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EXECUTIVE SUMMARY Since its initiation in 1976, the Medical Research Program has expended over ~45 million to basic biomedical research. The focus of this support has been on the processes of various chronic degenerative diseases. In addition, work has been supported in frontier areas of basic biomedical research, and recently an increasing emphasis has been placed on fellowship support of young scientists, immunology, and research into the interaction of selected lifestyles and chronic degenerative disease. The overview of the Medical Research Program is by the Medical Research Committee, chaired by Mr. Charles A. Tucker. Committee members include: Mr. John L. Bacon, Dr. G. Robert Di Marco, Mr. Donald Haver, Dr. A. Wallace Hayes, and Mr. Wayne W. Juchatz. Current scientific advisors to the Committee include: Dr. Dolph O. Adams (Duke University), Dr. Alvan Feinstein (Yale University), Dr. Frederick Seitz (Rockefeller University), Dr. Charles D. Spielberger (University of South Florida), and others as needed. In 1987, the Medical Research Committee focused its support of biomedical research on the following four areas. Focus One - Basic research on the part played by the 'N interaction of individual attributes and selected « lifestyle modes in the genesis and progression of chronic degenerative disease. Within this general area, programs of a multidisciplinary nature were funded in areas such as stress, hypertension, personality traits, behavioral patterns and genetic background. Focus Two - The biological basis of chronic degenerative diseases. This program includes studies that may be multidisciplinary and deal with such chronic degenerative diseases as cancer, coronary heart disease, and chronic lung disease. Most of these studies emphasize the immunology of chronic degenerative diseases. Future gifts in this area will focus on the immune system at the cellular-molecular level.

j~*r :F,W-a.aeKAaX eA. F ocus Three - New fr ntiers of basi.c biomedi.cal research. This program supports scientists working in frontier areas of research that appear basic, unusually innovative, and pioneering. In 1987, the Medical Research Committee supported such research in the areas of immunology, cancer, infectious diseases, and electron microscopy. Focus Four - Fellowship support for bright, young scientists working in frontier areas of basic biomedical sciences of interest to RJRN. RJR Nabisco, Inc. initiated its Research Scholars Award program in 1985. It is designed to foster the research careers of academic postdoctoral scientists in their formative stages. The award is designed to promote research that will yield new insights into pathophysio- logical responses of the lung and the airways. Each year, three Awards are made to academic institutions on behalf of individuals selected on the basis of scientific merit. Candidates are chosen by a Selection Committee composed of distinguished medical educators. A second Research Scholars Award program will be initiated in 1988 which emphasizes research on basic immunology.

SUMMARY OF THE RJR NABISCO, INC. BIOMEDICAL RESEARCH GRANTS PROGRAM FOR 1987 INTRODUCTION 1 v,•{ The Medical Research Program which was initiated in 1976 had an authorized budget of $5,500,000 in 1987. However, only $4,454,740 of this budget was committed during the calendar year. The totalr..~ommitment up to December 31, 1987 since the inception of the program in 1976 has been $45,611,636. During 1987, 26 research programs were funded and 6 young investigators received RJRN research scholarships. A total of 43 research investigators (excluding Scholars) have been supported up to the end of 1987 - 17 of the grants having been completed. Eighteen of those funded in 1987 will continue to be funded in 1988. While in the past, including 1987, considerable emphasis was placed on new frontiers of basic biomedical research on a very broad front, a decision was made to concentrate the program in the future into three areas as follows: Focus One - Basic research on the part played by the interaction of individual attributes and selected lifestyle modes in the genesis and progression of chronic degenerative disease. Within this general area programs of a multidisci- plinary nature will be considered in areas such as stress, hypertension, personality traits, behavioral patterns and genetic background_ Focus Two - Basic immunology research. This program is by invitation only. Within this area large multifaceted programs will be established at major medical centers with emphasis on molecular immunology. Immunology is broadly defined as host-resistance to foreign materials and to replicating cells or organisms.

Focus Three - Fellowship support for bright young scientists working in areas of basic biomedical sciences of interest to RJRN. This program is by invitation only. RJRN has established two research scholars award programs for young scientists who have limited or no federal support. One program focuses on basic immunology and the other deals with pulmonary diseases. One of the program's key objectives is to encourage the development of physician scientists. As in 1986, the overview of the Research Grants Program is carried out by a committee consisting of full-time employees of RJR Nabisco and outside advisors. The committee is chaired by Mr. Charles A. Tucker. The other inside members are: Mr. John L. Bacon (Staff Vice President and Assistant Secretary) Dr. G. Robert Di Marco (Senior Vice President, Research and Development, RJR Tobacco Company) Mr. Donald Haver (Vice President, Contributions, RJR Tobacco Company) Dr. A. Wallace Hayes (Vice President, Biochemical/Biobehavioral Research and Development, RJR Tobacco Company) Mr. Wayne W. Juchatz (Senior Vice President, General Counsel, and Secretary, RJR Tobacco Company) The outside members are: Dolph O. Adams, M.D., Ph.D. (Duke University) Alvan Feinstein, M.D. (Yale University) Frederick Seitz, Ph.D. (Rockefeller University) Charles D. Spielberger, Ph.D. (University of South Florida) Dr. Maclyn McCarty of Rockefeller University serves as a permanent consultant and participates in a number of on-site research reviews. Other professional advisors are called on as needed. The Committee suffered a very great loss in 1987 with the death of Dr. Leon Golberg of Duke University - an internationally distinguished toxicologist and a rare human being.

The Committee has had six meetings in 1987 and will continue to meet as required to serve the needs of the program. Each full meeting is accompanied by a separate meeting of the inside members who determine policy. Each group being supported by more than ab_oarz-C't150, 000 per year is expected to have its own outside advisory panel consisting of three or four experts in a related field of research. In general the review panel is expected to provide a report on the program both to the principal investigator and to the corporation. The participation of members of the Medical Research Committee is optional but usually provides an excellent opportunity to obtain a discussion of the goals and accomplishments at the professional level. Appendix A contains the budgets through 1987 of completed grants. Appendix B contains a list of current grants with anticipated expenditures through 1990. Appendix C contains the current Biomedical Research Focus Statement and Procedures for Submitting Proposals. Appendix D contains a chart of ongoing and completed biomedical research contributions since 1976. Appendix E contains a chart of ongoing and completed biomedical research contributions from 1985-87. c~ ~~'Appendix F contains a list of institutions to which RJR.,has contributed over *l million for biomedical research since 1976. Summaries of research in each of the areas supported in 1987 are given in the following four sections. As stated previously, the areas of support will be reduced to three as existing programs are phased out.

