Opportunities in Biology (1989)

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

10 Advances in Medicine, the Biochemical Process Industry, and Animal Agriculture ;.. The impact of biological research on health care has long been recognized by the medical and pharmaceutical industries. The rapidly expanding fields of cellular and molecular biology continue to generate possibilities for new pharma- ceutical products and medical practices that will have major impacts on the pre- vention, diagnosis, and treatment of human disease. The application of biochemical and molecular understanding of cellular pro- cesses to product development in plant and animal agriculture lags behind that for medicine. Where such applications have been made in animal agriculture, they have generally resulted from the transfer of biomedical breakthroughs made origi- nally in biomedical research. Currently, however, the biological knowledge is being exploited with increasing effectiveness in the development of new plant and animal agricultural products. ADVANCES IN MEDICINE The long and successful overlap of all areas of biology makes it impossible to separate their relative contributions to the development of medical practices and pharmaceutical products. Instead it is more fruitful to discuss some key examples in which the fundamental knowledge of biology is likely to lead to advances in . . medicine. 323

324 OPPORTUNITIES IN BIOLOGY Molecular Pharmacology and Human Disease A Primary Objective of Biomedical Research Is the Development of Chemical Agents That Can Selectively Relieve and Abolish Pathological Processes Traditionally, the discovery of new drugs has been based on the chemical synthesis of large numbers of analogs, which are then analyzed empirically in screening programs. The success rate has been low relative to the effort and expense involved. In contrast to these traditional methods, most drugs can now be discovered by a concerted effort with a limited number of compounds because the investigators proceed with a rational approach based on a clear understanding of the fundamental properties of the systems~hat is, the receptor-recognition prop- erties or the identity of key regulating enzymes. Some notable examples have been the development of propranolol, an agent used worldwide in the treatment of cardiovascular diseases; cimetidine, a histamine-2 agonist, which has dramati- cally altered ulcer therapy; and lovostatin (Mevacor3, a selective inhibitor of cholesterol production a powerful new tool in the attack against atherosclerosis. Scientific and analytical developments in the past decade have provided powerful tools for the dissection and understanding of many critical biological processes. For perspective, it took 40 years to elucidate the structure of the neurotransmitter norepinephrine after the demonstration in 1921 that adrenergic neurons released an agent that increased the rate and force of cardiac contraction. It took an additional 20 years to understand the sites and mechanism of synthesis, storage, release, and response to norepinephrine. These inquiries are valuable because the chemical manipulation of intrinsic norepinephrine production, re- lease, and action is the primary basis for the treatment of hypertension in millions of patients. In contrast to the time required to develop norepinephrine, the application of modem analytical chemical techniques (especially thin-layer chromatography, high-pressure liquid chromatography, and gas-chromatography-mass-spectrome- try) have resulted in the rapid discovery and identification of trace amounts of extremely labile but potent substances produced in the body from arachidonic acid that are involved in the regulation of cardiac, pulmonary, allergic, inflamma- tory, and blood disorders. In 1973, the prostaglandin endoperoxides were de- scribed; in 1975, thromboxane A2 (vasoconstrictor and platelet aggregator); in 1976-1977, prcstacyclin (vasodilator and inhibitor of platelet aggregation); and in 197S, leukotrienes (chemotactic and anaphylactic substances). Chemical agents that modify the synthesis of such arachidonate metabolites have already been discovered and proven effective in experimental animals and in clinical trials. The recognition Hat normal bodily function, cell-cell communication, and disease processes (such as inflammation, myocardial infarction, immune response, and allergy) are mediated by minute amounts of biologically active substances

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, ED ANIMAL AGRICULTURE 325 produced in the body and intricately regulated has provided targets vulnerable to chemical manipulation for therapeutic benefit. Such studies require the develop- ment of interdisciplinary programs that bring together cell and molecular biolo- gists, analytical and synthetic chemists, whole-animal physiologists and pharma- cologists, and clinical scientists, who collectively focus their research on under- standing the molecular bases of disease processes and on their therapeutic modifi cation. The efforts are directed at elucidating (1) the characteristics of the intrinsic biochemical pathways that mediate and modulate normal and pathologi- cal function, (2) the determinants and mechanisms of receptor recognition and coupling to intracellular pathways, (3) the design, synthesis, testing, and targeting of new chemical entities, and (4) the evaluation of human disease through the development of new diagnostic strategies, and (5) the application of newly dis- covered potential therapeutic agents. Thus, opportunities presented by modem analytical, cell, and molecular biological techniques will not only elucidate struc- ture, but when coupled to synthetic efforts, can provide large amounts of material for whole-organ and whole-animal pharmacology studies and ultimately to thera- peutic applications. Two specific examples of such efforts are illustrated below. Prostaglandins, Thrornboxane, and LeuLotrienes Have Great Potential for Therapeutics For the past decade, arachidonic acid research has emphasized the discovery and elucidation of locally synthesized potent metabolites that influence regional tissue function. Two primary metabolic routes have been characterized exten- sively. The first of these is the cyclooxygenase pathway, which produces prostaglandins and thromboxane; the second is the lipooxygenase pathway, which produces the leukotrienes. The cyclooxygenase products have been characterized for their role in renal, cardiovascular, and platelet function. The leukotrienes subdivide into two types: (1) those, such as leukotriene (LT)C/D, that are slow- reacting substances of anaphylaxis and that seem to be intimately involved in pul- monary smooth muscle during anaphylaxis and allergy; and (2) LTB4, which is an extremely potent chemotactic substance most likely involved in inflammation and . . . tissue injury. The manipulation of arachidonic acid metabolism provides an ideal approach for the development of new therapeutic modalities. The progression of these investigations has created a situation that dictates the metabolic targets that could be usefully modified by pharmacological agents. A fascinating discovery has arisen from the dietary manipulation of fatty acids in experimental animals. The depletion of arachidonic acid or its precursor from the diet produces essential fatty acid deficiency, which is life-saving in the autoimmune destruction of the kidney in mice genetically affected with glomeru- lonephritis (lupus). The lethal consequence of this disease arises from macro

326 OPPORTUNITIES IN BIOLOGY phage invasion and destruction of the glomerulus. Essential fatty acid deficiency blocks the leukocyte attack on the glomerulus, apparently by blocking local LTB synthesis. Although essential fatty acid deficiency is not a practical treatment for lupus, it provides a special insight on what might be a useful therapeutic ap- proach. Alternatively, substitution of fish oil for arachidonic acid or its precursors in the daily diet could achieve similar results if they were adequate to interfere with macrophage function. Thus, on the basis of a sound foundation of studies of arachidonic acid metabolism, specific therapeutic target areas can now be approached on a rational basis. Manipulation of the cyclooxygenase pathway could yield beneficial effects in immune response regulation and thrombosis, while manipulation of He lipoxy- genase pathway, especially leukotriene synthesis, could provide a major new class of agents for the treatment of asthma, allergy, anaphylaxis, and inflamma- tion (including myocardial infarction and renal disease). Atriopeptin Is a Cardiac Hormone Intimately Involved in Fluid, Electrolyte, awl Blood-Pressure Homeostasis Atriopeptin, a peptide hormone, also termed atrial natriuretic factor (ANF) is intimately involved in the regulation of renal and cardiovascular homeostasis. Figure 10-1 illustrates the way in which atriopeptin links the heart, kidneys, adrenals, blood vessels, and brain in a complex hormonal system that regulates volume and pressure homeostasis. Basal levels of circulating atriopeptin can be increased by atrial stretch caused by volume expansion, constrictor agents that elevate atrial pressure, immersion in water, atrial tachycardia, and high-salt diets. Once in the circulation, atriopeptin exerts a number of effects related to renal and cardiovascular functions. Because it suppresses elevated renin in the plasma and relaxes blood vessels, atriopeptin reduces vascular resistance, thereby lower- ing blood pressure. Atriopeptin-immunoreactive neurons have been observed in rat brains, espe- cially in a hypothalamic region that has extensive connections with structures that regulate cardiovascular functions. Animals with lesions that include this region show profound alterations in the regulation of fluid balance, and neither renal hypertension nor salt-induced hypertension develops. Thus, atriopeptin may serve as both a central neuromodulator and a peripheral hormone in the regulation of cardiovascular and renal function. Current evidence suggests that the major actions of the hormone are to promote loss of fluid and electrolytes and to reduce vascular tone. These com- bined actions would be expected to reduce atrial stretch and suppress the release and processing of atriopeptigen, the 12~amino acid prohormone that is the primary form of atnal peptide stored in atrial myocytes. Effects on Renal Function. Atriopeptin also alters salt and water metabolism. The infusion of atriopeptin into laboratory animals produces rapid and transient

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 327 Brain ~~~~ TV_ / Vasopr~ssin , ~ FIRM, electrolyte, Elevation Atrial arm vascular ~ stretch , .- __- / hi; Decreased blood pressure  Decreased aldosterone \ Increased urine volume, sodium excretion, etc. Heart ~3~- Blood vessels ~\ Adrenal\ it\ \1 L Circulating form an I FIGURE 10-l Schematic diagram of the atnopepiin hormonal system. [Adapted from P. Needleman and J.E. Greenwald, New Engl. J. Med. 314:828 (1986)] Atriopeptigen 126-amino-acid prohormone I Selectrve cleavage  excretion of water, sodium, and potassium. The glomerular filtration rate and the filtration fraction remain elevated throughout the peptide infusion and diminish abruptly when the infusion is stopped. At present, there is no strong evidence of a direct inhibition of sodium transport in the renal tubules. The atriopeptin-induced increase in glomerular filtration rate is striking, especially since it occurs at doses of peptide that decrease blood pressure and total renal blood flow. Autoradiography techniques have revealed high-affinity recep- tor sites for atriopeptin localized on the glomeruli and in the papillae of the kidney. Those data, together with the finding that cyclic guanosine mono sphosphate (cGMP) increases markedly in isolated glomeruli and medullary collecting-duct suspensions incubated with atriopeptin, suggest that the glomeruli and the medullary collecting duct are the sites that regulate volume and electrolyte excretion. Such action at these sites would be useful in situations in which conventional diuretics, which act through tubular sites, are ineffective. A potent natriuretic and diuretic agent that is a selective renal vasodilator would have therapeutic potential in pathophysiological states characterized by fluid and electrolyte imbalances. Some promising clinical targets for research on atriopeptin therapy include renal failure, hepatorenal syndrome, and congestive heart failure.

328 OPPORTUNITIES INBIOWGY Human Physiology and Pathophysiology. With the development of radioim- munoassays, it has become possible to study the release of human atriopeptin. As in other mammalian species, the concentration of atriopeptin in humans is ele- vated when intravascular volume and right atrial pressure are increased. Plasma atriopeptin is elevated in adults with chronic renal failure or with congestive heart failure, and a worsening of the condition is directly correlated with the concentra- tion of circulating atriopeptin. Thus, atriopeptin may have a unique role as a therapeutic agent, especially in critical-care situations in which patients are undergoing intravenous therapy. The demonstration that atriopeptin can increase the glomerular filtration rate in ani- mals with severe renal insufficiency suggests that it has the potential to reduce the frequency of dialysis. The doses of a number of currently used drugs, such as certain antibiotics and chemotherapeutic agents, are limited by their toxicity to kidney tissue. Concurrent administration of atriopeptin with such agents may increase their therapeutic efficacy by inhibiting renal deterioration. The maintenance of circulatory volume and salt homeostasis, despite changes in diet, fluid intake, posture, physical exertion, and stress, requires the integrated action of several endocrine systems that have contrasting functions. Imbalances of these complex compensatory systems become unmasked in certain disease states. One approach to the therapeutic manipulation of endocrine systems is to administer agents such as atriopeptin to mimic, release, enhance, or antagonize the intrinsic hormone. The discovery of nonpeptide mimics of the atrial peptides or of agents that could stimulate the synthesis and release of endogenous atrio- peptin would allow the testing of long-term atriopeptin therapy for hypertension as well as for numerous renal and hepatic diseases. New Approaches to Understanding Health and Disease: The Lipoproteins Atherosclerosis Is the Principal Cause of Death in the United States, and Heart Attack (Myocardial Infarction) and Stroke (Cerebral Infarction) Are Its Overt Manifestations Atherosclerosis, the disease process leading to the life-threatening events of heart attack and stroke, causes the progressive narrowing and eventual blocking of critical regions of the arterial bed. A major feature of the atherosclerotic lesion is the deposition of cholesterol in association with cell proliferation and connec- tive tissue elaboration. Extensive epidemiological and pathological studies have pointed to an important association of lipids and lipoproteins in this disease. Certain species of lipoproteins that accumulate in plasma as a result of aberrant production and catabolism are the source of the cholesterol that accumulates in the artery wall.

