/NCI 
Dr. Bruce Merchant, BOB/FDA 



COOPERATING UNITS (if 

Laboratory of Cell Biology, National Cancer Institute; Immunology Branch, 
National Cancer Institute; Bureau of Biologies, FDA. 



iny) 

:el] 



lab/branch 

Laboratory of Immunology 



INSTITUTE AND LOCATION 



National Institute of Allergy and Infectious Diseases, 
National Institutes of Health, Bethesda, Maryland 20205 



TOTAL MANYEARS: 

2 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 
G (a) HUMAN SUBJECTS 

Q (al) MINORS G (a 2 ) INTERVIEWS 



G (b) HUMAN TISSUES 



SUMMARY OF WORK (200 words or less - underline keywords) 

The long range objective of this project is to carefully re-examine the 
nature of binding specificity at antibody combining regions and other types of 
receptor sites. The effects of polyvalent binding on binding energy and spec- 
ificity will be considered, and together these properties will be related to 
the behavior of immune systems. In particular, multispecif ic binding of disp- 
arate structures by individual antibody species is being studied through 
screening of specifically purified, radiolabeled antibodies with affinity 
chromatography columns. The ligands are bound to these columns using large 
haptenic reagents synthesized specially for this purpose using methods of 
peptide synthesis . Multispecif ic binding will be used for producing and 
isolating homogeneous antibody by cross-stimulation and immunoadsorption . A 
number of chemical techniques and products that have been developed from the 
above work are being used in collaborative studies on molecular interpretation 
of cellular mechanisms in immune responses. Materials employed include syn- 
thetic thymus- independent antigens . 
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PHS-6040 
(Rev. 10-76) 



Z01 AI 00035-0 5 LI 

Project Description 

Objectives 

The overall objective of the project is to gain more complete under- 
standing of the molecular basis for specificity in immune responses. The 
binding properties of antibodies and other receptor-bearing molecules are 
to be studied and related to the behavior of immune systems. Specific 
objectives are the following: 

1. To assess the generality and frequency of multispecif ic binding by 
individual antibody combining regions by means of screening experiments 
employing many diverse, structurally unrelated haptens. 

2. To develop methodology required for such screening tests including the 
synthesis of numerous, large haptenic reagents. 

3. To employ multispecif ic binding and cross-stimulation in raising high 
titers of highly restricted antibodies for fine specificity studies and for 
use as highly specific reagents in immunological studies. 

4. To use the large hapten screen to find disparate structure cross-reactions 
useful in identifying antibody-secreting cell clones in studies assessing 
antibody diversity. 

5. To use the screen in studying the multispecif ic character of other types 
of receptors and to employ observed cross-reactions in a general approach 

to affinity separations of receptors. 

6. To employ methods and reagents developed in this project in collaborative 
studies dealing with specificity in immune responses. 

Major Findings 

The main emphasis during the past year has been placed on exploring 
strategies for the efficient synthesis of highly varied specificity probes. 
These probes are reagents for introducing large haptenic groups onto 
macromolecular carriers, and they play a key role in fulfilling the above 
research objectives. With the collaboration of a postdoctoral fellow, 
Dr. Suresh Shukla, and my assistant, Miss Barbara Duntley, I have evaluated 
a large number of synthetic intermediates, pathways, tactics and strategies 
leading to branched peptide derivatives bearing a variety of substituent 
structures and attachment functions. Our findings are as follows: the use 
of glutamic acid as a trivalent hub structure sometimes led to undesired 
by-products when the last substituent was introducted. This result seemed 
to preclude, for the time being, the glutamic diamide approach as being 
sufficiently general and facile to meet the demands of our study. Instead, 
we turned our attention mainly to another trivalent amino acid, L-lysine. 
We have successfully synthesized large haptenic reagents having attachment 
functions and spacing structures connected with either the alpha-carboxyl or 

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Z01 AI 00035-06 LI 

epsilon-amino group. About 20 of the latter type of reagent have been pre- 
pared in final form and are at present undergoing purification. Techniques 
for achieving final purity are being studied. We have explored adipic acid 
as a spacer for the attachment function on lysine and have concluded that the 
latter should be a protected hydrazide introduced at an early stage of 
synthesis. In the course of this work we introduced a new protecting group 
for acyl hydrazides, the 2-(methylsulf onyl)ethoxycarbonyl (Msc) function 
which is stable during the removal of other blocking groups. It can be re- 
moved under special, mild conditions just prior to activation and coupling 
of the hapten to a carrier (immunogen or adsorbent matrix) . The Msc group 
has not, to our knowledge, been used for hydrazide protection. This finding 
should have general application in peptide synthesis: Along with our 
experience in haptenic reagent synthesis, it was presented in a poster paper 
at the Sixth American Peptide Symposium on June 21, 1979. Currently we are 
evaluating haptens based on L-ornit