I. DEGENERATIVE DISEASES ASSOCIATED WITH SELECTED LIFESTYLE MODES It has long been suggested in medical circles that the psychological attitude of a patient can play a significant role in determining the course of a disease or, more generally, factors derived from temperament and lifestyle can influence the health of an individual. Quantifying such relationships is, however, not an easy matter because of variations of a statistical nature that arise from several sources. First, individuals selected from a random population, particularly at the human level, differ from one another. Second, even in a given individual, there may be fluctuations from one time to another. Finally, uncertainties may creep into measurements of both the psychological and physiological parameters depending upon the skill of the investigator, the time at which measurements are made, and the characteristics of the measuring equipment being used in an experiment. For these reasons, research in this area inevitably requires expert interdisciplinary talents at the psychological, statistical and physiological levels. Support of this field of research under the auspices of the Medical Research Committee was initiated in 1984 and is now a major focus to the extent that promising groups of investigators can be found. In 1987 six major programs were supported. 1. Dr. Robert Ader at the University of Rochester Medical School, who has been supported since 1985, continues to study the effects that stress arising from various sources, such as disease, the therapeutic use of drugs and overloading of %the sensory system, can have on- the immune system, particularly on the killer and suppressor cells. The latter play very important roles in the defenses provided by the immune system. In some of the experiments, for example, special strains of mice are subject to feed or drink in which a distasteful chemical is inserted to see if this induces changes in the number of natural killer cells. Particular.emphasis is given to the use of an additive agent (Poly I:C) which stimulates the production of interferon - one of the molecules which plays a role in the activity of the immune system - to see if differences in its action can be observed when this is incorporated in the feed or is injected directly into the subject. The group working with Dr. Ader has obtained particularly interesting results working with a strain of mice which have an overactive immune system and are subject to what are termed autoimmune diseases. The course of this

disease can be mitigated with the use of immuno-suppressing agents. It was found that if the initial treatments with the suppressive agents are accompanied by immunologically neutral agents such as saccharin or saline solution, some degree of immuno-suppression continues to be exhibited if the neutral agents are continued after the immuno- suppressive agent is stopped. This indicates that the neutral agents can have suppressive effects on appropri- ately conditioned animals. With the cooperation of the university, Dr. Ader has greatly improved his laboratory resources during the past year. While this process has slowed the experimental research to a degree, the ultimate effect should benefit the range and quality of experiments that can be carried out by the group. 2. Dr. Paul H. Black of Boston University, who has been supported since 1984, is studying the interrelation between the emotional framework and the state of both the immune system and neural hormones in a family of depressed patients in a psychiatric clinic. A portion of the earlier part of the research program was spent developing standards of measurements for the experiments. However, 1987 has been a productive year. The work shows that patients with major psychological depression have a suppressed immune system in the sense that the reactivity of the protective cells is lower than normal. Moreover, the number of killer cells is substan- tially diminished. These effects are greatly accentuated in individuals who have become so depressed that they have attempted suicide. In parallel with these changes are various shifts in some of the hormones. In particular, some of the hormones which can act as suppressors of the immune system are elevated in keeping with the observations on the immune system itself. Research on these findings is being extended in both breadth and depth. Along with the foregoing work, Dr. Black and his colleagues are carrying out similar measures on individuals who are responsive to hypnosis and in whom various emotional states can be induced during hypnosis. Special emphasis is given to anger, sadness, happiness and what might be called a state of "neutral" emotion. The study is repeated on each of the subjects during four independent sessions to determine the extent to which the results obtained in one session are repetitive. Thus far the work shows that in some of the subjects the states of anger and happiness are associated with increases in natural killer cell activity and in the

responsiveness of the entire immune system. It should be emphasized that this phase of the research program is still in its early stages. However the results obtained thus far seem to be consistent with the well confirmed observations on animals that those which display anger under stress are more highly immuno-responsive. 3. Dr. Jan Breslow of The Rockefeller University has initiated a program to study genetic abnormalities that, along with lifestyle, can lead to premature atherosclerosis. Prominent among the materials which transport fatty agents through the blood stream are the lipoproteins - a family of molecules in which the fatty agent is bound to a protein. The concentration of these in the blood is, in turn, controlled in part by enzymes which participate in the way in which the balance of such molecules is maintained. The portion of Dr. Breslow's work supported by RJR Nabisco deals with the study of the genes which serve as the template for generating the effective enzymes. The analysis of the structure of the relevant genes is being carried out both in selected strains of mice and in human subjects. In the case of humans the research involves normal as well as abnormal subjects in which the controlling enzymes are deficient. The research with mice has been developed in sufficient detail and precision to provide highly specific information regarding the gene structure and the effect of various agents such as insulin upon the effect of the enzyme. Fortunately much of the research can be done with the use of cell cultures. The work with human subjects must proceed much less directly. However, Dr. Breslow and his group have, through extensive surveys, found about a hundred individuals who exhibit abnormal control of the crucial lipoproteins because of abnormally low levels of the enzyme. As is common in a genetically mixed human population, the subjects exhibit a statistical range of physiological behavior. The aspect of the work dealing with human subjects is still in its early stages. 4. Professor Leo Herbette of the University of Connecticut Science Health Center is studying the way in which chemicals interact with protein receptors on the outer surface of artificial and cellular membranes in solution. His ultimate goal is to determine the relative speed with which such agents can reach and affect the properties of the cell. Comparisons will be made when the membrane is in various states of hydration or is influenced