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 329 Several Major Classes of Lipoproteins mist Plasma lipoproteins normally serve important homeostadc functions in bio- logical structure and hormone action as well as in energy production. They are spheroidal particles constituted of a central core of hydrophobic lipids (cholesteryl esters and triglycerides) surrounded by a surface layer of hydrophilic lipid (phospholipids) containing cholesterol and unique proteins called apolipoproteins (Figure 10-2~. The major classes of lipoproteins in plasma are the chylomicrons, very-low-density lipoproteins (VLDL3, low-density lipoproteins (LDL), interme- diate-density lipoproteins (IDL3, and high-density lipoproteins (HDL). Each major class contains particles that are structurally and compositionally heteroge- neous, are routed into different metabolic channels and may or may not give rise to potentially atherogenic particles. A detailed understanding of this heterogene- ity, therefore, is crucial to an understanding of the basic biology of lipoproteins UNFSTERIFIED PHOSPHOLI PI P~PR~TFIN ~-100 CHOLESTERYL FIGURE 1~2 Low-density lipoprotein. [Reproduced, with pennission, from M. S. Brown and J. L. Goldstein, Scientific American 251(5):58 66 (1984)]

330 OPPORTUNITIES IN BIOLOGY and to the establishment of a rational basis for the treatment of lipoprotein abnormalities associated with atherosclerosis. Cigarette smoking, hypertension, and diabetes increase the chance of devel- oping artherosclerosis. Most individuals who are predisposed to the disease do not show clear-cut defects in lipoprotein metabolism. They may, however, show characteristic features in their plasma lipoprotein profiles, such as high blood concentrations of LDL and low concentrations of HDL. The data suggest that both genetic control and environmental modulation of lipoprotein production, structural and compositional properties, metabolic channeling, and catabolism may produce a net accumulation of lipid, including cholesterol, in arterial cells. The detailed study of these features provides an exciting opportunity for acceler- ated progress in understanding the function of normal and abnormal lipoproteins. Research on the Structure and Synthesis of Apolipoproteins Will Lead to Insight into Cardiovascular Disease Apolipoproteins are determinants of major processes in lipoprotein assem- bly, secretion, extracellular processing, and catabolic removal, particularly via receptor-mediated pathways. Sequencing of many of the apolipoproteins has been accomplished, DNA clones for some of these have been obtained, and the mapping of apolipoprotein genes has proceeded. Altered forms of some of these genes have been associated with hypertriglyceridemia and with an HDL defi- ciency state with premature atherosclerosis. The incisive techniques of cell and molecular biology offer exciting prospects for increasing our understanding of apoliprotein gene regulation and expression. It is important to elucidate how dietary, hormonal, and environmental factors regulate apolipoprotein gene ex- pression. In addition, a host of other questions remain on topics such as the assembly of lipoproteins, the role of mutations, and developmental changes in lipoproteins. Studies like these should be linked to studies on heritability. In this connection, research on genetic polymorphism is very important. Information on Critical Aspects of Lipoprotein Assembly and Secretion Is Lacking Some of the general features of lipoprotein assembly and secretion are known, but many important components of these processes are poorly under- stood. For example, signal peptides on apolipoproteins are probably involved in intracellular targeting and secretion. The secreted nascent lipoproteins are ex- ceedingly heterogeneous in particle size and apolipoprotein and lipid composi- tion, and therefore in their metabolic fates. Analysis of the steps in assembly and secretion of the different species of lipoproteins can now be approached by the techniques of molecular and cellular biology. While the major emphasis in understanding lipoprotein assembly and secre- tion has been on the VLDL particles, basic information on the determinants of

MEDICINE, THE BlOCHEMIC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 331 HDL and LDL assembly and secretion by the liver is equally important but lacking. The processes involved in the assembly and secretion of nascent HDL are still little understood, and they gain importance in view of the role played by HDL in the control of cellular cholesterol. Knowledge of the effects of dietary, environmental, and pharmacological factors on lipoprotein assembly and secre- tion is rudimentary and in need of detailed molecular characterization. Insight into What Determines Which Lipoproteins Are Directed into One Metabolic Channel Versus Another One Is Crucial to Our Understanding of the Origins of Atherosclerosis Once they have been secreted, lipoproteins circulate in the bloodstream, where they interact with enzymes, receptors, and other factors that modulate their catabolic clearance from the blood. As these interactions occur, atherogenic products are sometimes produced. Organisms deficient in components of the extracellular channeling system (lipases, esterifying enzymes, or cofactor lipo- proteins) demonstrate extremes of abnormal lipoprotein distributions. For ex- ample, the investigation of the cellular biology of the LDL receptor has delineated a finely tuned system for regulating cellular cholesterol that affects plasma LDL levels. Receptor-mediated internalization of LDL and its lysosomal degradation produces free cholesterol within the liver cell which, in excess, can inhibit cholesterol synthesis and the synthesis of the LDL receptor. Intracellular storage of excess cholesterol is facilitated by activation of an esterifying enzyme. Some cholesterol is also secreted from the liver cells in the form of bile acids that enter the intestine and are partially recycled back to the liver. These control mechanisms have been exploited in pharmacological approaches to the control of plasma LDL levels in humans with hypercholesterolemia The oral administration of a resin that binds bile salts secreted from the liver into the intestine can remove some cholesterol from the body. This treatment, while increasing the number of LDL receptors, also steps up cholesterol synthesis, thereby reducing the the treatment's effectiveness. Administration of inhibitors of hepatic cholesterol synthesis also increases receptor synthesis, but to a greater degree than the resin. A promising approach for control of LDL levels in hypercholesterolemia is the use of combined drug regimes, which control choles- terol synthesis and reduce LDL in the plasma. The further molecular analysis of the regulation of LDL receptor action and cholesterol metabolism is of high priority. Such studies need to be linked with others at the level of the whole organism, so that dietary and environmental factors in heart disease can be evaluated. Mechanisms of Atherogenesis Involving Lipoproteins Are Being Determined In the bloodstream, the diverse classes of lipoproteins and their catabolic intermediates and products make contact with platelets, endothelial cells, macro

332 OPPORTUNITIES INBIOLOCY phages, and smooth muscle cells. Mounting evidence indicates that the interac- tion of these cells with elevated levels of specific lipoprotein species, such as beta-VLDL (a cholesterol-enriched VLDL remnant) and LDL, directly or indi- rectly promote changes similar to those observed at various stages in the develop- ment of the atherosclerotic lesion. The frequent observation of lipid-laden macrophage cells in lesions has prompted intensive research on macrophage interaction with lipoproteins. These studies have contributed valuable information on the major features of the macro- phage receptors and their function. In addition, a role for the macrophage in converting smooth muscle cells to foam cells (lipid-laden cells) has been sug- gested by in vitro studies describing incorporation by smooth muscle cells of lipid inclusions derived from the lysis of macrophage foam cells. Detailed information on such systems will greatly enhance our ability to deal effectively with athero- sclerosis. New Opportunities Exist to Prevent and Treat Diseases of Cell Proliferation Atherosclerosis can serve as an excellent example of a repair process in the artery wall, which is modified in different ways by chronic hypercholesterolemia and by factors generated from cigarette smoking, hypertension, diabetes, or other factors associated with an increased incidence of this disease process. In the United States and western Europe, chronic hypercholesterolemia is the risk factor associated with the highest incidence of atherosclerosis. The study of chronic, dietarily induced hypercholesterolemia in monkeys and pigs has permitted a chronological assessment of the cells involved in the development of the lesions of atherosclerosis. Within a few days after cholesterol concentrations equal to those seen in humans afflicted by the disease have been induced, white blood cells become increasingly sticky and adhere to the surfaces of the endothelial cells lining the artery. The high cholesterol concentrations induce changes both in the circulating white blood cells and in the endothelium. These white blood cells are scavenger cells that take up foreign substances, including some forms of lipid. White blood cells take up lipid in the innermost layer of the artery wall and become foam cells. In so doing, they accumulate and create the first and most common lesion of atherosclerosis, the "fatty streak." In the United States and western Europe, such fatty streaks are found in individuals of all ages, including infants, and represent the precursor lesion, which in many instances goes on to become the advanced, proliferative, occlusive lesion of atherosclerosis, the fi- brous plaque that causes myocardial and cerebral infarctions. Thus, experiments with pigs and monkeys were of critical importance in establishing the connection between hyperocholesterolemia and atherosclerotic lesions. The next change that occurs in chronic hypercholesterolemic monkeys may represent further"injury" to the endothelial cells. These cells seem to separate from one another at particular sites in the artery. After the endothelial cells

MEDICINE, THE BIOCHEMICAL PROCESS INDU=RY, AND ANIM~ AGRICULTURE 333 ~ ~:~ ~PORRI~:REE~:~NK:BE~E~EN ~POP~E~INS~AND~ ~ ~ :~ :; i: ~ :: ~::~:~:~:~:::: ~ ~ ~ ~;:~ ~:~TH~E~:~C~LorrlNG~SYsTEM~:~:~:~ ~:~:~:~ ~:~ ~ ~:~:~:~ ::: ~ ~: ~: :: ~::~:~:~An ~ :LDL~ variant ~:n~am~sd~::~ ~:~Lp~a)~ ~ ~h~as~bben~shown~to:~poss~ess ~ An Amen ah:::: ~ ::: : ~ :: : : :: :: :: :: : : :: ~ ~ : :: ~: ~ : : :: ~ ~ :: ~ : : : : : ~: ~:~ ~applipoprdtein ~called :~ apo ~A. ~:~:lThis ~apolip~protain~ is ~bttached ~ td:~the norma ~:~LDL:apolipoprotei~a~apQ~ B-1:~0~0~ b~r~a~:~sU~:llide~li~nkag~e.~:~::~:Hig~h~pl~asma~=nce~n~-~ :~::~ trab~ons:~(a3~ ~are~stron9ly~as~soci~d~with~accelerdi:~ d - elopment~of ~:::~dtheroscle~rosis~ ~:~:~e :~n~an~ner i:n~:wh~ich~::~Lp(~a)~:infl~ue~n~cas:::~:ath~eroselerosIs de-~:: ~::~ ~ ~.~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ ~,~ ~ ~ ~ ~ ~ ~ . ~ .~ ~ ~ ~ ~ ~ ~ ~ ~:~ ~ ~ ~: ~ ~ ~: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~:~ ~ ~ :~ ~ ~ ~: ~ ~: ~ ~ ~ :~ ~ ~ ~ : ~ ~ ~ : ~ ~ ~ : ~ v Q I o : p m ~ e n t ~ ~ ~ ~ : i s : : : ~ ~ ~ ~ s t ~ l I : ~ ~ : a : ~ : ~ ~ m ~ y s t e ~ r y . ~ ~ ~ : ~ ~ : : ~ : ~ : H o w e v ~ r ~ ~ ~ ~ : ~ o : n : ~ : ~ ~ : ~ t ~ h e ~ ~ ~ ~ ~ ~ ~ ~ : b a s ~ l s ~ : : : ~ ~ ~ ~ o f ~ ~ ~ ~ t h ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ a r n l n o ~ ~ a c l d : : : : ~ ~:~ ~ ~ ~ ~ ~ ~:~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~,~ C~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~, :~ ~ :~s~eq~u~en~ce::~ :af ~apo A: ~sCfe~nbsts~h:ave ~ :recently~:~:~shown~:~ that~ ~tl1:~'s apolipoprote':n: ~:~::~::; ~ ~:~:~:~ ~ ~:~ ~ ~ ~ ~ ~:~ ~:~ ~,~ ~ ~ ~: ~ ~ ~ ~:~ ~ ~ ~:~ ~: ~ :~ ~:~:~: ~ ~ :~: ~ ~: ~s:hares cons'~d:e:rable~:s~eq~u~Q~nce ~ho~m:ol~ogy~w~th:~:plas~m~noge:~n.~ ~P~I~asm'~n~gen~ ~is~a~ ks~f~ comp~nen~t~ ~i~n~ihe~ ~cluttin9:~sysiem,~whi~binds~to~fibrin~and~:when ~: :adtivatdd~ by: tissue~ plasminogen~=ivatbr~can enzy:rr~bt:~ally~dissolves~fibrin ~:~c~s~.~ ~: ~:lt~is~ ~interssting~to~:~s~c~ul~to o~n~the ~ ppssibld ~ ¢onn~ection~ between~ th~e~ ~ :plasminoge~n-likb :strutture~of ;~apo :~;~:an~d::~:~4s~:role: i:n::~bth~:rosclerosis:.~ ~Thers~is~:::: ~:~: ~avid~e~nce~ ~th~at~lip(~) is~ ~associdted~ w~th~ ~init~ial bthero~lerot:i£~plaques :~w~hich~ ~ ~ :;~i i :~:~ cobtai~n:~fib~ri~nog~e~n~(fib~rin ~precU~rsor)~and~fibri~n~i~n~ro~gh~r~the~sam~e~oonca~ntra~:~: ~:~tion~as;c~holesterol~.~ ~ It~h~s~been ~propo0~thdt~(a)~.~mediatted~by~th~e~apD~A:~:~:~ ~:mino ~ac~d~ seq~uences~th~are~homologous~plas~minoge~n s~fibrtn~ ~binding~ ~:~s~eq~uences ~:~::ca~n~:~he~Q:~ ~to:~the: :hth~eroscle~rot~;c~:pl~aqu:e~.~i~: :~EJucid:dt~io:n: ::6f~:~pos-~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : ~ ~:~s:ible~:apo~:~A~:~wit:h: co~m~pon~e~nts~of:~e clotting~sy~tem :awa:~:~:add~itio~nal r~search~ :~:~:~ ~to~ dete~rmine~: the ~ex~= role~ ~ ~:Lpfa3 ~and ~:aDo~A ~in~ ~e~ accele~ted~develop-:~:~ ~:::~::~:~: mQ:nt~nf ~h~aros£1erosis:.: ~:~ separate from one another, they then retract and expose the underlying white blood cells that have become foam cells. In many instances, another circulating blood cell, the platelet, attaches at these sites of exposure. Platelets seem to release substances that stimulate the localized proliferation of smooth muscle. We now know that the proliferation of smooth muscle cells at sites where platelets interact can be partly due to the powerful growth factors produced by platelets, endothelial cells, macrophages, and the smooth muscle cells themselves. One of these, the platelet-derived growth factor, is a potent mitogen that stimu- lates the proliferation of cells that form connective tissue, such as smooth muscle cells in the artery wall. Perhaps of equal importance is the secretion of other growth factors into the local environment by activated macrophages or platelets. Like platelet-derived growth factor, these factors could have a major impact on the local multiplication of smooth muscle cells and macrophages and set the stage for the development of advanced occlusive lesions of atherosclerosis. These lesions could enlarge over a period of months or years and eventually sufficiently

