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PRODUCED IN BUTLER V. PHILIP MORRIS, ET AL. DISSEMINATION IS PROHIBITED BY PROTECTIVE ORDER ENTERED BY THE COURT.

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Bio-Research Institute Incorporated 9 Commercial Avenue John Langone, former Medical Science Writer The Boron Herald Cambridge, Massachusetts 02141 Telephone: (617) 864-8735 Third Report 1966 Drawings and Photographs Thomas Am$1er This brochure woa made [m.ible by funds from the Sciences and ,4rts Foundation. Dedham, Ma.~. and Irom B,o-Research Consultants, Inc.

The Front cover symbolizes the tree of life. Aerial photograph shows Institute location. Gifts and Bequests to Bio-Research Institute, Inc. are Tax Deductible to the extent prescribed by law Under present U. S. Public Health Service cost-sharing policy, funds from private sources are indispensable to obtain government health research support. Each dime obtained from private sources can attract dollars of government support. ~Nithout private money no Public Heal|h Service support can be obtained. L

Contents Foreword .............................................................................................. 6 Scientific Achievements ........................................................................ 7 Muscle Studies ...................................................................................... 8 The Heart Muscle in Muscular Dystrophy ......................................... 9 Studies on Diseases of the Vascular System " 10 Cancer Research .................................................................................. 11 Genetic Studies .................................................................................... 14 Histochemistry ...................................................................................... 14 Enz)a'nology Studies .............................................................................. 14 Endocrine Research .............................................................................. 15 Background .......................................................................................... 17 The Seeds of Enterprise ...................................................................... 19 Facilities ................................................................................................ 19 Educational Programs .......................................................................... 20 Extramural Staff Activities .................................................................. 20 Statement of Financial Research Support .......................................... 22 Director~ and Ol~..cers .......................................................................... 23 Research Associates .............................................................................. 23 Advisor~ ................................................................................................ 23 Current Bibliography ............................................................................ 24

i Foreword The continuous publication of our research findings in scientific journals and through papers read at meeting~ of national scientific societies keep our colleagues informed on our work. Periodic comprehensive reports such as the present one are intended to provide information to the public who supports this Institute through private donations and through grants of private and government foundations and agencies. In addition, these reports endeavor to give an overall picture to the public and scientists of the purpose, organization, method of work and achievements of this Institute, the type of information which is not contained in scientific publicatiom.. Since publication in scientific journals establishes credit for the individuals who have carried out the published work the present report denotes the Institute's studies collectively without acknowledging individuals. A professional medical science writer ha~ interviewed our staff and reports things as he sees them. From an experienced *cience journalist we thus learn how others ~ee us and obtain a better view of the whole than we may have ourselves. On reviewing what John Langone has written whi!e he still was Medical writer of the Boston Herald, we read about an impressive institution which has accomplished much in a "..hort period of tirt,e. Muck, remains to be done to realize o**r fondest dream~. This Institute ha., already made significant contributions a,:d is showing the way to a new and fruitful approach to the solution of Bio-Medical problems. On the occasion of the opening of the AMA Institute for Biomedical Research Irvine Page noted that "After a decade or two a body of knowledge with some in- ternal structure should begin to emerge." Here at Bio-Research Institute this has already happened during our first eight ),ears and it will be the task of the coming years to consolidate and to expand this structure of new knowledge, hoping that by doing so some of the problems which we have discovered and defined will be solved for the benefit of mankind. F. Homburger, M.D. President and Director o o

Scientific Achievements Bio-Research Institute, Inc is an aggregation of scientists with a wide raoge of interests. Their motivation ~s the inquiring mind of the scientist and the necessity of increasing mankind's knowledge of the healthy and diseased bodies of all living crea- tures. The basic research they perform falls within the spheres of experimental pathology, endocrinology, genetics, biochemistry, immunochemistry, nutrition, histo- chemistry and organic chemistry. These studies have some bearing on consti- tutional diseases of man such as chronic heart failure, aging in general, muscular dystrophy and related neuromuscular disorders, environmental hazards such as carcinogens, the relationship of certain antigens to cancer, the relationship of serum llpoproteins and lipids to arterial and cardiac disease, certain physiologic factors affecting reproduction, and endocrine studies bearing on health and disease. Some solid successes after eight year~ of operation are listed below, achievements of which Bio-Research Institute may justifiably be proud. Some of this work already has brought forth results which are clearly applicable to the problems of human health, and the Institute's achievements have been widely recognized : • Bio-Research Institute has discovered, for the first time, a spontaneously and predictably occurring heart disease, congestive heart failure, occurring in a laboratory animal, the golden hamster. In essence, the discovery means that scientists can now "take apart" the syndrome of heart failure. • The discovery, of a dystrophy-like myopathy (muscle disease) in Syrian golden hamsters will go a long way toward advancing the understanding of httman mus- cular diseases. • Findings made by Bio-Research Institute relative to the liver carcinogenlc~ty of Safrole and to the mode of action of tobacco smoke condensates when al~lied to the skin are clearly relevant to the serious problems of man's relationship to his environment. • Following the catastrophic Thalidomide episode which left in its wake thou- sands of malformed children, Bio-Researrh Institute under a contract with the Food and Drug Administration studied the possible usefulness of inbred hamsters for the study of drugs for teratogenicity, the causation of fetal malformation. This work continues under a grant from the National Institutes of Health. • Bio-Research Institute studies have resulted in the development of a method for the formation of water-insoluble, biologically active antigens. The water-insoluble antigen is a new tool in immunology. It makes it possible for the first time to obtain specific antisera against minute amounts of antigens free from contaminating antibodies against other antigens occurring in large excess. • The development of an analogous t~hnique perroitting the transformation of any enzyme into a water-insoluble yet biologically active form. This new principle not only allows easy removal of enzymes from reaction mixtures with multiple appli- cations in industrial and basic research, but also permits the study of certain phases of the mechanisms of enzyme-catalyzed reactions. o