in other ways. Initially the program was started to see if one could determine what effect the alcohol in normal beverages might have upon the behavior of the cell membrane. In the meantime, the scope of the work has broadened considerably. The influence of external agents on the inner chemistry of the cell normally begins with the direct interaction of such agents with protein receptors situated on the outer membrane of the cell. These receptors can, in turn, transmit an informative signal or a chemical constituent to the inner cell and start metabolic changes. Using a variety of techniques, Herbette and his colleagues have demonstrated that some agents are sufficiently soluble in the cell membrane that they can dissolve into its mid-layers and migrate laterally through the mid-layer by diffusion to the protein receptors. Other agents are sufficiently insoluble that, in the main, they react with a protein directly. The physical-chemical condition of the membrane will influence the relative rate of migration in the first case. Moreover, the route by which the agent reaches the receptor can determine both the form and tight- ness of the binding to the receptor and have an effect on the duration of its influence on the inner chemistry of the cell. Herbette and his colleagues have used a variety of techniques to demonstrate the principles involved in the processes described above. 5. Professor Stevo Julius of the Department of Internal Medicine of the University of Michigan Medical School is investigating the correlation between neurogenic markers and the development of hypertension in individuals. He has been supported since 1985. The group is studying a mixed population in Tecumseh, Michigan for which extensive family records are available. The number of subjects involved thus far is about 1500. Work carried out to date suggests that hypertenstion follows a dynamic course which is partly genetic in origin but which in young subjects can be ascertained to a significant degree by investigating behavioral as well as indirect physiological markers. The group believes, for example, that a hyperkinetic state of blood circulation in a young individual presages the development of high blood pressure. This could be a particularly useful indicator since it could be measured by non-invasive means which have been developed in his laboratory. Additional work indicates that the response of the individual to certain drugs such as the alpha blockers and to a special family of -hormones also provides useful information. One of the most interesting results of the work suggests that individuals who internalize anger without

giving a clear external indication of their emotional state are highly prone to develop hypertension, a condition which can be detected by appropriate psychological and physiolog- ical studies. Dr. Julius emphasizes that his group is paying special attention to markers which indicate the potentiality to develop hypertension in young individuals so that they can be given early warning and take corrective measures. Later on in life biochemical factors can become dominant and require a different, more direct type of testing. It should be emphasized that the involvement of such large numbers of patients requires a combination of a field office, home visits and clinical studies at the university. 6. Dr. Sandra Levy of the University of Pittsburgh Cancer Center is being supported to study the interrelation of behavioral characteristics, the levels of neurohormones and the characteristics of the immune system during the course of cancer in patients under treatment at the University of Pittsburgh Hospital. Particular emphasis is given to patients who have breast cancer and melanoma. The patients under investigation have varying levels of psychological stress in addition to natural differences in personality. Some are highly responsive to professional encouragement. Others are less responsive and even show depression. It has been found that those who either lack or are unresponsive to social support have lower natural killer cell levels and fare less well in the treatment of the disease. Consecutive followup studies on individuals of the group over a period of months show stability in trends of killer cell levels. Dr. Levy has been joined in her work by some highly experienced colleagues who are providing analytical and advisory support in relation to studies of the immune and hormonal systems, as well as patient care. Two programs which"' art , ~.,i:ri ~ear-1,~ in 1988 were funded from the 1987 budget. They are: 1. Professor Arnold H. Greenberg of the Department of Pediatrics of the Manitoba Institute of Cell Biolo and his co eagues are stu ying t e e ect on t e immune system in mice of otherwise inactive agents which are given simultaneously, or in association with, agents which do affect the immune system. Particular emphasis is being

placed on circumstances in which the immunoeffective agent is discontinued after an interval but the otherwise inert "cue" is continued. For example, experiments have been carried out with the simultaneous application of a stimulant of the killer cells (Poly I:C) along with the addition of an otherwise neutral psychological cue, such as the odor of peppermint or illumination with strong light. If the immuno-stimulant is used alone and continued sufficiently, the immune system of the mice eventually becomes tolerant to it in the sense that the immune system no longer responds if there has been an extended rest period (for example eight weeks). On the other hand, if one of the cues accompanies the stimulant and continues alone during the rest period, the tolerance associated with continued injection of the immunostimulant disappears. The group in Manitoba is extending such research to examine the effects of altering the sequence of cues and stimulants to see if a better understanding of such effects can be obtained. Dr. Greenberg's research program is earmarked by a very careful combination of controls of environmental factors and a high level of expertness in dealing with the immune system. 2. Dr. R. N. Rosenman of SRI in Menlo Park, California is cooperating in the reactivation, after twenty-seven years, of a study of the medical and psychological history of the survivors of a large group of individuals who were classi- fied with respect to behavioral type (Type A, Type C and so forth). This study was originally termed the Western Collaborative Group Study (WCGS) and involved 3,154 adult males between the ages of 39 and 59. In the follow-on which is estimated to involve about 1800 survivors, particular emphasis will be placed upon cancer morbidity and mortality but in combination with measurements of such factors as dietary and smoking habits, blood constitution and pressure, and heart rate. Special attention will be given to members of the group who were rated as Type A (the hyperactive group) in comparison with the more sedate Type C group. A part of the follow-up program dealing with physiological health and morbidity will be supported by the National Institutes of Health which has had a more or less continuing interest in the cohort. In fact, some information is available for the group for intervening years such as 1969 and 1982.

II. THE BIOLOGICAL BASIS OF CHRONIC DEGENERATIVE DISEASES Initially, this aspect of the research program was interpreted fairly broadly. However, it was decided that, beginning in 1988, priority will be given to research that focuses on studies of the immune system at the cellular- molecular level. Support of other forms of degenerative disease related to the immune system will be phased out on a scale commensurate with prior commitments. Studies of the structure and action of the immune system which protects us and other species against foreign disease- producing agents are in the forefront of present day biomedical research. In the case of the higher verte- brates, the complexity of this system is so great that it will probably take several decades to obtain anything like a complete picture of its characteristics. The system is both intricate and self-interactive forming a network of diverse cells and biochemical entities which inter- communicate as circumstances require. Should this network fail in any one of us we would succumb to a fatal disease in a matter of days, as does occur when individuals are subject to large but non-fatal doses of radiation which destroy the integrity of cells of the immune system. The mature cells of the immune system exhibit great diversity, playing a number of specialized roles as circum- stances demand. Among other things, they can produce a variety of biologically active agents, including antibodies, that are able to play aggressive roles, such as attacking bacteria or,^ _neutralizing the action of viruses. Or, they can act as messengers which activate normally quiescent members of the family in order to stimulate them to pursue and destroy an invading agent. One of the major objects of pharmaceutical work in the field is to suppress or enhance portions of the immune system or products of the cells of which it is composed. For example, the common antihistamines suppress the activity of an agent designated as IgE which produces inflammation in order to enhance blood flow to an infected or wounded area. Similarly the development of more complex immunosuppressive agents is the object of intense research since they can be useful in inhibiting the rejection of grafts of foreign tissue or organs from an individual other than self or an identical twin. The suppression of any part of the immune system evidently involves a calculated risk since a corresponding component of the defensive system is then weakened or muted. As commented earlier, we would be unable to survive the normal activities and experiences of life for more than a few days without the defense provided by the immune