334 OPPORTUNITIES IN BIOLOGY occlude the lumen of the artery so that the vascular supply of blood and thus the oxygenation of the tissues are compromised. Such a set of conditions leads to the clinical sequelae commonly associated with heart attacks and strokes. The approaches of cell biology have permitted us to define the cells that take part in the process and to understand which cells interact, at what points in time, and where. Thus, it has been possible to analyze these cells and determine whether genes are expressed for substances such as growth factors, and, if so, to determine whether these growth factors can be responsible for the observed tissue responses. In addition, cells involved in these tissue responses are being isolated, grown, and puriSed, making possible their direct investigation in culture. These approaches represent exciting research opportunities both in basic biology and in the medical application of this knowledge. ARTHRITIS Since the 1930s, considerable progress has been made in distinguishing such entities as osteoarthritis and rheumatoid arthritis through the use of clinical and biochemical criteria. This progress has led both to an improved understanding of pathogenesis and to the development of new therapies. The Greatest Success Has Been in Understanding Hyperuricemia and Gout The major cause of hypen~ncemia is decreased excretion of Orate, which can occur spontaneously or be induced by drugs, particularly the diuretics used to treat hypertension. Drugs that increase the excretion of urate were introduced nearly 40 years ago and used successfully, but the introduction of the drug allopurinol has revolutionized the therapy of gout and almost completely elimi- nated chronic gout. Allopurinol, which decreases urinary urate by inhibiting the final reaction in its generation, is effective in lowering serum urate levels. This prevents the onset of gouty arthritis and the formation of uric acid renal stones. Inflammatory Joint Diseases and Seronegative Polyarthritis Remain Major Problems Although we know a great deal about its pathogenesis, we still do not understand the cause of the inflammatory joint disease rheumatoid arthritis. Specific antibodies called rheumatoid factors have been found in rheumatoid arthritis patients. These antibodies are specific for the Fc portion of other antibody molecules. The interaction of the rheumatoid factors with other antibod- ies amplifies inflammatory reactions. The increasing work on rheumatoid factors, with molecular cloning techniques, should provide results far beyond the study of rheumatoid arthritis itself.

MEDICINE, THE BlOCH~IC~ PROCESS INDU=RY, ED DIME AG~CULTU~ 335 In patients with rheumatoid arthritis, considerable attention has been directed toward the study of interactions of T lymphocytes with cells in the joint lining. T lymphocytes interact with other cells by cell contact mediated mechanisms and by the production of cytokines such as interleukin 1, tumor necrosis factor, lympho- toxin, transforming growth factors, platelet-derived growth factor, and certain colony-stimulatory factors. These interactions could result in the proliferation of the joint lining cells that leads to joint fibrosis and in the production of proteinases and lipid metabolites (prostaglandins and leukotrienes) that are responsible for swelling and pain in and around joints. The proteinases and cytokines are being studied in detail, in the hope of identifying new sites for therapeutic intervention. Factors Influencing Genetic Susceptibility to Rheumatic Diseases Are Being Determined In 1973, an exciting report indicated a strong association between suscepti- bility to ankylosing spondylitis and related disorders to the HLA-B27 allele of the class I histocompatibility locus. Since the HLA-B27 gene has now been cloned and sequenced, a new approach to understanding its role in pathogenesis has evolved. This approach involves searching existing protein sequence data bases for homology between HLA-B27 protein and proteins in suspected bacterial pathogens. A sequence has been identified that codes for six consecutive amino acids in Klebsiella pneumonias nitrogenase. Some patients with ankylosing spondylitis have circulating antibodies that specifically recognize this six-amino- acid peptide, which suggests molecular mimicry. In addition, rheumatoid arthritis has been associated with class II histocom- patibility antigens. The use of restriction fragment length polymorphisms (RErLPs) should lead to a more precise genetic analysis and more extensive use of specific probes to define genetic susceptibility. Lyme Disease Might Be a Good Modelfor Understanding the Pathogenesis of Rheumatoid Arthritis - Lyme disease was f~rst described in 1975 on the basis of observations of children with what was initially thought to be a form of juvenile polyarthritis. Since then, the disorder has been shown to be caused by the spirochete Borrelia burgdorferi. Lyme disease begins with a characteristic skin lesion that is fol- lowed by systemic symptoms. Polyarthritis may occur early, but more persistent joint swelling, particularly in the large joints, may take place weeks to months later. The histopathology of the persistent joint swelling is remarkably similar to that of rheumatoid arthritis. The demonstration of small amounts of Borrelia antigens in the joint lining suggests that some persistence of infection could be responsible in part for the arthritislike symptoms. Furthermore, the organisms are

336 OPPORTUNITIES IN BIOLOGY susceptible to antibiotics; high doses of penicillin not only prevent late complica- tions if given early in the disease, but also produce responses in patients with established arthritis. Thus, when details of the pathogenesis of Lyme disease become known, the information should aid in understanding the pathogenesis of rheumatoid arthritis. Osteoarthritis Is the Major Joint Problem in Terms of Number of People Affected and Overall Morbidity The etiology and pathogenesis of osteoarthritis are still undefined. In pa- tients with osteoarthritis, extracellular matrix components degrade through mecha- nisms that are probably basically similar to those operating in typical inflamma- tory joint disease. In some individuals, genetic factors may be responsible for abnormalities in matrix proteins (collagens and others). Because of the persis- tence and unpredictable course of osteoarthritis, it is extremely difficult to evalu- ate the effects of possible therapeutic agents. For these reasons, the major approach to this disabling disease is joint replacement. CANCER: CURRENT STATUS AND SOME NEW APPROACHES TO ITS CONTROL Cancer Has a Number of Causes Environmental and life-style risk factors such as the use of tobacco and alcohol, exposure to occupational carcinogens and pollutants, and diet are major causes of cancer. Only a small number of cancers are thought to be entirely genetic in origin. Environmental and life-style risk factors are believed to con- tribute to the development of about 90 percent of cancer incidence. For example, a major reduction in tobacco use alone could prevent 30 percent of cancer deaths. In comparison, improvements in cancer diagnosis and therapy have resulted only in a 5-year survival rate of 49 percent. From this, one can see that cancer prevention by avoidance of risk factors has the potential to play a more important role than therapy in the overall reduction of cancer deaths. However, much more study is needed before the full benefits of risk avoidance can be realized Even though cancer has a number of causes, many and possibly all cancers seem to have a common component, namely, alteration in the DNA of the transformed cell. That genetic damage is involved in the pathogenesis of cancers is suggested by several lines of evidence including (1) the existence of a subset of cancers influenced by hereditary predisposition, (2) alteration in chromosome structure specifically correlated with certain types of cancers, (3) impairment in mechanisms involved in DNA synthesis or repair of damaged DNA correlated with susceptibility to cancer, (4) the relation between mutagenic potential and carcinogenic activity of venous substances, and (5) the discovery that proto- oncogenes, which are normal cellular genes that in an altered form become

MEDICINE, THE BlOCHEMIC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 337 oncogenes, are linked to tumor development. Cancer is fundamentally a disorder in the regulation of cell proliferation and differentiation, reflecting genetic change that leads to loss of normal constraint. The process of carcinogenesis has many steps; when a genetic abnormality is present it probably represents only one necessary, but not sufficient, step in producing the malignant phenotype. Envi- ronmental factors such as viruses, radiation, or chemical carcinogens can also play roles in causing cancer. The remarkable rate of advance in cellular and molecular biology is leading to an understanding of the causes of cancer and providing more effective ap- proaches to prevention and therapies. This section summarizes certain areas in which this increased knowledge is being applied or is likely to be applied to the problems of human cancers, and it indicates some lines of necessary further investigation. Cancer Therapy Advances in Cancer Therapy Are in Surgery, Radiotherapy, and Chemotherapy Two major premises underlie current treatments of cancer. The first is that the disease, and especially solid tumors, spreads progressively, in discrete steps. It begins locally, permeates local tissues, and then extends to distant sites. For some solid tumors, however, this description is inappropriate. Further, even within tumor types, there may be wide heterogeneity, with some tumors demon- strating early distant metastasis, others showing no proclivity for metastasis, and others behaving according to the premise of progressive spread. A major limita- tion of present treatment and an opportunity for the future is to develop techniques that distinguish these types of tumors. The second premise is that tumor cells proliferate more persistently than normal cells. This has given rise to the development of cancer chemotherapeutic agents, which take advantage of putative proliferative differences between normal and malignant cells. Although this has been a useful approach, we have learned that tumors of tissues that are constantly renewing themselves, such as the hematopoietic (pertaining to or affecting the formation of blood cells) system and cells of the epithelial lining, violate this rule. These cell-renewal tissues are usually the most rapidly proliferating cells in the body. Therefore, they become the sites of dose-limiting toxicity when tumors of cell renewal tissues are treated by chemotherapy. Surgery. Surgery cures more patients with cancer than any other treatment. Examples of its success are in the treatment of colon cancer, cervical cancer, many head and neck tumors, and uterine cancer. Surgery fails when the tumor has already metastasized beyond the bounds of surgical resection or when the volume of the tumor is too great. Efforts for improvement are concerned with developing techniques that meet the same goal for cure, but are less mutilating because of

338 OPPORTUNITIES IN BIOLOGY better reconstruction and graft techniques, improved prosthetic materials, and the use of transplantation. Most importantly, surgical results are improving as sur- gery is combined with more effective diagnostic techniques as well as techniques such as radiotherapy and chemotherapy. Radiation Therapy. Radiation therapy is also based on the hypothesis that ablation of tumor can be curative. Its limitations are different from those of surgery. When radiation therapy fails, it usually fails in the center of the tumor. This has given rise to techniques of radiation that administer moderate doses of radiation to larger volumes, but restrict the high doses to the areas with proven involvement. Radiation has the advantage of potentially less functional impair- ment. Examples of treatment success include the treatment of prostate, cervical, and a variety of head and neck cancers, as well as Hodgkin's disease. New efforts in radiation therapy are concerned with techniques (1) to better localize tumors so that the treatment can be more effectively applied and (2) to deliver radiation more precisely, minimizing the irradiation of normal tissues. Such techniques take advantage of the major advances in electronic and computer technology. Biological approaches are concerned with increasing tumor sensitivity to radia- tion, and sensitizers of hypoxic cells to radiation are being studied. Chemotherapy. Cancer chemotherapy has often been based on the second hypothesis, that tumor cells proliferate more rapidly than normal. These therapies have been most successfully applied in those tumors that were associated with rapid proliferation, such as acute leukemia, the lymphomas, and testicular carci- noma. These drugs act at sites shared by malignant and normal cells, thus limiting their usefulness because of their toxicity to normal tissue. Further, these agents may produce alterations for which the cell can develop alternative mechanisms; selection of resistant clones of tumor cells seems to be the most prominent cause of failure, resulting in the development of cells resistant to the action of the chemotherapeutic agent. Mechanisms for this resistance include gene amplifica- tion and changes in membrane transport of chemotherapeutic drugs either into or out of the cell. Increasing the drug's dosage and therefore its efficacy has been made possible by the vigorous use of blood products to treat the hematopoietic toxicity. The logical extension of this treatment is the transplantation of bone marrow, which is being attempted with bone marrow obtained either from the patient and stored or from appropriate donors. A most important type of drug resistance that limits efficacy is pleiotropic: Cells become resistant to a whole host of apparently unrelated agents by one or a few changes. New antitumor cytotoxic agents are being developed that may be less toxic and also more effective against tumor cells that are resistant to available agents. Additional approaches to the treatment of cancer are being based on advances in our understanding of immune mechanisms and of the control of cell proliferation and differentiation.