• By use of insoluble antigens, minute amounts of a strange antigen were detected at Bio-Research Institute in the blood of virgin ,nice susceptible to certain tumors, whereas this antigen was not found in non-susceptible virgin mice. • Systematic research on the chemical composition of mouse uterine secretions has resulted in the isolation of a mucin-llke glycoprotein with a ve,y high content in carbohydrate and which contains a novel carbohydrate component. • A new approach to the study of human serumlipoproteins has made it possible at Bio-Research Institute to draw conclusions on the complex molecular structure of low-density lipoprotein, a substance which occurs in human serum and which is responsible for the transport of most of the fat in blood, and which may be implicated in the deposition of fatty materials in patients with vascular diseases. • Fundamental studies in en~'mology at Bio-Research Institute ha~'e given further insight into the behavior of enzymes at high dilutions and into the mechanism. of their inhibition by macromolecular polyanionic substances. • Bio-Research Institute studies have demonstrated that the incidence of lung adenomas (tumors consisting of glandular material) in certain strains of mice was reduced in groups of animals having received one or more intravenous injections of from 2 to 4.5 mg of a denicotinized cigarette tar condensate. The findings, therefore, suggest the existence of a tumor-growth-inhibiting substance in cigarette smoke condensate. • Bio-Research Institute studies of anticoagulant and clot-dissolving materials in secretions of the mouse uterus may apply to problems currently encountered with certain widely used hormonal drugs. Some of the Institute's other research projects have yielded new and valuable knov,ledge, the practical significance of which may not yet be apparent. Muscle Studies During the early year~ of the Institute a study was begun on the inheritance of coat color in hamsters, as well as on the anatomical features of the various inbred lines with different coat color~ which had been developed. It was soon noted that one line, identified as BIO 1.50, had a shorter life span than all the others, and ~hat animals in this line showed evidence of premature aging, becoming weak and dying at about 200 days of age. Subsequent microscopic studies revealed cardiac lesions, and shortly thereafter typical lesions in skeletal muscle were observed and recognized as resembling muscular dystrophy. This discover7 of a dystrophy-like myopathy in Syrian golden hamster~ ~ an achievement which has made Bio-Research Institute a leader in the field of muscle research -- is significant became the heart muscle also is involved and a unique opportunity is provided for the study of spontaneous heart muscle necrosis. The ~nly dy.~trophy-like mu.~cle dls.-.a~e which was noticed ~-x't urring in animals up to the time of this discovery was spontaneous dystrophy in mice and chickens. Myopathies induced by vitamin E. deficiency have also been described in rabbits, hamster~ and desert rats. The newly-observed myopathy evoked considerable interest in the scientific world. (including two editorials in the British Medical Journal "Lancet") because it appeared to have a simple genetic mode of transmission (three separate genetic studies indicate transmission of the disease or the suscep- tibility to the disease by an autosomal |-ccessive gene). "['he animals are not severely int'apacitated until late stages of the disease, which means they are able to breed and are available for study over extended periods of time. o 1"-4