system. Yet this complex, ever-alert system can become harmful if it is triggered in the wrong way and thereby encouraged to attack parts of the tissue that it is intended to protect. The reasons--for such triggering can apparently be quite complicatedf=however the damage to otherwise normal tissue can be caused directly by the action of some of the immune cells or by chemical agents which they produce as part of their defensive acitivty. It has also been demonstrated that complexes formed of antibodies and the antigens they combine with by way of neutralizing the latter can become attached in normal cells and cause damage. Such autoimmune diseases can take various forms and induce afflictions such as colitis, rheumatoid arthritis, rheumatic fever, cystic kidney disease, childhood diabetes and many other ailments. It is suspected that diseases such as multiple sclerosis and amyotropic lateral sclerosis (ALS) are' the result of an autoimmune reaction linking the immune system to the products of as yet unknown viral agents. Unfortunately, the progress made in unravelling the detailed nature of the source of such diseases has been slow. It is interesting to note that the rapid growth of the disease AIDS (Acquired Immune Deficiency Syndrome) has given increased impetus to the studies of special aspects of the immune system since it, or prominent components of it, is the target of an attack by the family of viruses responsible for the disease. 1. The support of the research program at Harvard University under the leadership of Professor Barger, who has been supported since 1978, was phased out in mid-1987 with his retirement. This activity, which was notable both for its interdisciplinary character and for the high quality of the research produced, started with the goal of examining the influence of hormones produced by the kidney on high blood pressure. Dr. Barger had developed experi- mental techniques for controlling the blood pressure by altering the flow of blood to the kidneys by mechanical means and proceeded to investigate the associated changes in kidney hormones, particularly renin and its physiological products. By the time of Dr. Barger's retirement, the group had exceeded the goals of the original investigation and had uncovered a wealth of information related to such topics as atherosclerosis, infant death syndrome and several forms of cardiac disease, including premature thrombosis in a rapidly growing network of coronary capillaries. In addition, they had discovered that the key hormones controlling blood pressure are produced in the heart as well as in the kidney. Moreover, brain cells contain

receptors for such hormones indicating a link with the nervous system. Many of these major discoveries would have been delayed without the support provided by RJR Nabisco. R N~.. 2. Professors ;Y. G. Farquhar and G. Palade of Yale University, who have been supported since 1986, are studying the special features of the transport of albumin through the circulatory system and into the intercellular region of the body'. Albumin is a complex molecule containing a protein,base which transports fatty acids, simple hormones and some drugs at special openings in the blood vessel wall where transport is possible. The albumin molecules become attached to such transport sites in the vessels and control as well as assist in the transport of various agents, including albumin, into tissues. As part of the study they have investigated the nature of the protein binding sites in the inner vessel wall near the transport openings to which the albumin becomes attached. The most likely candidate turns out to be a special unit which is formed by combination of a sugar and a protein (glycoprotein). The investigators have been greatly aided in the study of albumin transport through vessels by the discovery that bovine albumin behaves much ,like mouse albumin in mice and that antibodies which b''ori~d.'to bovine albumin and serve as markers for its pathway do not bind to mouse albumin. As was noted in the report for 1986, this research has given us new insights into the reasons why some arteries serve as loci for the development of arterio- sclerosis and others do not. Moreover, it also provides information on ways to deliver drugs to selected regions of the organism. In addition to studying the transport through and across the capillaries associated with albumin, the group at Yale has focused a great deal of attention on the structure of both the capillaries and base membranes in the kidney. In this organ the capillaries serve as a massive porous filter of the blood-and the base membranes, among other things, hold back some-blood constituents such as albumin. Particular attention has been paid to the position and structure of special molecules which are bound to the surfaces in the filtering region and which are presumed to play an important role in the filtering process. The Yale group has used great ingenuity to _obtain exquisitely detailed pictures of the structures associated with these regions and, is proceding to study the function of the molecular constituents.

3. Dr. Paal Klykken of the University of Mississippi Medical Center, who has been supported since 1986, has instituted a study of the influence of biochemical agents (neurotransmitters) derived from the autonomic nervous system upon component cells of the immune system, placing emphasis on selected families of the macrophages. The goal behind the work is to see if biochemical substances associated with activation of the autonomic system are capable of altering the immune system in a direct way. His work has the advantage that the initial research can be carried out at the test tube level. In testing a number of substances derived from nerve tissue or which stimulate the nervous system, Klykken has discovered that low concentrations of nicotine can activate the macrophage systems with which he works so that they become much more responsive to challenges from foreign proteins. 4. The program at the Wistar Institute in Philadelphia under the leadership of Dr. Hilary Koprowski has been supported since 1983. The object of the work to date has been to determine the effects produced over a long period of time by viruses which take up permanent residence in tissue of the host and remain dormant or quasi-dormant. Typical examples are provided by the viruses responsible for cold sores (herpes simplex) and shingles (herpes zoster). The latter is identical with chicken pox virus. As a result of a request emanating from the Medical Research Committee, the funds derived from RJR Nabisco have been used to support research on the effect of such resident viruses on the immune system. In order to satisfy this request and to add practicality to the experiments, they have been carried out with the use of segments of the principal proteins associated with the viruses - proteins that can serve as antigens for eliciting responses from the immune system. Among other things such a study offers the possibility of developing synthetic proteins related to those associated with the viruses which can act as vaccines, that is, produce immunity, without the hazard of inducing an infection. Thus far in the experiments, particular attention has been given to proteins associated with the influenza and herpes simplex viruses. In the case of the influeriza virus it was found, somewhat surprisingly, that only a small portion of the natural protein associated with the virus actually stimulates the immune system. In contrast, a much larger segment of the protein in herpes simplex serves as a stimulating antigen. However, the group has found a distinct difference in the ability of fragments of