MEDICINE, THE BIOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 339 Combined Use of Therapeutic Modalities Has Evolved Gradually Surgery, radiation therapy, and chemotherapy have different toxicities and limitations. Surgery is limited by the volume requiring resection in order to remove all subclinical disease. Often surgery fails because of unappreciated microscopic disease beyond the limits of the surgical procedure. Radiation therapy rarely fails at the margins of the treatment for subclinical disease. Rather, limitations in radiotherapeutic treatment efficacy are often caused by a large central tumor: Its toxicity is due to the high dose required to ablate such gross tumor masses. These two forms of therapy can be combined. Surgery removes the gross tumor and radiation therapy is applied to the larger region containing lower densities of cancer cells. Chemotherapy appears to be effective only against small volumes of tumor. It offers the best opportunity for application as a part of a multidisciplinary program where the two regional therapies have eradi- cated the gross tumor. Examples of combining the three modalities in this fashion are the treatment of Wilm's tumor of childhood, soft tissue sarcomas, breast cancer, a variety of tumors of the head and neck, and osteosarcoma. In some of these, only two of the three modalities are required. All have significantly increased survival and improved the functional and cosmetic results. Similar potential for multidisciplinary treatment is seen in bladder cancer; chemotherapy seems to reduce tumor volumes so that they are amenable to surgical procedures or radiation therapy. Where chemotherapy has the major role, radiation or surgery may be useful to destroy tumors in sites to which the chemotherapeutic agent cannot penetrate. This appears to be true for the central nervous system, where radiation therapy of the brain has improved the results in the treatment of some patients with leukemia and lymphoma. Surgery has a variety of new uses when combined with radiation and chemotherapy. The use of surgery to evaluate the extent of tumor has proved important in Hodgkin's disease, where removal of the spleen provides vital diagnostic and therapeutic information. A standard hypothesis that may not always be supported is that once the bloodstream has transported cancer through the body, the cancer becomes multiple and therefore resistant to a local therapy. Surgical treatment of limited metastasis in the liver, lung, or even brain has increasingly been attempted with good results. Further improvement may be achieved in combining such surgery with systemic agents. Newer Approaches to Cancer Control Prevention of Cancer Involves, in Part, Persuading People to Limit Their Exposure to Environmental Agents That Seem to Be Associated with Increased Risk The most outstanding example of increased risk for cancer associated with an environmental agent has been the relation between cigarette smoking and lung

340 OPPORTUNITIES IN BIOLOGY cancer. On less firm experimental grounds, but with sufficient evidence to be provocative, are suggestions that dietary factors are important causes of human cancers. Viruses Cause Some Types of Cancer, Especially in Developing Countries It is estimated that some 10 percent of cancers in the United States and as many as 30 percent in China are associated win virus infections. Among these are carcinoma of the uterine cervix, which has been linked to Be human papil- loma virus; primary liver cancer (prevalent in Africa and Asia), linked to hepatitis B virus; acute leukemias, linked to human T-cell leukemia viruses; and Burkitt's lymphoma and nasopharyngeal carcinoma, which may be carried by the Epstein- Barr virus. With the identification of such viruses, the possibility of developing immunizing vaccines is being actively pursued, although in each instance this approach is in an early stage of development. Perhaps most advanced is preven- tive immunization to block transmission of hepatitis B virus. Since the lag period between viral infection and onset of clinical cancer is many years, this program will require some time before its efficacy can be evaluated. All of the DNA tumor viruses mentioned above (hepatitis B virus, papilloma viruses, and Epstein-Barr viruses) infect differentiated cells that cannot be propa- gated readily in culture. This inability is a major limitation in studying these viruses. Some cell lines can be infected by human T-cell lymphoma virus 1 ~V-1) and human immunodeficiency virus (HIV), but the former virus is cell- associated and therefore infection can be most readily accomplished by cocultiva- tion. A major goal for advancing the study of all of these viruses is to develop efficient and easy modes of infecting cells in culture. For the DNA viruses, this goal will be achieved either serendipitously or by developing more and easier methods of culturing epithelial cells than those available today. It is also most important to identify those viral genes that predispose infected cells to develop Into tumors. Oncogenes. Tumor viruses were first found in chickens more than 75 years ago. Some of the genes coding for the proteins that control growth have been found in a mutated form in certain highly oncogenic retroviruses of animals; the mutated genes are called oncogenes, and the normal form of the genes, proto- oncogenes. Although no virus containing an oncogene has been found in humans, altered proto-oncogenes have been found in several human tumors. The proto- oncogenes are altered in different cases by base-pair mutations, amplifications, or translocations. The presence of these altered proto-oncogenes in human cancer has suggested that they might have a role in the genesis of the tumors. Certainly, the correlation of the location of proto-oncogenes with the break points of chro- mosomal translocations specific for certain cancers supports this hypothesis. For example, in Burkitt's lymphoma, one of a specific set of translocations involving Be c-myc proto-oncogene is invariably present (see Chapter 7~.

MEDICINE, THE BlOCHEMIC~ PROCESS INDUSTRY, ED ANIMAL AGRICULTURE 341 ~ :~ ~ ~: ~ ~ ~ MOLECUI>R GENETIC~ANA~INSIS ~OF~::INM~PH~O~ID~:: 71~MORS~ ~ ~ ~ ::: ~ lint ~ addition to providing ~ detailed information: about ~ the mechanisms of ~spec~if~ity generation lathe ~ immun~e~syste:m,~ molecular~ge~net~:analysis :of ~ ~;~ ~:k~mphocyt~es has: :~prov~ided :some~:im~po~rtant~in~s~ights~into~th~e classification I: ~lym~phoid~m~a~lig~nanc~ies~and,~perhaps~more~im~portant~ly,~into~the~mech~an~is~ms~ i: aft: aft ~disord:ered~::~:gro~'th~regulat:ion: Lint ~th:e~se~tumors.~ The determination: :of~th~e~:~ ~ :cell:~of~:~origi:n~ and th~e~d~eg:ree~::~:6f~d~iffere~ht~iation soft: the: malig~n:ar~t~ cell i~n:~many~ ~:~ ::: ~ ~ ~: ~ ~ ~ ~ ~ :~ ~ ~ :: loukemias: ::an~d:: iymphom~as~:~is~ made ~ possible: :by~establish~i:ng:~ :whethe~r~: the: If: :: ~im~munoglobu~l~i~n (19~ genesis or The ~71~e:1~1-r~cep~r~ genes Of That cell have been rearranged and how far ~the: rearrangement Process has~:~proceeded. :Such information often has:::cons~'de~rable~valu~::'n the~cho~e of:therapy~:a~nd~ ~:in~d~bte~rminingth~eprognos~is~ofthese~9mphoid~m~align~ancies. Abe~rrarii genera rearrangements sometimes playoff an Important Opt in : 'orming~hum:an~umors~from ~white~blo~ Cellist In the~oou~rse of detailed ::~ Examination ~of: the ~rearrangem~e~nts~of~lg~genes~ in Burkm's ~m~phoma~ cells ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ , ~ ~ ~ :: :~ and An: mused asmacytomas,~ A non-~lg~gene~::was~toun~a Stop ~ rave ~:~Deco~m~e~ ~associdt~ ~w~h~t~he~ ~:~9 genes as a rssu~lt~ of ~a: rec~iprmal~ exchange~f~ma;ter~al : :~ ~:between:~distinet~:c:hromoso~mes.::~ ~:~:lThat~no~n~-lg gene~provad~to fibs: threes cellular: Aft: ~ :onooge~ne ~c-myc, which lists crit~i¢al ~for~gro~h ~regul~tion. Mitts Changed lion ~: :~ ~:is~be~lieved~to ~be:crit:ical::to tha~process ~of:mal~ign~an~:in B~u:rkitt's Iymph~ma~ ~:~ ~ ~an~d~:~plasm~ytomas,~poss~ibly~ Cause the normal regu ation of ~ c-~7~ex- ~:~ ~ ~ : ~ ~ ~ ~ : ~ ::: p~ression has be:en:~d~isturb~ed: by: Rs:: using ~pl:ac31g ~ y:actlYe~ ~g:e:net~c: ~ ~ ~: ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ .~ :. ~ ~ .~ ~:~ ~:~:e:nviron:me~nt. Other oncog:snes are: translocatad::~into tn:e::reg~on ot~tn~e:~'g :ana ::: el~l-receptor~genes ~in~ot~her:types~ By- End Flkell ~;mai~ignan~¢;es,~ stm~ngi~y~: ind~icating~that~t~he process~of~d~iso~ered~oncogen~e regulation resulting from ge~n~etic~:trans~location infix :Iymphocytes~is~an~ important :mechanism in ~mal~ig-~ :~ ~:~nant transformation. Continued a:nd::~expan~ded: : efforts :to: determine The ~ :: : mechanisms of ~o~ncoge~:ne ~m~:dd~i~at~ion~ ~f~normal~and~abnorm:al~ growth :r~gula- :: :: ~;::::tion~:wil~i be ~:::fu~nda:me~ntal v~alue:~;n~ ~a~nde~rstanding~:~:~normal: growth :~reg~ulatio~n~ :: : ~:~:a~n~d:~:may lead~to~the development :of~drugs:that~can:contraI::th~e g~ro*Ith::::~df ~ ~ ~ malignant ~:ells. ~ ; :: :: i: : : : Other genes that have not been identified in detail may also place an individ- ual at increased hereditary risk for cancer. If a person who carries a gene associated with increased risk for a specific cancer could be identified, minimiz- ing exposure to environmental hazards associated with induction of such cancers (for example, skin cancers attributable to ultraviolet light) or applying early detection methods to individuals at risk (for example, familial development of polyps and colon cancers) might help in preventing cancer or treating it more effectively.

342 OPPORTUNITIES IN BIOLOGY New Developmental Therapies Utilizing Biological Response Modif ers, Monoclonal Antibodies, and Other Molecules Over Prospects for Treatment Biological Response Modif ers. These modifiers are substances that stimu late or modulate the host's cellular or immoral responses that may play a role in killing cancer cells. Examples include the interferons, interleukins, colony- stimulating factors, and tumor necrosis factors. The genes for most of these factors have been cloned, and the application of recombinant DNA techniques has made these substances available in sufficient quantities to permit evaluation of their clinical usefulness. A few years ago considerable publicity attended the potential of interferon as antitumor therapy. The availability of abundant amounts of interferon has made adequate investigation possible; it has now become apparent, however, that most cancers do not respond to interferon. One rare malignancy, hairy-cell leukemia, does respond to interferon-a. It is not clear why this leukemia is so sensitive to this interferon. Interleukin-2 activates killer lymphocytes. Recently interest in it has been rekindled by the demonstration that removing a person's lymphocytes, activating them in vitro with in~erleukin-2 OAK cells), and then injecting them back into the patient, along with substantial doses of interleukin-2, results in tumor regression in some patients. The most impressive results have been seen in patients with renal cancers and melanomas. The ultimate role of interleukin-2 in cancer therapy awaits controlled clinical trials along with further studies to increase efficacy and decrease toxicity and the complexity of administration. These studies raise the question whether the human immune cellular system can be manipulated to enhance normal function in dealing with tumor cells. Turrwr Necrosis Factor and Lyrnphotoxin. These modifiers were initially recognized because of their cytotoxic and antitumor properties. The dramatic effect of tumor necrosis factor on animal tumors aroused considerable interest in its potential clinical application, but initial clinical trials in patients with cancers have not shown similar antitumor effects. Lymphotoxin has limited amino-acid sequence homology to tumor necrosis factor, and, because it has been available in only small quantities, its biological characterization is less advanced than that of tumor necrosis factor. The general consensus is that tumor necrosis factor is part of a network of interactive signals for host immunological and antiinflammatory responses and in maintaining hematopoietic cell development in balance. Growth Factors. Growth factors regulate the proliferation and differentiation of cells in the body; many of these proteins have been isolated, their genes cloned, and their biological activity defined. Among these growth factors, the hematopoi- etic colony-stimulating factors are of particular clinical interest. These factors may be clinically useful after bone-marrow transplantation, in cancer chemother

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, ED DIME AG~CULTU~ 343 apy to ameliorate toxic effects of drugs in suppressing bone marrow, and in treatment of infectious diseases for which stimulation of hematopoietic cells can be clinically important. Monoclonal Antibodies (MAbs). Antibodies are proteins secreted into the blood by immunoglobulin-producing cells as part of the body's complex immune response against foreign substances. With the availability of hybridoma technol- ogy for the production of individual antibodies in large quantities, it is now possible to evaluate the usefulness of antibodies in the diagnosis and treatment of cancers. Hybridoma technology has made it possible to develop MAbs against many Apes of tumor cells. Some of them are useful in improving the precision of pathological diagnosis in a number of cancers. Radioactively labeled MAbs have been used as diagnostic tools to identify sites of primary tumors and metastatic deposits within the body; nuclear medicine imaging devices detect the radiation they emit. The factors limiting the usefulness of MAbs in diagnosis are similar to those limiting their application in therapy: (1) inadequate access of the MAb to tumor cell surfaces; (2) heterogeneity of tumors with respect to the amount of surface antigens on individual tumor cells to react with MAbs; (3) alterations in antigen on tumor cell surface causing the cells to be no longer recognized by the MAb; and (4) expression of target molecules for the MAb that are not unique to tumor cells, In that there may be cross-reactivity of an antitumor MAb with portions of molecules expressed on nontarget cells that lead to undesirable toxic side effects. Rarely does an antibody in isolation kill tumor cells. For example, in order to kill a tumor cell, the MAb can recruit natural effecters such as complement, killer lymphocyte cells, or phagocytes. MAbs can also be used as vectors to deliver to the target tumor cell a lethal agent such as a bacterial or plant toxin, a cytologic drug, or a radioactive isotope. MAbs can also potentially suppress a function in tumor cells, which decreases the viability of the cells. Clinical application of MAbs in therapy of malignant disease has had very limited positive results. Aside from one prolonged complete remission of a human B-cell lymphoma, the thera- peutic results for the dozens of patients with various cancers that have been treated have been transient and partial at best. Promising areas of current and future research are based on advances in molecular biology that provide a means to tailor antibody fragments for targeting to tumor cells. Other Molecules. Smaller molecules that cause cancer cells to differentiate have been discovered. Studies with these inducers are based on the concept that cancer frequently blocks differentiation and results in increased proliferation. Growth need not be fast to cause a tumor, only inexorable. The neoplastic state need not destroy the potential of tumor cells to differentiate to an end-state cell that would have lost its malignant potential to multiply. Differentiation of many tumor cells can be induced by a variety of structurally diverse agents, including