Work carried out in the Bio-Research Institute since this discovery has dealt with the detailed descr;ption of the disease, its genetics, histopathology., histochemist~', and its enz.vmology. Attempts have been made to clarify its pathogenesis, and to evalo uate the position of hamster myopathy among the many experimental myopathies and its relationship to progressive muscular dystrophy in man. These studies have demonstrated that in the inbred lines carr)'ing the dystrophic gene, all animals develop myopathy. When animals beyond weaning stage are sub- jected to the severe exercise test of swimming, all individuals of the dystrophic lines show weakness of the adductor muscles of their hind legs. This occurs after as little as one hour of swimming. By four hours it is evident in all animals of the dystrophic lines, whereas this type of weakness of the hind leg~ is not seen in equally stressed individuals of non-dystrophic lines. Animals allo,ved to live a normal life and to be. bred have relatively few litters, the females usually dying after the second or third delivery.. The average life-span of breeding females is 144 days,, that of males 130 days, and that for the combined sexes of the dystrophic 14.6 line is 146 days. Death usually occurs from cardiac failure with decompensation. These studies have shown that until the animals die they suffer from progressive muscle weakness, not growing as well as animals of other lines. Their "normal" or abnormally high body weight at later ages is the result of fluid retention, while their organs and muscle mass are actually normal or reduced in size, with the exception of liver and heart which are often markedly enlarged. Since dystrophy-like myopathy and cardiac necrosis of hamstem are transmitted by an autosomal recessive gene or genes, it is possible that the genetic constitution causes a primary enz2,'matic defect similar in its general nature to other inborn errors of metabo- lism. In that case, the problem is to discover the primary molecular disturbance as distinct from the secondary changes which result from muscle breakdown. It is also possible that only the susceptibility to the disease is inherited and that unspecific and perhaps ubiquitous environmental factors could be responsible for triggering the disease in susceptible animals. This situation would be analogous to the combination of inheritance and milk agent which in mice cause cancer of the breast. In that event, it would remain to discover in addition to the primary molecular disturbance, the environmental agent or agents. Attempts have so far failed to transmit the myopathy to animals of non-suscep- tible strains by direct injections of crude muscle and Organ extracts, supernatants and cell-free filtrates from muscle and kidney tissue cultures as well as by injection of the cell cultures themselves into newborn and weanling animals. The Heart Muscle in Muscular Dystrophy The acquisition of knowledge concerning heart involvement in progressive mus- cular dystrophy and in other forms of primary myopathies is important from several viewpoints. Ignorance of this subject may lead not only to i**correct clinical classifica- tion of the cardiac symptoms, but also to inaccurate etiological diagnosis and treat- ment. From a pathophysiologic viewpoint, cardiac involvement is of great interest in respect to both its hemodynamic effects per se and the underlying mechanisms of heart failure. Furthermore, studies of this type of cardiac condition provide an oppor- tunity to gain information concerning the difference between the disease susceptibility

l 10 of the cardiac and skeletal muscles, since virtually nothing is known of the factors that determine which of these two structures will undergo degeneration in any given set of pathogenic circumstances. From a biochemical viewpoint, an analysis of the altered myocardial metabolism may elucidate the primary biochemical derangement responsible for the disease of striated muscles. In addition, animals which invariably develop progressive fatal heart failure constitute a uniquely valuable model for the study of drugs that may benefit the failing heart and circulation. Studies on Diseases of the Vascular System Over the years, great emphasis has been placed on the blcod serum l'evel of cholesterol in relation to heart and blood vessel disease. Cholesterol v~as singled out among all the blood lipid components simply because an easy clinical method for measuring it has been available for some 40 years. It is accepted that cholesterol occurs in the serum only as an integral part of the lipoproteins. Therefore, the relation of these serum proteins to heart disease should be at least as close as that of cholesterol. In fact, certain researchers feel that lipoproteins may be even more important than cholesterol. Bio-Research Institute has been engaged in studies of beta-lipoprotein and its relation to disease. With experience gained investigating the interactions between certain large, charged molecules and beta-lipoproteins, the first practical, simple and accurate method for the clinical determination of beta-lipoprotein in serum was developed. Statistical evaluations on large populations without costly and intricate instrumentation, thus, became practical for the first time. Subsequent work has led to new ways of purifying serum beta-lipoprotein with charged macromolecular poly- anions as precipitants. At the present time, human serum lipoprotein is being reacted with proteolytic enzymes, i.e., protein-splitting enzymes, including trypsin. Lipoprotein which contains 75 per cent fats and 25 per cent peptides has been found to be only very slowly degraded by trypsin, and this degradation stops at about a point when only a quarter of the peptides is removed. The lipid does not separate from the lipoprotein during this procedure. - Amino acid analysis showed that the amino acids or peptides which are re- moved have exactly the same composition as those which remain on the lipoprotein. From this, it is concluded that the trS'psin does not take off a small part of the protein from the surface of the lipoprotein, but that certain particles of lipoprotein are com- pletely broken down, while others are not degraded at all. This led to a closer look at certain structures making up the giant particles of beta- lipoprotein. It is believed that instexd of having a single large spherical particle contain- ing all of the lipids and a thin coating of peptides at the surface, there are small spherical particlm of lipids coveted with peptides, and that many of the small ,~pheri. cal particles clump together to form a large spherical particle. Thh is the structure which, at the moment, explains best the experimental findings. Modified lipoprotein can be prepared by proteolytic digestion, with the resulting products being useful for study/ng the steric organization of the water-soluble lipoproteins. Degradation of certain lipoproteins and of chylomicrons by the enzyme lipo- protein lipase is an important step in the lipid metabolism of the human body and lipoprotein lipase has been known to be generated in serum upon administration of the mucopolysaccharide, heparin, a substance which prevents clotting of the blood,