the antigen to provide protection against the disease in animals and to stimulate immune cels (T cells) in the test tube. This difference is being studied further. Interestingly enough, it has been found that if a sufficiently large dose of influenza antigen is presented to the immune system the latter fails to generate a normal response as if, in some way, it were de-stimulated. The laboratory also has devoted a great deal of attention to cellular-molecular phenomena associated with multiple sclerosis in an attempt to determine whether the disease is linked to a resident virus. The group has demonstrated that constituents in the cerebral spinal fluid of patients with the disease do stimulate the immune system, however, the source and nature of the antigens (gangliosides) in the cerebral fluid is not yet clear. 5. Dr. Steven B. Mizel of the Bowman Gray School of Medicine has been supported since 1986. He and his colleagues are in the initial stages of studying the effect of biologically active messenger molecules produced by cells of the immune system upon various cells derived from pathological tissue, such as cancer cells or cells from patients with rheumatoid arthritis. One family of such messenger cells is that of the so-called lymphokines or cytokines which are known to influence the migrational properties of immune cells and to affect the development of cells from other tissues. Special attention is being given at a molecular level to the interaction of one of the purified lymphokines (interleukin-1) with controlled cell lines. Mizel was previously involved in the work which led to the purifica- tion of interleukin-1 and is, as a result, in an excellent position to carry out such experiments. We should expect to report interesting results at the end of 1988. During the past year he was able to demonstrate that cells from tissues derived from patients with osteoarthritis and rheumatoid arthritis do have receptors for<"Interleukin-1 and that this agent has a major stimulating effect on such cells. He also found that the stimulation can be prevented with the use of a well-known blocking agent Propranolol. When it is used, the cells ceased generating a group of molecules which are known to help produce inflammation (prostaglandins). It is hoped that this knowledge will eventually aid in providing therapeutic help for the diseases. -16-

6. Dr. Moser of the Medical Center of the University of California at San Diego is one of the international leaders in the field of pulmonary disease. He has been supported in connection with his studies of degenerative diseases of the lung, with special emphasis on emphysema, since 1979. This work has included a focus on pulmonary hypertension and the effect of foreign bodies, such as asbestos fibers, in the lung. The work has been carried out both with laboratory animals such as rats, dogs and sheep, and with human patients suffering from lung related diseases. Since the lung is one of the central organs of the body, providing oxygen to the blood, and is the site of the removal of some of the byproducts of metabolism, it is not surprising that many of the results of Moser's workChas' Y -. influence on other forms of disease such as those of the cardiac and arterial systems. The elasticity of healthy lung tissue is maintained as a result of a close balance between agents which produce a turnover in the system, that is, dissolution and renewal. Emphysema occurs when enzymes which destroy molecules that maintain elasticity in the tissue gain the upper hand in the process of turnover. Moser has purified the agent (designated A-l-Pi) which inhibits the enzyme that acts to degrade elasticity. Work during the past year has demonstrated that it can be administered both intravenously and directly into the lung in the form of an aerosol without causing toxic side effects. The initial work was carried out with animal models, however subsequent work has demonstrated that the experimental treatment is also safe for human patients. The inhibitor remains present in the lung for several days in both animal and human systems. As a result individual doses can be administered several days apart in cases in which the inhibitor is to be tested over a period of time in order to see if it will arrest, or possibly reverse to some degree, the damage associated with the progess of emphysema. In brief, Moser's research on the cause of emphysema has finally reached a point where it may offer a useful therapeutic agent. In association with the foregoing studies, Dr. Moser and his team have achieved a number of other successful results. For example, they have made a great deal of progress in developing a non-invasive method of determining the blood pressure within the pulmonary artery and the flow of the blood from the heart to the lung. They also have developed special agents which make it possible to determine the presence of blood clots in the systems of veins of interest in their work.

7. Dr. Robert J. North of the Trudeau Institute in Saranac, New York is studying the way in which the immune system in selective strains of mice reacts to the presence of cancer cells. In fact, he and his research team at the Trudeau, aided in a major way by support from RJR Nabisco, have finally separated fact from fiction by demonstrating conclusively that the killer component of the immune cell system will destroy tumors very effectively if it is allowed to carry through its attack on such cells without the mitigating influence of the suppressor cell component. Normally the killer cells respond vigorously to the presence of cancer cells but their attack is terminated by the suppressor cells which play the role of controlling the killer cells before they can become so numerous and potent that they may damage host tissue. This safeguard plays a reasonable and effective role when the organism is combatting normal foreign material such as infectious agents but it prevents the immune system from completing its role when the invasive agent is a tumor. North and his colleagues have developed a number of methods of suppressing the suppressor cells under conditions in which the killer cells continue to remain active. Under such circumstances, the destruction of the tumor is complete. The procedures followed most intensively so far, and with remarkable success, are based respectively upon the use of x-irradiation, a toxic drug (cyclophosphamide) and a chemically engineered version of a natural hormone, interleukin-1 mentioned previously, which can stimulate the action of the killer cells. Careful investigations with the use of mice have shown that the timing of application of any one of these agents is of primary importance. In all cases the agent must be used during the proper interval of time. In the case of x-rays, for example, the appropriate time to use irradiation lies in the period in which the killer cells are aggressively attacking the cancer. In this situation the x-rays will decrease the effectiveness of the suppressor cells without damaging the killer cells. In contrast, the toxic agent must be used before the killer cells become highly active. The use of interleukin-1 in turn is most effective when the killer cells are very active and just before the suppressor cells have become effective.. 8. Dr. Barry Pierce of the University of Colorado in Denver has been supported since 1978. His initial program cen:Ee-red about the study of agents which can act upon cancer cells in such a way as to compel them to undergo normal, regulated behavior as though they were a part of the tissue in which they reside. The factual basis for