344 OPPORTUNITIES IN BIOLOGY polar planar compounds, hormones, vitamin derivatives, low doses of certain cytologic drugs, physiologically active growth factors, and tumor promoters. Among the several cytodifferentiation agents, the polar planar compound hexam- ethylene bisacetamide has been most extensively studied with respect to its cellular and molecular effects on transformed cells. It induces differentiation in a variety of cancer cell lines in vitro: leukemia, neuroblastoma, teratocarcinoma, colon, and bladder cancer, among others. Phase 1 and phase 2 clinical trials are attempting to define the toxicity and efficacy of this approach to the treatment of cancer. The possible "reversibility" of the neoplastic state provides new possibili- ties for therapies as alternatives to treatment based on use of cytotoxic agents. Substantial Progress Is Being Made in Our Understanding of the Nature of Cancer It is becoming increasingly possible to identify alterations in gene and gene products in transformed cells and factors that interact with transformed cells. These factors distinguish them from normal cells and cause abnormal cell prolif- eration and differentiation. New approaches to prevention, earlier diagnosis, and better treatment will contribute to progressive improvement in our ability to control cancer as a major source of human suffering and death. ADVANCES IN UNDERSTANDING HUMAN GENETIC DISORDERS Many Genetic Disorders Are Now Being Successfully Investigated Genetic mapping the localization of a gene or trait to a specific area on a chromosome-has created a great stir in the popular media and the biomedical literature alike during the 1980s. Diseases of interest include Huntington's disease, cystic fibrosis, polycystic kidney disease, neurofibromatosis, myotonic dystrophy, familial Alzheimer's disease, and Duchenne muscular dystrophy. The excitement is entirely justified. In all of these disorders, the nature of the basic biochemical defect is (or was at the time of mapping) unknown; consequently, no reliable diagnostic method could be designed for prenatal and presymptomatic diagnosis, and no definitive method for carrier detection was available. Further- more, aside from the current impossibility of gene therapy, treatment strategies directing the fundamental pathogenic processes could not be devised. Knowledge of the location of the chromosomal change responsible for these and many other disorders opens possibilities for diagnosis by genetic linkage analysis or by direct identification of the alteration in the DNA. It also opens prospects of approaching the pathophysiological mechanisms of these disorders through the back door, as it were a strategy that has come to be termed reverse genetics. It is hoped that elucidation of these mechanisms will suggest ways of interrupting the pathogen

MEDICINE, THE BIOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CUL== 345 etic chain that connects gene to disease phenotype or of compensating for genetic deficiencies. Elucidation of the Primary Genetic Defect Two General Methods for Elucidating a Prepay Genetic Defect Are the Candidate Gene Approach and Reverse Genetics What is the protein gene product that is absent or altered in disorder x? The candidate gene approach, based on the gene map, often provides a tentative answer (or excludes a possibility). If the map location is known, the gene defect (and thus the abnormal gene product) may be identified through analysis of the segment of chromosome where the mutation lies, recognizing the mutant gene by a difference from the normal, and then characterizing the normal gene in deter- mining the nature and physiological function of its protein product. A second approach involves reverse genetics. Usually, in unraveling the pathophysiology and primary genetic defect in disorders inherited through classi- cal Mendelian genetics, medical geneticists start with a phenotype that shows a "simple" pattern of inheritance; they identify a biochemical abnormality such as an abnormal metabolite in serum and urine; then they demonstrate the deficiency of an enzyme that could plausibly account for the abnormal metabolize; they clone the normal gene for the enzyme in question; and perhaps ultimately they deter- mine the change in the gene in patients with the disorder. In many disorders, however, no clue exists to the nature of the defective protein gene product. In these conditions, going directly to the gene (located by mapping studies), determining the structure and function of its normal counter- part, and then working back to the phenotype-reverse genetics is the approach that has been taken. The approximate location of the gene is determined by genetic mapping methods. When markers are at hand when defined DNA segments have been cloned they can be used for locating the disease mutation. In addition to cloned markers, the chromosomal location of genes can be facili- tated by cloning large segments of DNA through the use of the newly developed yeast artificial chromosome cloning vectors, followed by new electrophoresis techniques Hat can separate large DNA fragments. These techniques allow heritability studies of genetic disorders to be readily linked to the techniques of molecular biology. As an alternative, exploitation of deletions and chromosomal rearrangements can narrow the location of the segment of DNA containing the mutation. With high-resolution cytogenetics (examination of banded prometaphase chromosomes) it has been possible, in X-linked disorders in particular, to find microscopically visible deletions in patients with Mendelian disorders such as Duchenne muscular dystrophy or chronic granulomatous disease. Cloning the normal X-chromosome

346 OPPORTUNITIES IN BIOLOGY segment that is absent in the affected male is accomplished by subtractive hybridi- zation: DNA from cells of the male with the deletion is repeatedly hybridized with DNA from cells of a normal male; the DNA that does not hybridize is that which was deleted in the affected male. This strategy was used in cloning the genes for Duchenne muscular dystrophy and chronic granulonatous disease and in identify- ing the gene for susceptibility to retinoblastoma However, even after the relevant segment has been cloned (in multiple pieces), identification of the specific gene may still be difficult. Granulonatous disease provides the first example of full utilization of reverse genetics. Demonstrating that the normal gene cloned by these methods is indeed the one that is mutant in the given disorder can be accomplished through several approaches. Finding the mutation in a protein gene that has been linked to the disease by additional lines of evidence is a good indication that the mutation is responsible for the disease. The demonstration of different types of mutations in the same gene in different patients can also help. Tissue distribution of the corresponding messenger RNA (if it matches the distribution of the clinical phenotype) indicates that the gene is relevant, and evolutionary conservation of the gene in question indicates that it is functionally significant. Correcting the cellular defect with the cloned gene is convincing evidence that the gene is the relevant one. Gene Diagnosis Gene Diagnosis Can Take an Indirect Approach by Linkage or a Direct Approach by Demonstrating the Lesions in DNA Diagnosis by Linkage Analysis. Information on the chromosomal location of a disease-producing mutation can provide a method for diagnosing that disorder by the linkage principle. The approach can be used to diagnose prenatally or before symptoms have appeared, or to detect carriers. The method has shortcom- ings: To give reliable results, multiple family members must be studied, a linked marker (or preferably more than one) must be segregating into different gametes in the family, and the markers must be closely linked. Direct Gene Diagnosis. Any genetic disorder exhibits changes in the physi- cal structure of DNA that can be used for diagnosis. By a process that might be called diagnostic biopsy of the human genome, DNA can now be prepared from the circulating white cells in a sample of peripheral blood and appropriately studied for the presence of the lesion characteristic of one or another genetic disorder. A probe of the normal gene is required for direct DNA diagnosis. Genetic modifications in the altered gene can be detected by a variety of labora- tory procedures. For example, a specific synthetic oligonucleotide probe can be used when a specific nucleotide substitution is known in a given disorder, such as sickle cell anemia.

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, AND ^IMAL AGRICULTURE 347 ~ ~ ~::~ ~GENE:r:ICS~:~OF~::NEU~3~P~SYCHIATR~IC DISORDERS ;: act: ~:~ ~:~ An~impo~hant~applicat~ton~recebt~advances~i~n~mo~t~ecu~lar~g~e~n~es~is~in~ ~u~nderstanding~the~causQs~ol~ne~uropsychlainGdiso~ders~such~as~fam~i~JialAlz-~ ~: ~heimers~rr~entia~or~biDolar~manic ~er~These~d~isoide~rs~ ~ ~ are fan~ilrat but their exact Elude; of ~in~her~tance~a~n~d~4r~ly~i~ng pratein~d~efect ~ ~ ~ ~ ::~ i: :~: :: it: ~ ::~ i:::: :: ~ : :~::: ~ :: ~ ~ :~ :: ~ ~ : ~ : ~ ::: i: : :: ~ : i: :~ ~a~re~u~nkno~n.~Nev~ertheless ~by~u:~si~g~hypervari~abl~e~13NA~ma~rs~ of:~the~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ · :~ ~ ~ ~ . :, ~ ~ ~ . ~ ~ ~ ~ :::: human :~g~e~no:me~:'t~ has:: Demean Pass: ~ e My In ~age:~:~an~a ysis~to:~m:ap~t ~e~suses~p-:~ : ::: : : :: : : : ::~ i: ::: i: : :: : i: ~ ~ :~::: :: ~ : ::~ : :~: ~ ~ :: : : :~ i: ~ ~: : i: i::: ::~: :: ~ : ~ : : : :~ ~ : : : : ~ ::::::: :~: : ::~ : :: i: ::: ::: i:: : ax: I::: : ~ i: :: : : i: ~ it::: :: :~:~::~:: ~ ~ :~ i: i: : :::: : libility~gene~fo~r~Alitl~eimtQ~r s~disorder~an~d~for~ ~man~i~d~epress~nfe~d~isorde~r. :~l~n~se~of m~d4Drd*:er~iesli - Was :~ ~:~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ :: ~ ~:~.~:~:~:~ ~ :~ ~:~ ~:~ ~ 3: G ~ hi: - ich~'s~hie~r~e~sy~h~esis~of~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I~ ~ ~ ~ ~ ~:~:~th~e~n~e:urotran~s~mittQrs~dopami:ne:~an~d~no!~repin~e~ph:rtn~ thems:et'es~lmpl~cated~:~:~ :~in~m~s~of~affective~disord:er.~Ho~weve~r~recent~experim~e:nts sugg~t~: :~ ~ : :: i::: :: :~ :~::~: ~ ~ ~ ~ :: ~ : ~ aim: ~ ~ ~ ~ :: i: ~ :: ~ ~ ~ ::: ail: ~ ~ ~ ~ ::: ~ ~ ~' ~: ~ :::~: ~ ~ ~ ~ :~ ~ i: ~ if: ~ :~ :~ ::::that~::th~Q:~:~:~;bilitv~:~l£,cu:s~id:~:~bi:Dolar ~d:iso~rd~e:r~ is~:~not~:~the~:h`::rosin~Q~h~d~r~lase: ~:~ ::: i: ~ ~ ~ ~ You so ~ sear. ~ ~ ~ I: ::: ~ ~ aft:: :: :~:~::Similarly~the~:~g~e~nel~:~idefect~cau:s~i~ng~:~fam~ilial :~Alzhai:m~s~:d~is~eas~e~:~::~was~:~:~:~ ~ ~ ~ ~ ~ ~ . ~ . . ~ , ~ ~ ~ ~ ~ . ~ ~located~n:~e~ar~the :: ocus Tort eel gamy oar: ~eta~protel~n:~p~cursor~g:ene: glow bloc '~ If:: ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~: . ~ ~ ~ ~: ~ ~ :: ~ ~ ~ ~: ~ ~: ~ ~ ~ ~ ~ ~ ~: ~I:~:~:~I~h:as:~bee~n~::~:£~n~ed~:~a~::::~m::appe~:::~::~::1nitially~it~:~was~:s:ugge~§tdd~thdt~dupliGatio:n of::: ~ ~ ~ ~ ~ ~: ~ ~ ~ ~ ~ ~ ~: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~: ~ ~ ~ ~: ~ ~ ~ ~ ~ ~ ~:~th~Q~amylaid~:~prote:in~:loc:us~:~:~::~may~ca~u~sQ ~fam~ilial~:and~spprad::ic:~cases~of:~Al:~-~:~::~:~ :~::~h~eim~e~es~:~:diseass.~:~However~,~:~recent~exper~imentsi~haAre~fou~nd~no:~evidence~of~:~:~:~:~ ~ ~ ~ . ~ ~ ~ . ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~d:~upllcat~on of: this: locus:~ln~:elth~er ~:f~amilial:~:~or~:::~sporad:l£~:~ cases Of ~AIzhel~m~e~(s: ~ ~:~ ~ ~ ~ ~ ~ ~ ~ :: ~ ~ ~ ~ ~ ~ ~ ~:~ ~:~'se~ase.~ ~:~rthermo:re~ ~:genetic~linkage~::st:udies~ Have ~now~:~excluded~:~th:e:~::~ ~:~:~:amyloid~l~prote:in :~g:ene~:~:locus~:~as~:~:th:e~:~site I ~for:~l~:~fam~'lialL:~ Alzhei~mer so m:~atio:n~ :: ~:~:~:~ the his a ts~dou~ont~be~:orig~i I~ncl:u~s: .~:~:~:~:~::~:~ ::::: ~:~::~:~:~:~ ~::~:~:~ :: :::::: ::: ::::::::: :::~::::::~:::: :: ::::: :::::::::: :::::: :: :::::::: ::: :~ i::::: :~: ::: :: ::::::::::: : i:::: ::~:: ::::::::: i:::::::: : :~: :: : ~:~:::~:: ::: : ~:~:::~:: ~::~::::: :::: :::: :::: ~ :: ::.:: ::::::: :: :::~::~:: :: ~::~::~;::~:~ hi: These ~Qxamples~::show~:the~:~:i~nteractive:~u~se:~ of ~ca:ndidate:~g:en~e:::~and~re-~:~:~: ~ :: . , . ~ ~ ~ ~ · ~ if:::::: :~v~erse:~genetic:; strategies. :: ~appin:g a susceptive ity~ locus Toga particular ~::~ :~:~mg~ion~suggests:~st:ud~ies:~6f ~n~ea:rby~:candidate~ge:n::es~.:~:~St:ud~ies~ of~these~g:enes:~:~ i:: ;~:~he~lp::~to :~narrow~:~:~the~possibl~e~:sites~i~:~for :~t~h~e:~susceptibil:ity~locu:s.~:~:ln~:~th~e:~ case~of~:~::::~: :: ~ ::: : ::: :: : ::::: ::::::::: :::: ~ ~ ~ : : ~ ~ ~ ~ ne:u:ropsych~iatric~d'lso~rders,~:~the~ genetric~rTegulatIon::~:~of~mo:re~:than::~t~:~OO~ne~u~ro-:~ If: :~ ~:~ ::::::: ::: ~:~transmitte~:rs~p~rovi:de~s:~specif~ic:~:~candid~at:es~fQr~inv:estigat:ion~:because~:~these~:~ ::::: ~ :: : : ~ : ~ ~ ~ ::: : :: : ::: ~ :: : : : ::: :: ~ :: ::: ::::: :: : ::: : : :~: : : ::::: :: :::: :: :~ n:Qu:rotrans~mitters~ ~a:r:e~:~:DeDtid~as ~:that:~::= n tire I ::th~e ~::che~mt:ca1:: c om~m`~:n~icat lo n ::: ~ wllnln:lneoral:n.~:: Gene Therapy Replacement or Repair of Genes Is No Longer Science Fiction Introduction of cloned genes into cultured cells (transfection) by methods of calcium or electrical enhancement or into the developing embryo by microinjec- tion (as in ~ansgenic mice) indicates that somatic cell gene therapy of some

348 OPP0~171NITIES IN BIOLOGY genetic disorders may not be far off. For example, it may be possible in vitro to introduce genes into stem cells of a patient's bone marrow and reimplant that marrow. All of these techniques, however, are still in their infancy, requiring much more research to establish effective and safe therapeutic procedures. Scien- tists are exploring the possibility of gene therapy studies in patients suffering from a rare genetic disease resulting in severe combined immune deficiency caused by defective production of the enzyme adenosine deaminase. However, major ob- stacles still exist, such as low levels of expression of the gene and possible virus contamination during treatment. In addition, the chromosomal location of the integrated donor genes is rele- vant to gene therapy. The experience gained with transgenic mice indicates that integration of the foreign DNA into certain locations in the genome may cause the new gene to fail to be expressed, or worse yet, if it becomes integrated in the wrong place it may cause abnormality. More complete maps of the human genome, now being prepared, will enhance our ability to deal with such problems. In some disorders in which an abnormal protein leads to abnormality for example, sickle cell anemia and amyloid polyneuropathy merely introducing a normal gene may not solve the problem. Ablation of the abnormal gene may be necessary. Research on site-specif~c (or directed) mutagenesis possibly appli- cable in this connection is progressing (see Chapter 4 for a discussion on homolo- gous recombination). NEW APPROACHES FOR CONTROL OF MICROBIAL INFECTION One of the Great Achievements in American Medicine in the Twentieth Century Is the Reduction in the Incidence of Infectious Disease Many of the old epidemic diseases have been controlled ore like smallpox, eliminated. Polio and measles are clearly on the road to elimination. The course of this revolution in infectious disease began with dramatic improvements in sanitation, housing, and nutrition. More recently, antibiotics and vaccines have aided in control. Antibiotics have been particularly successful in treating bacte- rial diseases, although they have been less effective in controlling diseases caused by parasites, viruses, and fungi. Since viruses use many of the host cell's synthetic pathways for replication, it is often difficult to specifically inhibit viral replication without affecting the host. However, significant breakthroughs have been made in antiviral compound research. For example, the antiviral drug acyclovir is converted into a nucleotide by enzymatic phosphorylation only in cells infected with herpes virus. After acyclovir is converted into the nucleotide, it can then exert its antiviral properties by selectively incorporating into replicating viral DNA, which inhibits viral

MEDICINE, THE BlOCHEMIC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 349 multiplication. Since phosphorylation is a prerequisite to DNA synthesis, acy- clovir is active only in cells infected with herpes virus. This limits its potentially toxic effects on uninfected cells. Vaccines The Development of an HIV Vaccine to Prevent Acquired Immune Deficiency Syndrome Is Progressing Slowly A new disease of humans, acquired immune deficiency syndrome (AIDS) was recognized in 1981. Since that time, more than 30,000 cases have been reported in the United States; it has been estimated that more than 1.5 million people are infected in this country alone. In certain Is of the world, especially in regions of Africa, AIDS is a major and growing problem. Worldwide, 10 million or more persons may be infected. Projections for the future growth of the epidemic indicate continued increases and, despite the uncertainties of these projections, disease and death resulting from AIDS will continue to increase for at least the next decade. Our understanding of AIDS and its viral cause, HIV, have progressed re- markably because of basic research in molecular and cellular biology, carried out in thousands of laboratories over the past decades. HIV infection has a wide variety of effects on the immune system, resulting in opportunistic infections (infections caused by microbes that rarely cause disease in the healthy host) and cancer (such as Kaposi sarcoma and non-Hodgkins lymphomas). In addition, it has deleterious effects on the nervous system. Although most of the early cases have appeared in homosexual males and intravenous drug abusers, heterosexual transmission has been increasing and is the most common form of spread in Africa. HIV was isolated in the early 1980s. It is a member of a group of retroviruses that causes many naturally occurring, progressive nonmalignant disorders in animals. Much has been learned about the structure of HIV. It has several genes in addition to the usual retrovirus virion protein genes. The activity of these additional genes probably causes HIV to remain latent in infected cells, and virus infection can thus persist even in the presence of neutralizing antibody in the victim's serum. The enormous effort currently under way to develop an effective vaccine or drug to control AIDS has thus far produced only drugs with limited effects. Many vaccine candidates are being evaluated, but there is little hope for an effective vaccine in the near future. A major challenge of modern biology is the better understanding of the immunopathogenicity and eventual control of HIV infection. In this regard, an animal model for HIV infection is of utmost importance.

350 Conventional Vaccines Have Been Among the Most Successful of All Biological Products OPPORTUNITIES IN BIOLOGY Smallpox, for example, has been eradicated. Vaccines against poliomyelitis, measles, rubella, and mumps offer the best means of preventing these diseases. For other viral, bacterial, and parasitic diseases, however, effective vaccines still have not been developed. Biotechnology has opened two major new avenues for vaccine development. First, biotechnology provides tools for understanding virulence and the nature of the microbial immunogens. Second, biotechnology offers new strategies for . . . engineering vaccines. Inducing an effective immune response requires precise knowledge of the antigens that stimulate and are recognized by humoral and cellular immunity. A number of new strategies exist for engineering vaccines. When a protein is identi- fied as an important antigen, the gene encoding the immunogenic protein can be inserted into a suitable host (bacteria, yeast, or plant), which can be grown to yield large amounts of the immunogenic protein. The protein engineered and prepared in this manner may be used directly as a subunit vaccine. An alternative to proteins so prepared is the chemical synthesis of immunogenic regions of the protein (synthetic peptide vaccine) based on the DNA sequence of the gene that encodes the immunogen. In addition to using isolated genes or portions of genes to make immunogenic proteins, isolated genes can be inserted into vaccinia or other DNA viruses. Inoculating the host with such hybrid viruses, can stimulate immunity to the inserted "foreign" proteins in addition to stimulating immunity to its own pro- teins. For example, the U.S. Food and Drug Administration has approved clinical trials of a HIV-vaccinia vaccine. Hybrid viruses can stimulate immunity against a number of foreign proteins simultaneously. Manipulation of live microorganisms may also reduce virulence while maintaining their capacity to induce immunity. For example, a gene for a toxin can be modified or deleted, causing it to become less virulent while retaining its colonizing abilities. Such a mutant of the cholera vibrio has been shown to immunize human volunteers effectively after oral inoculation with the live, attentuated (less virulent) mutant strain. Selection of viral mutants can lead to nonvirulent mutants that are immuno- genic. It is feasible to reduce the virulence of live microorganisms and acquire live, attentuated vaccine candidates. A special type of attentuated vaccine strain may be generated for segmented genome viruses since new combinations of genes may result in attentuated viruses by reassortment of genome segments. Finally, it has been possible to mimic the antigenic structure of certain parasites and viruses with anti-idiotypic antibodies. Such "anti-antibodies" have a configuration that resembles the immunogenic protein. Anti-idiotypes prime or actually stimulate an effective immune response. A number of anti-idiolype vaccines stimulate immunity to pathogens such as ~ypanosomes, rabies virus, and . - pO 10 VlrUS.

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, AND ANIMAL AGRICULTURE 351 ::: i:::: ~:~ ~IN~S~FRTION~:OF~:AN~: ANTIGEN:~G:E~hIE~ INU)~:V:lRl6JS ::~VECU):RS~ ~ ~ : :~ ~ Vaccin ia Virus, as well ~ as ~ adenoviru;ses and ~ heaps viruses, have ~ Boeing used Basil v~tors~for~ carrying ~ other genes. ~ The Rationale ~ is ~that;~part~ of These Vera I o~enomes~can~be~:re~D~laced~without~siq~nifi£a~ntly~alIering~t~he~capaciti~of~ ~ The virus ~ to grow In Ace cultures ~: ~u~£n~ ~ grown ~resurts~'n~ a eve recom~'na~n~ ~ ~ ~ : ~ ~ ~ ~ ~ ~ . ~ ~ ~ . . ~ . ~ ~ . . ~ ~ ~ _ :~ ~ virus.;: ~ ~ ~ Inch: the case~:~:vaccin;ia:~;:;~vi~rus,~: ~ t~h:sse~; remnant: :~v:'ruses Are fess ~ :; Virulent In Experimental ~ ~ animals Than ~ ~the~va~'n~a ~ v~rus~ ;~ parent, Diana ~ to E ~recom~binants can pote;ntially indu~¢e~th;~eel;lu~lar~a~nd~ Femoral m~mu;nib.~An~ : ~ ::: :: : : : ::::~: ::::: : : : ~ :: ::::::: :::: ::::: :~ ~ increasing Number of viral. bacterial.~;~and~parasitic arittg~ens (including Pro- ~ ~ teins~from~ ;~hepatitis~B,; influenza, rabies, Epstein-Barr, and human ~i~m~mu-~ ~ :: :: :: :: :: :: :: : :: :: : :~:: :::: :: : ::: ~ ~ :~: ::: : :: i:: ~ : : ~ : a: :~: : ::::: : nodbficiency Viruses And from Plasmod~£~m~ ~3a~u71 ~h~ave~been;~insett~ into vamin~ia virus In this manner. ~ In many ;instances,~animals~inocu~lated~ with These ~ ~re¢ombi~nant~ Viruses ~ rave demon~strat"~a~ ~protect~ve;~;resp~n~se. ~ ~ ~ ~ One striking Advantage of this approach Lathe possibili1:y~of~;~elicitin~broad ;;; ~ ~ · ~ ; ~ immunity ;w~4h~a~sing~le~ dose. However, ~;t~he~ approach Still needs to be ;~ ~;~ carefully eval~uated~in~te~rms~of~hum~an Safety,; ~since~the~toxicity~and ~possible~ : ~ ~u~n:anticioated~ Q# - So remain u~nknow;n~.~ ~: Antiviral Agents Most Approaches to the Treatment of Viral Infections Relate to the Fact that Viruses Are Intracellular Pathogens Viruses use the host's normal cell machinery; therefore, it is often difficult to develop effective and nontoxic drugs that are virus specific. However, recent advances in molecular biology, immunology, crystallography, and drug design have initiated a new era of treatment of viral infections. The theoretical possibil- ity of rational drug design based on an understanding of specific steps of viral replication is becoming a reality. In discussing a rational approach to antiviral chemotherapies, it is useful to divide the viral cycle into a series of steps: early stages (including adsorption, penetration, and uncoating); transcription and replication; and late stages (includ- ing assembly, maturation, and release). The development of specific drugs that block each of these stages has been facilitated by many studies that have identi- fied details of the strategies of different viral groups. Selected drugs that affect early events include heparin and other anionic polyelectrolytes, which prevent attachment; oligopeptides, which by mimicking part of the fusion polypeptide of paramyxoviruses inhibit fusion; and amantadine and rimatadine, which inhibit early steps in the growth of type A influenza. Drugs that affect the synthesis of