pursuing this line of investigation rests upon the well-substantiated observation that cells taken from cancerous tissue and inserted into the early-stage embryo of an animal of the same species will become a normally behaving constituent in that embryo. His work has thus far involved frogs, mice and rats. In the course of this research, and in the very recent past, he has found two types of natural agents in embryonic tissues. One can act as a regulator of cancer cells, returning them to normal behavior, and the others are lethal for such cells. Both types of agents are of great interest for possible application in cancer therapy, particularly as replacements or supplements to the highly toxic compounds now used in conventional chemotherapy for cancer. Dr. Pierce is pursuing this avenue energetically. 9. Dr. Theodore T. Puck of the Eleanor Roosevelt Institute in Denver, who has been supported since 1979, has devised a family of new methods for analysing the structure of human genes, determining mutations and linking the results to important areas of human genetic disease. He is a remarkably creative scientist in more ways than one. Among other things, he has remained ingeniously active with a continuously' new flow of discoveries over a forty year period since he first developed techniques for growing cells in culture - a major milestone in modern biology. In one of his recent programs, he has used techniques of cell fusion, developed by others in the 1970's to pro- vide large quantities of identical antibodies, in order to replicate selected portions of the genetic material in human cell lines essentially indefinitely. In this pro- cedure a human cell from a cancer patient is fused with another cell of a different type (Chinese hamster ovary cell) and made to replicate in culture. Some of the progeny of this process will contain fragments of the parent human cells and can be multiplied independently. In this way he has been able to build up a quantitative library of sections of selected human chromosomes for both normal individuals and those suffering from genetic diseases. This has permitted him to locate defective genes and chromosomes by comparing molecular sequences in the two. In an extension of work of this type he has developed methods for detecting changes (mutations) induced in chromosomes at very low levels of x-irradiation - a tenth or more below those which kill an appreciable fraction of the cells. The method is much more sensitive than any other developed so far and makes it possible to detect not only changes in the composition of genes but also -19-

rearrangements of the genetic material. The results obtained from such studies indicate that there is no significant threshhold below which x-rays and similar ionizing radiations do not produce genetic changes. This result, if substantiated by others, must be tempered by the knowledge that all forms of life on earth have lived in a substantial field of such radiation since life first came into being on our planet some three and a half billion years ago. Puck's techniques are now being extended in attempts to understand why some chemical agents which cause cancer are able to reinforce one another in a manner out of proportion to the effects of either alone. It may be added that he is also extending the experiments described in the Annual Report for 1986 in which he is searching for biochemically active materials which will transform cancer cells into normal ones. 10. Professor Russell Ross of the University of Washington, Seattle has been supported since 1977. Such support is now in its final phase. Over that decade, and to a major extent as a result of the support from RJR Nabisco, he and his group of young colleagues succeeded in generating entirely new insights into the origin of atherosclerosis. He has demonstrated that the malady originates in a wound to the inner layer of cells of the artery. This wound attracts cells of the immune system which actively promote cell growth in the neighborhood of the injured site. As a result of over-reaction, the immunological agents incidentally promote growth in the smooth muscle layers underlying the endothelium and encourage a rupture of the tissue. Once the integrity of the layers is broken, fatty molecules including cholesterol can deposit there forming a plaque. In earlier work Ross and his colleagues demonstrated clearly that a component of the immune family of cells termed macrophages play a role in the disruption of the smooth muscle layer. More recent work shows that other members of the immune system (T-cells) also play a role. This suggests that in many ways the development of the atherosclerotic lesion can be looked upon as a form of autoimmune injury. 11. Dr. Gordon H. Sato and his colleagues at the W. Alton Jones Cell Science Center in Lake Placid have been supported since 1986. At present their interest is focused on the study of atherosclerosis. In a sense they

are following in their own way the leads provided through earlier research by Dr. Russell Ross described previously. As mentioned above, it is now known that the lesions associated with atherosclerosis start with an injury to the inner wall of the artery (endothelium) and is followed by abnormal growth of the underlying smooth muscle tissue accompanied by the deposition of fats and related materials such as cholesterol. The process is stimulated by agents termed growth factors which, under normal conditions, are involved in the repair of the initial injury but which are over-responsive in cases in which the lesion is followed by the deposition of an atherosclerotic plaque. The group is studying the origin of the growth factors and the agents which regulate their generation and action both in the normal and abnormal cases. As noted above, the development of the plaque requires access to excess fatty material. The work underway indicates that the fatty precursors also play a role in stimulating the acceleration of growth of the smooth muscle cells. It is well known that in normal individuals any wound which leads to a rupture of blood vessels contains substances which produce clotting of the leaking blood. One of the remarkable features of the cells in the inner wall of the blood vessel is that they have substances which prevent such clotting when there is no wound. The laboratory group at Alton Jones has discovered at least one of the anti-clotting agents produced by the endothelial cells and is seeking to determine its composition and structure since it could be a very useful pharmaceutical agent. One notable characteristic of the Alton Jones Center (shared incidentally by the Wistar Laboratory in Philadelphia) is that it has a system of core service laboratories led by experts in various areas of chemistry. For example, some of the core laboratories deal with gene coding, lipid (fat) analysis and protein chemistry. The heads of the core laboratories are encouraged to generate their own research programs but at the same time be prepared to cooperate as needed with those engaged in the main activities of the center. The laboratory devoted to gene coding is deeply involved in studying the genes which code the growth factors that play a role in atherosclerosis. 12. Dr. Richard Stankus of the Tulane University Medical Center has been supported since 1986 in the study of chemicals which promote the production of immature fiber-producing cells termed fibroblasts in the lung. The

proliferation of such cells is associated with diseases such as silicosis and asbestosis. Stankus hopes to collect sufficient lung fluid from rats which have artificially induced silicosis to determine the nature of the responsible agents. He has demonstrated that the collected fluid does promote fibroblast growth in culture. It remains to be seen if he is successful in isolating the promoters. 13. Dr. Christopher Taylor of the Medical College of Pennsylvania has been supported since 1986 in a study of newborn animals (mice) which have been subject to mild stress through confinement in order to investigate the status of the immune system relative to unstressed control specimens. As was reported last year, he has found that the stressed animals had lower immune resistance both to infection and to a bacterial antigen. The effect appears to be stronger in young females than males. In fact, the differences for males were hardly of statistical significance. Interestingly enough, the suppression resulting from this early stress disappears once the animals mature under conditions in which they again live in the same way as the controls. The transition from the suppressed to the normal state will be studied in more detail in the next stage of the research program. . N -22-