352 OPPORTUNITIES IN BIOLOGY viral nucleic acids include purine and pyrunidine, nucleoside analogs that are active against herpes and other viruses (such as adenine arabinoside, acyclovir, and ribavirin); single-stranded polyribonucleotides, which are thought to inhibit retrov~ruses by blocking the reverse transcriptase reaction with single-stranded polyribonucleotides; thiosemicarbazones, agents active against pax viruses; and phosphonoacetate and phosphonoformate, which are active against herpes. Drugs active against late events include rifampicin (blocks pox virus assembly) and 2- deoxy-D-glucose, which blocks functions attributed to virus-specif~c glycopro- teins, such as cell fusion, hemaglutination, and hemadsorption, by interfering with the glycosylation of virus-specif~c polypeptides. ADVANCES IN THE BIOCHEMICAL PROCESS INDUSTRY Biological Discovery Is the Driving Force in the Biochemical Process Industry The biochemical process industry began thousands of years ago with the production of wine, beer, and bread. By the mid-nineteenth century, the technol- ogy underlying this industry began to advance in response to a better understand- ing of microbiology and biochemistry. The discovery that actively growing yeast cells were responsible for the active component in baking and also for the production of alcohol in brewing led to the observation that control of oxygen and sugar supply had greatly improved both processes. These discoveries in the biological sciences led to major process improvements in the evolving biochemi- cal process industry. It was not until the mid-1940s, however, that a true collaboration between the biological scientist and the process engineer began to develop. It has recently become apparent that biological discovery provides the driving force behind process development: The new discoveries in biology during the past decade have become the enabling technologies that underlie a modem and rapidly growing biochemical process industry. Recent important discoveries include techniques of genetic engineering, cell fusion, nucleic acid and protein sequencing and synthesis, techniques for visualiz- ing protein structure, and an understanding of the molecular basis for enzyme catalysis. With these discoveries, it has become possible to isolate and produce a new set of therapeutic proteins, explore new regimes of biocatalysis, improve diagnostic procedures and plant species, develop better methods for plant protec- tion, develop new biomaterials, and begin to address the effective use of biologi- cal systems for hazardous waste disposal and of natural resources. The rapid advancement in biological discovery has created new opportunities and, as a consequence, new problems in industrial biotechnology. The opportunities relate to the possibilities for new products and services in industries devoted to human health care, agriculture, chemicals, and environmental services. The problems relate to the need to translate new discovery in science to technological practice. Problems, such as efficient recovery of therapeutic proteins of high purity or

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CUL=~ 353 large-scale cultivation of animal cells, were not problems a few years ago; it is only the recent discovery of products requiring these technologies that have forced us to develop advanced manufacturing processes. Goals in Industrial Biotechnology The Overall Coal in Industrial Biotechnology Is to Translate New Scientific Discoveries into Processes That Provide Products and Services Many opportunities for industrial biotechnology are coming from progress in biology. However, for discoveries to be translated into products the techniques for developing biochemical processes must be available and manpower must be adequate. To fully appreciate and understand the problems and opportunities associated with industrial biotechnology, it is essential to examine the barriers inherent in product development. Reduction or removal of these barriers is essential to maintaining and even accelerating successful translation of science into technol ogy. Difficulties with the Identification, Isolation, and Characterization of New Products Are the Primary Impediments to Product Development The primary initial milestones in product development are discovery of new and useful functions followed by the isolation, purification, and characterization of the chemical entity responsible for this function. In many cases, the respon- sible chemical entity is masked by the complexity of biochemical systems. Many molecules of interest have a regulatory function in stimulating or inhibiting the specific actions of other cells. As a consequence, these molecules are present in very low concentrations in the cell. This makes the identification of their function and the determination of their structure exceedingly difficult. The hundreds of man-years required to isolate and characterize the interferons is a good example of this problem. Even though we have isolated and purified the interferons and characterized their molecular structure, we do not fully understand their mecha- nism or implications of their function. Realizing that a product is not truly a product until it can be manufactured in a form suitable for use is an important first principle. The discovery of an exciting new chemical entity with important functions is only the first step in product and process development. For example, recombinant DNA techniques permit us to make a new class of therapeutic drugs composed of pure, highly active proteins. Because these proteins are produced in complex mixtures of similar molecules and require an extremely high degree of purification, developing suitable manu- facturing processes has been a major challenge.

354 OPPORTUN171ES IN BIOLOGY Acquisition and Interpretation of Analytical Information Is a Major Problem A major problem that has plagued efficient process development has been the scarcity of analytical techniques for measuring the product of interest. The biological activity of many new products is difficult to measure, and their struc- ture is often poorly defined; thus, routine measurement of these molecules is difficult. Assays for their biological activities are often cumbersome and impre- cise. A powerful analytical technique that has recently evolved is He use of monoclonal antibodies as the basis for molecular recognition and analysis. New sensor technology is need, including biosensors, instrumentation, and software, that will provide the power needed to support process development more effec- tively. Process Synthesis, or Manufacturing, Requires Multiple Steps from Compound Production to Purification Manufacturing technology for the production of biochemical products re- quires a multiplicity of unit operations. A typical process for manufacturing a highly pure therapeutic protein is diagnosed in Figure 1~3. The primary unit operation in which the raw materials or nutrient medium is converted to a desired product is carried out in a bioreactor. This reactor may be a fermenter for growing microorganisms or a device for propagation of animal cells; in either case, the cells may have been genetically engineered to overproduce the desired producL Once formed, the product must be separated, concentrated, and purified to its active form and formulated to stabilize, and perhaps enhance, its activity for eventual use as a drug. A process for manufacturing therapeutic proteins derived from recombinant microorganisms is expensive and complex, not only because of the multiple unit operations that must be carried out to recover highly pure materials, but also because the final product must be made in its native form that is, with a single three-dimensional structure. Thus, in the case of a protein that has a large number of possible folding configurations, only one is permitted in the final product. This will ensure its efficient use as a therapeutic agent and help prevent the body's immune response from rejecting the molecule as a foreign protein. Both biologi- cal and structural integrity must be retained. The ability to achieve this integrity depends on developing a better understanding of how proteins behave during their production and purification and as well as on developing high-resolution analyti- cal techniques that are able to measure structural properties of biological mole- cules. Other problems that hinder successful process development include the pres- ence of proteases that modify the product, insufficient reduction in nucleic acid content, and the presence of isomeric forms of a protein product. Glycosylation of proteins is a useful and important posttranslational modification implemented

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, AND ANIMAL AGRICULTURE 355 Medium preparation Bioreactor (Fermentation or cell culture) Cell harvesting Cell disruption Separation of cell debris Concentration of product Multistep purification (Chromatography, extraction, and precipitation Product concentration (Interaction with quality control) Formulation FIGURE 1~3 Bioprocessing. [Charles Cooney, Massachusetts ~sutute of Technology] by cells, the function of which we need to better understand; we also need to improve the techniques for characterizing, modifying, and recovering properly glycosylated proteins. Additionally, a substantial number of proteins of interest form insoluble aggregates or "inclusion bodies" inside genetically engineered cells, and we need to better understand procedures for recovering these proteins. Such recovery of many proteins could be enhanced by high levels of secretion; however, the molecular basis of secretion is unlmown in many microorganisms.

356 OPPO~UNITIES IN BIOLOGY Further advances in understanding the biology and biochemistry of protein secre- tion will have a major impact on process development. In recent years, many of the improvements in recovery of new biochemical products have come about through scale-up of known techniques and the use of a wider variety of materials that enhance purification. Improved scale-up has occurred as a consequence of automation of many procedures, especially chroma- tography, the availability of new chromatographic resins and other absorbents, the wide use of membrane processes for concentration and desalting, and improve- ments in mechanical devices such as centrifuges, cell homogenizers, and mem- brane filters. Improvements are still needed. New techniques, such as affinity chromatography and electrokinetic separations, are still not applicable to large- scale purification because of scale-up cost. There thus remains a need for innovation in both the development and implementation of new recovery proce- dures, the development of improved materials to enhance unit operations such as extraction and chromatography, and the development of techniques for achieving process integration. As part of this process, computer-aided design software and flow-sheet simulation techniques are likely to become major tools in the hands of the process biochemist and the engineer. In response to these needs, substantial research has been done to delineate the necessary components of the complex and ill-de6'ned nutrient media that are used for the growth of mammalian cells. From this effort has developed a set of better defined and less expensive cell-culture media that avoid the use of animal sera. These defined low-protein media not only enable efficient and controlled growth of mammalian cells, but also facilitate the recovery of products excreted by these cells. Because these products are often secreted into the medium in very low concentrations (<10 milligrams per liter), the use of media containing high con- centrations of protein makes subsequent downstream processing for product re- covery difficult. Thus, the use of well-defined, low-protein media.areatly en- hances efficient product recovery. c ~_ ~ Modern Biology Coupled to the Biochemical Processing Industry Has Enormous Potential Human Growth Hormone. Human growth hormone is a protein produced by the pituitary gland that stimulates the liver to produce somatomedins, which stimulate bones to grow. Children produce large quantities of growth hormone, whereas adults normally produce very little. Pituitary dwarfs represent a popula- tion with a defect in the production of the pituitary growth hormone. Children judged deficient in growth hormone used to be injected with the hormone ob- tained directly from the pituitary glands of cadavers, which they received three times per week. The National Hormone and Pituitary Program processed as many as 50,000 pituitaries per year, supplying the growth hormone derived from them

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CUL=M 357 to treat 3,500 individuals. Since the hormone was difficult to obtain, it was always in short supply; the criteria for deciding who to treat were stringent. In 1985 three people who had been treated with the cadaver-derived human growth hormone in the 1960s and 1970s died of Creutzfeldt-Jakob disease, a rare disease that results in dementia and death. The proposed cause of the disease was infectious virus derived from the pituitaries of victims of Creutzfeldt-Jakob dis- ease. At the time of these deaths, biotechnology companies had been working on the application of recombinant DNA technology to produce synthetic human growth hormone. In this effort, the gene coding for growth hormone was isolated from pituitar- ies, and genetic expression vectors were developed to produce the hormone by fermentation. This was followed by several years of careful research on the isolation and purification of this rare protein. The successful completion of this task in the fall of 1985 resulted in Food and Drug Administration approval for a genetically engineered version of human growth hormone. Since the growth hormone is manufactured in a bacterium, no human pituitaries are needed and the threat of Creutzfeld-Jakob infections has been eliminated completely. In addition to solving a major health and social problem, the development of a commercial process for the manufacture of recombinant human growth hormone has made major contributions to the identification of fundamental problems in protein structure and function. Challenging problems such as determining the factors that influence proper folding of proteins derived from the linear assembly of amino acids, determining the crystalline structure of growth hormone, and understanding its mechanism for effecting the observed physiological responses are receiving widespread attention. The study of growth hormone will lead to a greater understanding of the biology of other regulatory and mediating proteins, as well as to the design of smaller and more effective molecules. We can antici- pate improved treatments for hormone deficiencies, wound healing, osteoporosis, bone fractures, and obesity. Tissue Plasrrunogen Activator. Both strokes and heart attacks can occur when a blood clot lodges in a blood vessel, blocking the flow of bloom Tissue plasminogen activator is a naturally occurring enzyme that binds to fibrin clots and is activated to convert plasminogen to plasmin, the latter being capable of dissolving the blood clot. The primary initial applications for this protein are in the treatment of heart attacks, with minor applications for other conditions such as unstable angina, pulmonary embolism, and arterio-venous occlusions. More than a score of companies are currently involved in the research and development of tissue plasminogen activator. The story differs considerably from that for human growth hormone produc- tion. Tissue plasminogen activator is a glycosylated protein; that is, it contains oligosaccharides (sugar complexes) attached to the backbone of the protein mole- cule. Since bacteria cannot glycosylate proteins, the manufacture of tissue plas

358 OPPORTUNITIES IN BIOLOGY minogen activator, or its variants, must be in higher organisms or cells representa- tive of those organisms. At present, major production is achieved through mammalian cell culture technology. This approach has initiated a new cycle of chemical and biological research to develop analytical methods for characterizing and analyzing the sugar attachments to the protein as well as basic biological studies to develop an understanding of the role of these oligosaccharides in the structure and function of tissue plasminogen activator. Other thrombolytic agents such as urokinase and streptokinase are also being marketed and intensively investigated. Second-generation clot-dissolving mole- cules are being designed. It is hoped that new products with enhanced specific activities, greater specificity, and fewer side effects (such as nonspecific bleed- ing) will be generated. The challenge to increase the survival rates of the 900,000 heart attack victims in the United Stems who reach hospitals each year is great, and the tools available today provide scientists with the means for solving these problems. Industrial Growth in Biotechnology Is Predicted, but International Competition Will Be Great Over the past 5 years, biotechnology, defined as the application of biological organisms, systems, and processes to the manufacturing and service industries, has developed dramatically, and commercial products have been made. Although the number of products that have entered the market at this time is limited (for example, insulin, human growth hormone, interferons, tissue plasminogen activa- tor, and hepatitis B vaccine), the momentum to introduce more products contin- ues. Within the next decade we should see the introduction of many new products for medicine, agriculture, and environmental management. Economic considerations and analyses have replaced some of the euphoric hopes for products derived from biotechnology. This change has resulted in a significant focus toward high-value specialty products and away from large-scale production of general-use products. Combinatorial evaluations of gene engineer- ing, mutagenesis, fermentation optimization, and downstream processing have intensified as economic dictates have become more obvious. Regulatory issues remain a major question worldwide, particularly with regard to the deliberate environmental release of plants and microbes developed through the use of recombinant DNA technology. As a result, there now exists a regulatory patchwork with little uniformity from country to country. In the United States, regulatory issues are currently under study, and debate continues with regard to what risk-benefit information will be required and who will regulate what. Worldwide, there is little doubt that biotechnology is seeing major develop- ments in Europe, Japan, and the United States. Other complementary and syner- gistic technologies that will influence the economic trends and impact of biotech