III. NEW FRONTIERS OF BASIC BIOMEDICAL RESEARCH In the initial stages of the Medical Research Program, a substantial amount of support was given to new frontiers of biomedical research. A decision has been made to phase out this portion of the program and to concentrate on other areas. The programs currently underway are as follows: 1. Professor polph Adams of Duke University is studying the development and differentiation of an important member of the family of cells of the immune system, which have been mentioned several times in the preceding sections, namely the macrophages. These cells are probably best known as major scavengers (phagocytes) since they can demonstrate great flexibility in engulfing and destroying by digestion foreign agents such as bacteria or inanimate organic matter. In pathological cases, they can also fail to destroy such agents and serve inadvertently as shielded domiciles for infectious agents. It is now known that they actually can play a much more complex role both by sending signals within the network of the immune system and acting upon signals which they receive. They also can produce and emit agents which are toxic for foreign cells, including cancer cells derived from the host. Professor Adams and his colleagues have devoted much attention at the molecular level to the results which occur when macrophages are activated by interferon gamma, an important natural immunological agent and stimulant, as well as when this stimulus is reinforced by a second signal with a biologically active agent, such as a bacterial toxin or a member of a special family of biologically active molecules termed lipopolysaccharides. The result of such stimulation is to induce changes in the metabolic processes in the macrophages, which in turn prepare them for various forms of defensive action governed by the nature of the stimulus. As a result of work in the recent past, the group has determined that the stimulation of the cell occurs in three successive stages, each of which has its own time scale. The first stage which occurs relatively rapidly involves readjustments of the ongoing metabolism. The later stages, which are conditioned by the earlier ones, involve new expression of the constituent genes with the production of new molecules that can be employed in various ways such as for external stimulation or as lethal agents.

The ultimate goal of the program is to obtain sufficient information concerning the way in which macrophages operate to develop pharmaceutical agents which can assist them in their task. This program clearly could be regarded as belonging in Section II. 2. Dr. Nicholas Heintz of the Universit of Vermont has been given an award that is intende to provide an i.nsight into the mechanisms of gene emplification. This is a process whereby certain genes, termed oncogenes, which are known to play a role in the induction of some forms of cancer, are amplified under the influence of a toxic agent, such as a chemotherapeutic chemical, and are able to assist in providing resistivity to such an agent and, occasionally, lead to uncontrolled growth. This problem is of very great interest at the present time because of its relationship both to the generation of cancer and the development of immunity to anticancer drugs. Heintz hopes that he will be able to add special insight as a result of his experience in the field of molecular biology. 3. Dr. Stanley Prusiner of the Medical School of the Universit of California at San Francisco has been supporte since in 1980 an in- ept stu y of infectious diseases of the nervous system termed slow diseases. As an animal model he selected scrapie, best known for contagions in sheep and goats but which is transmitable to rodents such as mice, rats and hamsters. The human diseases kuru found in New Guinea cannibals and Creutzfeld- Jacob Disease are prototypes. In selecting an animal model Prusiner has, in the main, focused on the development and analysis of scrapie in the brain of hamsters for which it has a lethal cycle of about two months. It now appears essentially beyond reasonable doubt that the infectious nature of the disease involves a mechanism completely different from conventional infectious diseases that originate from bacteria, viruses or viroids (an exceedingly primitive form of virus which does not possess a protein coating). What he and his group have found is that the infectious agent is a protein produced by the brain cells of infected animals which in turn is closely related to a normal protein found in the cells of healthy brain cells and in other tissues of the animal. The role of the normal protein is as yet unknown. The infectious version of the protein has the property that it compels the cells which it invades to produce its kind of protein instead of the normal one. Moreover, this production is so prolific that the cell is destroyed.

Until the cell is destroyed it appears that the errant form remains either in the cell or on its surface membrane but it is released upon destruction to spread the infection. Prusiner's research has been carried out with such thoroughness and depth and with such a high degree of ingenuity at the molecular-cellular level that many remarkable properties of the infectious agent have been analysed, including the chemical structure of the backbone of the infectious protein, some of its side chains and the structure of the gene in the cell nucleus which is the source of replication for the protein. This remarkably innovative research program,;which may well represent a major new landmark in the study of infectious diseases._i..would not have been possible without the help provided by RJR Nabisco. 4. The program carried out at Duke University under the leadership of Professor David Robertson since 1983 was completed in 1987. The funds were used to support a group of young investigators who use the electron microscope and other facilities available in his laboratory to investigate the structure and properties of membranes which occur in a number of organisms and tissues. Professor Robertson has been one of the world's experts on the development and use of the electron microscope for studying biological systems and has contributed substantially to the advancement of cellular biology through exploitation of this form of technology. Robertson's personal research, which he is continuing in retirement, has focused on biochemical studies of the nervous system of the octopus. The animal learns both through sight and by touch. Professor Robertson has discovered biochemically active agents that can affect the two learning processes in different ways and hopes to gain further information on the learning process through such work. 5. Dr. Aziz Sancar of the University of North Carolina at Chapel Hill, who has been supported at a modest level since 1986, is studying the way in which cells in different tissues react to cance " r<causing chemical agents. He and his colleagues hope to relate this type of specificity to the circumstance that the cells in different tissues derive their special characteristics from differences in the activation of genes which are common to all cells. The members of the group plan to break down the chromosomes in damaged cells from different tissues and examine the extent

to which the genes which are modified as a result of the cancer--catsing chemical agent differ from the normal genes. They--hope in this way to obtain a much more detailed picture of the changes induced in the genetic material. In connection with this work he has discovered that extracts from a special line of cancer cells widely used in cancer research (HeLa cells) contain an agent that can repair the damage caused to genetic material (DNA). The effect is reproducible and suggests that there is a substance in the cancer cell line that can reverse damage to genes. A detailed search for the agent is underway.