MEDICINE, THE BIOCHEMICAL PROCESS INDUSTRY, AND ANIMAL AGRICULTURE 359 nology must be considered. Examples are more traditional plant breeding, so- matic cell culture techniques, and computer science. The Japanese have organ- ized to become a major force in biotechnology. This movement has involved government, industry, and universities and has focused on key target areas such as plant cell culture, hybridomas, bioreactors, and large-scale mammalian culture. New opportunities to service the growing activities in biotechnology have led to new businesses such as those that provide computer support for molecular biology, build equipment for nucleic acid and pervade synthesis and analysis, and produce mammalian cell cultures. Various government initiatives have led ~ broader joint ventures and venture capital operations on an international scale, which eventually should have a significant impact on intemabonal trade. ADVANCES IN ANIMAL AGRICULTURE Challenges Lie Ahead to Increase the Efficiency and Productivity of Domestic Animals Under Widely Disparate Environmental, Economic, awl Political Constraints Of the thousands of animal species that might have been domesticated, human beings have utilized only a handful. These have been selected over the last 10 millennia to occupy increasingly narrow ecological niches. These niches are highly structured management systems designed to increase the efficiency of our domestic species by removing negative environmental effects and maximizing the nutritional status of these animals as they grow to maturity. This intensified approach to domestic animal agriculture has reached its peak in the temperate regions of the world, where the "developed" economies and, generally, a surplus of agricultural products exist. Great discrepancies are found between He agricul- tural productivity of the temperate regions and those of the tropics and subtropics, where 60 percent of the world's population and half of its domestic animal species reside. For instance, it requires from two to four times as many animals to produce equivalent amounts of meat and milk in the tropics as can be produced by domestic animals in the temperate regions. Molecular biology has helped provide the ability to address these production problems by unlocking the vast information found within the genome of each animal. Using this information, we can increase productivity of specific tissues, provide new weapons against disease, reduce inefficiencies caused by overpro- duction of fat in growing animals, increase the efficiency of rumen fermentation, and increase the genetic diversity of the domestic animal gene pool. Since the amount of available information present in the gene pool of the domestic animal population far exceeds our ability to interpret it, it is critical to define those areas most likely to produce real gains in the productivity of domestic animal agricul- ture.

360 OPPORTUNITIES IN BIOLOGY Growth Regulation Growth Occurs As a Result of an Increase in Cell Number, Cell Size, or Both Increasing growth rate has a number of potential benefits to agricultural productivity. Among these are decreased interval to reproductive maturity or market weight, increased feed efficiency, and reduction of maintenance require- ments. In addition to increasing the rate at which animals grow it is also possible to consider increasing the growth rates of specific tissues (such as muscle) and decreasing the growth of others (such as adipose tissues. Regulation of the Somatotropin-Somatomedin System. The growth of whole animals can be accelerated by growth hormone (somatotropin) and insulinlike growth factors (somatomedin). The release of somatotropin into the circulatory system is correlated with a concomitant increase in the circulating concentrations of somatomedin. The release of somatotropin can be effected by the use of a growth-hormone releasing factor, inhibitors of somatostatin, release, or antibod- ies against somatostatin. Exogenous supplementation with the homologous so- matotropin or somatomedins is also an available approach. Largely unexplored is the regulation of the receptors or binding proteins that influence the rate of the degradation and the degree of responsiveness of the target tissues for soma- totropin or somatostatin. The approach of designing drugs by studying the receptor-ligand interaction has been successful in the human pharmaceutical area, but it is only recently that a similar approach has been attempted in the domestic animal industry. A recent example is the p-agonists, which stimulate p-adrener- gic receptors to partition energy away from adipose tissue and toward muscle. An approach that has been envisioned for the future is to produce peptide mimics of steroids by studying the steroid hormone receptor and building peptides that will activate it. This would be an important breakthrough, since it would alleviate the problem of steroid residues in meat. To date the use of recombinant homologous somatotropins and chemically and or recombinantly produced growth-hormone releasing factors has been most carefully studied. Since growth hormone does more than produce somatomedins, it is likely that this approach or the use of releasing factors will be the method of choice for the immediate future. However, the use of somatomedins and epider- mal growth factors has been proposed for wound healing in valuable animals. Regulation of Growth of Specific Cell Types. The ability to favor or retard the growth or development of a single cell Me has obvious advantages in increasing the productivity of domestic animals. The specificity required to achieve either of these results dictates a greater understanding of He biology of these cell types than may be currently available. However, progress is being made with two cell types: the adipocyte of adipose tissue and the myoblast and myotube of muscle.

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 361 Excess storage of lipid is a major cause of reduced feed efficiency in domes- tic animals and a major health problem in the human population. Reducing excess fat storage in animals addresses both problems simultaneously, since the meat marketed will be of higher quality. Present approaches include the exogenous use of somatotropin and p-agonists during the finishing period to partition nutrients away from adipose tissue and toward muscle synthesis. A more direct approach being tested is the immunization of animals against membrane proteins of adi- pocytes. This approach has had dramatic effects in laboratory animals, but is largely untested in animal production systems. Much additional work is needed to learn to regulate specific metabolic pathways in adipocytes by regulation of the genome. Increasing muscle mass in animals may be accomplished by increasing the number of muscle cells or the amount of muscle protein present in cells. Two basic in vitro cellular models are the fetal myoblast cells and myotubes. Two- dimensional mapping of proteins during proliferation and myotube formation has identified specific proteins produced during these processes. However, we do not yet understand the regulation of the synthesis and degradation of these molecules or their specific functions in proliferation and maturation of these muscle cells. Additional basic research is warranted in these areas to provide tools and more sophisticated approaches to increasing the availability of high-quality protein for human consumption. Reproduction It Has Been Difficult to Increase the Reproductive Performance of Domestic Animals A great deal of basic research has been carried out in the past three decades on the regulation of the reproductive process in an attempt to increase reproduc- tion performance. Widespread use of artificial insemination and embryo transfer has increased the rate of genetic gain in certain specifically desired traits, such as milk yield. However, a reliable pregnancy diagnosis is not yet available. In addition, the average litter size in the swine population has not increased in the U.S. herd, although certain breeds in other countries have much higher litter numbers. For example the average litter size of the Chinese hog is 24, versus 10 for the United States. A major constraint in regulating the reproductive process is the large number of complex cell types that influence reproductive success or failure. In addition, the management systems in place for most domestic animals stress the reproduc- tive process. For instance, increasing mink yield in cattle through genetic selec- tion has had detrimental effects on reproductive performance because of the large metabolic demands to increase mink production. Immediate gains in production could be realized if sexing of semen were accomplished and if pregnancy-specific peptides were identified that could be used to diagnose pregnancy on farms.

362 OPPORTUNITIES IN BIOLOGY In the domestic fowl population, some potential exists for manipulating egg formation to alter the lipid composition (reduce cholesterol), to alter the protein content of eggs, or to express novel proteins that could be harvested. It is also possible to harvest antibodies from eggs of chickens that have been immunized against a foreign protein. Lactation Increases in Milk Production Will Occur with the Use of Bovine Somatotropin and Increased Mammary Growth It is generally believed that use of bovine somatotropin in lactating cattle will increase average mink yields 10 to 15 percent across a 305-day lactation. Bovine somatotropin increases the rate of milk synthesis, but does not increase the amount of secretory tissue present. Increases in the growth of mammary tissue would also increase milk yield since each gram of mammary tissue produces approximately 1.6 grams of milk per day. Although several potential mammary growth factors have been identified, the specific roles these various factors play in regulating tissue growth are not well understood. If specific mammary growth factors could be identified and put to use, it would benefit not only the dairy industry, but also other animal industries as well, since milk production is a major [imitating factor on the growth rate of neonatal swine and beef cattle. Alteration of gene expression in mammary tissue to produce a milk with a higher solid content is a possibility. Milk is 87 percent water, and it is therefore expensive to transport. The major osmotic determinant of milk is lactose, the mink sugar. In marine mammals, the production of lactose is greatly limited, producing a milk that is extremely high in protein and fat. It would be possible to achieve the same effect in cattle by inhibiting the expression of alpha lactalbumin production. This protein, produced only in mammary tissue, is the rate-limiting protein for lactose synthesis. Another possible approach to manipulation of milk synthesis is the production of novel proteins that either improve the quality of milk or are of economic significance. Infectious Disease Molecular Biology Will Provide Major Advances in the Diagnosis, Treatment, and Prevention of Some of the Major Diseases That Threaten the Livestock Industry Disease is a major cause of reduced performance in domestic animals. Some diseases that cause major losses in the livestock industry are foot-and-mouth disease, neonatal enterotoxicosis, ~ypanosomiasis, mastitis, and respiratory dis- eases. At present, molecular biology approaches are being used to develop methods of diagnosis and treatment for these diseases.

MEDICINE, THE BlOCH~IC~ PROCESS INDUSTRY, ED DIME AG~CULTU~ 363 Some general approaches are the production of animal interferons for treat- ment of respiratory diseases and the use of monoclonal antibodies for use in diagnostic aids or as treatments against certain bacteria or viruses. Recently, a vaccine against foot-and-mouth disease was developed; it uses a protein-surface antigen encoded by a synthetic gene, which was cloned and expressed at high levels in Escherichia colt. Similar approaches might be productive in the search for a weapon against ~ypanosomes, which, by causing sleeping sickness in cattle, are a major deterrent to increased productivity in the African cattle industry. Use of antibodies against adhesion factors prevents the bacteria that cause neonatal enterotoxicosis from attaching to gut epithelial cells. A similar approach has been successful in preventing the attachment to mammary epithelial cells of certain bacteria that cause mastitis. Production of Feedstocks Some Feed Additives, Such As Amino Acids and Vitamins, May Be Efficiently Produced by Recombinant Techniques The two major amino acids now used as feed additives are methionine and ~- lysine. It is doubtful that methionine made by recombinant techniques would cost less than that made by chemical synthesis. However, lysine, which can be absorbed across the gut only in its ~-form, can be produced by fermentation that uses coryneform bacteria. It is believed that recombinant techniques will make it possible to produce bacteria that can make 130 g of ~-lysine per liter. At these levels of production, the price of lysine will be lower than the equivalent soybean meal source. Lowering the cost of lysine production may permit the addition to feed of the next two limiting amino acids, ~-'ryptophan and ~-threonine. Success in making recombinant organisms that produce these amino acids in large quantity has been reported. The production of most vitamins will not be affected greatly by recombinant technology since chemical synthesis will remain financially competitive. How- ever, it has been proposed that vitamin Bit, riboflavin, and niacin might be produced competitively by these techniques. Livestock Improvement Genetic Engineering and Expanded Breeding Programs Should Result in More Efficient Production of Livestock The insertion of foreign DNA into animals has received much publicity recently. The challenge is to obtain tissue specificity of gene expression. Most current work in domestic species involves manipulation of the somatotropin

364 OPPORTUNITIES IN BIOLOGY system by inserting extra copies of the somatotropin gene into embryos. This approach, which has as its goal improving feed efficiency and meat quality, has its highest chance for success in litter-bearing species such as the pig, where the number of embryos is high. Studies are also being canted out in sheep by using embryo transfer from superovulated animals to increase the odds of success. Although it has been possible to get expression of these extra copies of soma- totropin genes in animals, the results to date have been disappointing. For example, the health and reproduction capabilities of these animals have been impaired, suggesting the need for a tissue-specific approach to gene expression. Ideally, one would like to insert the extra copies of the desired gene into the target tissue to avoid side effects in other tissues. It is likely that this will be a major barrier to the practical use of this approach. One novel approach to this problem has been to use lactating mammary tissue to produce a foreign protein. This was accomplished by joining the desired gene to the control region of the gene coding for lactoglobulin. This recombinant gene is expressed only in lactating mammary tissue. Therefore, by using an appropriate tissue-specif~c promoter, investigators can obtain tissue-specif~c ex- pression. Another possible example of the same approach would be to insert the gene- regulating expression of the mediator of somatotropin action on mammary tissue. This would permit autostimulation of secreting mammary tissue to increase yield. As mentioned earlier, the productivity of animals in the temperate regions of the globe far exceeds that of the same species in the tropics and subtropics. Moreover, when the temperate breeds are exported to tropical regions, their productivity falls rapidly, and they often die of diseases to which they have no resistance. An obvious solution is to use local breeds in these regions, provide exogenous hormone treatments to increase their productivity, or to insert genes coding for metabolic enhancers. For example, adaptation to thermal stress is associated with a reduction in circulating somatotropin concentrations. Growth rates and mink yields of cattle in these regions might be enhanced markedly with exogenous supplementation of somatotropin or insertion of extra copies of the growth hormone gene. The diversity of the domestic animal gene pool is larger than most scientists realize because the predominant breeds of livestock represent only a small portion of the total number of breeds. For example, 90 percent of the dairy cow popula- tion in the United States consists of Holsteins, even though more than 40 different dairy breeds exist throughout the world. No doubt many of these breeds have genetic information of great value to the world cattle industry. In Asia alone, there are five species of nondomesticated ruminants, which are close relatives to cattle although poorly known. Another example is in the swine industry. What genes make it possible for the Chinese sow to produce twice as many piglets in a litter as the average U.S. sows? Southeast Asia is the home of a ruminant pig (Babirusa) that might be used to produce a new pig breed, one more efficient at meat production. We should consider not only the relatively few highly utilized domestic animals in our search to increase domestic animal productivity, but also their underutilized relatives.