IV. RJR NABISCO, INC. RESEARCH SCHOLARS AWARD RJR Nabisco, Inc. initiated a Research Scholars Award Program in 1985. It is designed to foster research careers of academic scientists in their formative stages. The award is designed to promote research that will yield new insights into pathophysiological responses of the immune system. Basic science approaches to research problems are encouraged as are clinical investigations, provided the latter encompass rigorous methodologies that focus on mechanisms underlying the immune process. Investigative approaches may emphasize the techniques of biochemistry, immunology, molecular genetics/biology, and similar disciplines. Successful candidates for the Research Scholars Award are those who have demonstrated scientific independence as well as outstanding potential for research contributions in basic immunology which encompasses host reactions to foreign invaders, intruders and rapidly replicating cells (i.e., microbial or cancer.) Scientific merit rather than programmatic emphasis is the single most important criterion in the peer review process of applications. The Selection Committee is particularly interested in seeking individuals who conduct excellent innovative research and who can be expected to make significant contributions to the field during their careers. Each year, three Awards are made to academic institutions on behalf of individuals selected as RJR Nabisco Research Scholars. Each Award is for up to t225,000 ($75,000 per year per candidate for each of three years). Distinguished medical educators comprise the Selection Committee for the RJRN Research Scholars Award. Committee members are responsible for selecting three RJRN Scholars annually. Listed below are members of the Selection Committee for the 1987 Awards. John E. Salvaggio, M.D. Selection Committee Chairperson Department of Medicine Tulane School of Medicine New Orleans, Louisiana 70112 H. Benfer Kaltreider, M.D. Department of Medicine University of California San Francisco San Francisco, California 94143 Herbert Y. Reynolds, M.D. Pulmonary Section Department of Internal Medicine Yale University New Haven, Connecticut 06510-8057 -27-

Peter A. Ward, M.D. Department of Pathology University of Michigan Medical School Ann Arbor, Michigan 48109 The following researchers were RJRN Research Scholars in 1986 and 1987. 1986 RJRN Research Scholars Jack Angel Elias, M.D., University of Pennsylvania Dr. Elias's laboratory is studying the processes that control the scarring response in the human lung. In particular, he is attempting to understand why certain people with lung inflammation heal without excess scarring while others develop pulmonary fibrosis. To answer this question, Dr. Elias has been studying how mononuclear cells and fibroblasts interact with one another. These studies have provided insight into the way inflammation regulates scarring and into mechanisms by which the scarring process in turn regulates inflammation. David H. Perlmutter, M.D., Washington University School of Medicine Dr. Perlmutter's research is designed to determine how lung macrophages control the activity of destructive enzymes during inflammation and/or tissue injury. He has demonstrated that lung macrophages synthesize and secrete an important inhibitor of destructive enzymes,--l-antitrypsin. Expression of-l-antitrypsin in lung macrophages is markedly increased by a prototype mediator of inflammation, bacterial endotoxin, and by a prototype destructive enzyme, neutrophil elastase. Dr. Perlmutter is studying the distinct molecular and cellular mechanisms which are involved in the expression and regulation of -l-antitrypsin by lung macrophages during inflammation. Additionally, Dr. Perlmutter's laboratory is examining the characteristics of other mediators of the host response to inflammation, including interleukin-1, interleukin-2, and interferon-gamma, as well as other specific genes activated during inflammation. -28- @

G. Scott Worthen, M.D., National Jewish Center for Immunology and Respiratory Medicine and the University of Colorado School of Medicine Endotoxins from gram negative bacteria, which circulate in the body during blood poisoning, injure the lung by mechanisms that are currently unclear, but which seem to involve the blood neutrophil. It is suspected that the neutrophil is activated by the endotoxin and is induced to adhere in the blood vessels of the lung, where it causes injury. Dr. Worthen is attempting to determine: (a) why the lung is one of the most frequently injured organs during blood poisoning; (b) how endotoxins induce neutrophils to become "stickier" (Dr. Worthen has found that some part of serum or plasma is involved in this event, and he hopes to isolate the factor involved); and (c) where the neutrophils lodge in the lung. it appears that the capillary is the site of action. This is particularly significant since this is also where gas exchange occurs. 1987 RJRN Research Scholars Jeffrey S. Berman, M.D., Boston University School of Medicine Dr. Berman is studying the regulation of T lymphocytes, which organize and regulate the immune response to foreign materials, certain bacteria and viruses, and tumors. An important part of the T lymphocyte response is the local production of hormones called lymphokines. One of these, interleukin 2(IL2), regulates the growth of stimulated T lymphocytes and has been used in the treatment of cancer. Dr. Berman is investi- gating the means by which IL2 interacts with its receptor to cause its effects on lymphocytes. Dr. Berman has found an IL2-binding complex in the membranes of T lymphocytes, and he is beginning to study its structure. Tomas Ganz, M.D., Ph.D., UCLA School of Medicine Neutrophils, the most abundant human white blood cell, are thought to participate in lung injury in several lung diseases, eg. emphysema, pulmomary fibrosis, and adult respiratory distress syndrome. Dr. Ganz's laboratory is exploring the mechanisms

whereby the neutrophil injures lung cells. In particular, he is studying the effects of a toxic neutrophil protein, defensin, on various lung cell types. Dr. Ganz has found defensin to be capable of injuring and killing several lung cell types in tissue culture. This indicates that defensin may contribute to inflammatory tissue injury in lung disease. John Joseph Murray, M.D., Ph.D., Vanderbilt/Duke University Medical Centers White blood cells, which are normally an important part of the body's immune system, are at times activated inappropriately, either by substances within the body as in autoimmune disease or by environmental agents, to produce a group of inflammatory factors known as leukotrienes. During the past year Dr. Murray has been investigating the mechanisms by which the white blood cells generate these leukotrienes. Dr. Murray has demonstrated that the production mechanism is kept in a separate compartment within the cell and only with certain signals does this compartment become available for the synthesis of leukotrienes. This insight may lead to new ways to regulate the generation of leukotrienes and to examine how different disease processes may be related to the activation of the leukotrienes.