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Am. J. Trop.Med. Hyg., 35(2), 1986,pp. 221—233 Copyright ©1986by The American Societyof Tropical Medicine and Hygiene

FILARIASIS: PROBLEMS AND CHALLENGES*

PAUL P. WEINSTEIN Department of Biological Sciences,University of Notre Dame, Notre Dame, Indiana 46556

Being President of the Society during the past other areas of biomedical research such as cancer year has been an enriching experience and I thank and heart disease does not fare well, considering you for the privilege and opportunity to serve. the harsh impact of tropical diseases on world My special thanks go to Dr. John Scanlon who populations. shoulders the work of the Society so effectively. However, even a cursory perusal of the liter The last time I addressed the entire Society' ature of the last 15 years indicates that activity was in this city in 1972 at a joint meeting with in research has not only increased in the American Society of Parasitologists. I dis quantity but more importantly in scope and in cussed the relationship of government, parasi the application of new research disciplines. As tology and tropical medicine and ended with an has happened in other fields of biomedical re assessment of funding for research for these fields. search, the newer technologies and analytical ap In reviewing the history of extramural funding proaches of immunology and molecular biology by the National Institutes of Health for filariasis are beginning to be applied more rigorously to research it was interesting to note that in 1965 biological and medical aspects of the filariases only two grants existed for the total sum of with interesting implications for the future. Lab $15,150. Seven years later, largely due to the oratory studies and field operations in endemic efforts of the U.S.-Japan Cooperative Medical areas appear to have increased. Some progress Science Program, the number of funded grants is evident in the control of in lim had increased to eleven for a total sum of ited areas of Africa. But the massive problem of $314,975. the endemic filariases, particularly that of lym I do not intend to review the problem of fund phatic filariasis, tropical pulmonary eosinophilia ing for filariasis research in this address. Analyses and onchocerciasis, for the most part remains of funding for research in tropical medicine and unyielding in the vast areas of the tropics and in parasitology, including the Illaniases,were made subtropics. Discouragingly, rapid urbanization is in recent years by Cook2 and Delappe.3 A few occurring worldwide. Lack of facilities for the weeks ago a detailed report on funding for trop proper disposal of sewage and waste is expanding ical disease research was released by the Office the vector breeding sites not only in the newly of Technology Assessment of the United States created city slums but also in the ruralareas which Congress.4 I recommend this publication to you more and more come under the domination of not only for the coverage of this aspect but for the towns. Populations of humans as well as vec an important overview of the status of biomed tor populations are increasing at an astonishing ical research and related technology for tropical rate, creating new hordes of susceptibles. Cou diseases. It indicates in a summary table of com pled with the flair of Culex quinquefasciatus to bined annual funding by United States sources develop resistance to insecticides, and the lack and by the World Health Organization that the of effective drugs without toxic side effects, there filariases receive 3% of the total available funds is little basis for optimism at this point in time. for research and training in tropical diseases. As The complex ecological interplay of humans, was pointed out by Dr. Cook,2 comparison of vectors and environment that gives impetus to research support for parasitology with that for transmission may yield ultimately to continued efforts in the laboratory and field, but only if support for basic and applied research is avail s@idential Address given before the 34th Annual able in adequate amount and expended in an Meeting of the American Society of Tropical Medicine intelligent manner. Our ignorance is fairly pro and Hygiene,Miami, Florida,5 November 1985. found and our efforts groping. 221 222 WEINSTEIN

All is not bleak, however. Excellent reviews against particular stages would similarly become have recently appeared on significant advances much more reliable and significant. These ap being made in research on chemotherapy,5 on proaches have been commonplace in microbi immunological aspects of lymphatic filaniasisand ology and protozoology for decades and defined onchocerciasis6 and on epidemiological, vector culture systems have been of inestimable value and control aspects.7 Consequently, for the brief to these fields. Such types of in vitro studies as time available, I will only touch upon these mat I have mentioned, of course, have been per ters and instead concentrate on a few other in formed in the past with various fllaniid species. teresting problems which I believe are of im Of necessity, however, they have been short term portance to our understanding of the filariases. experiments using media and physico-chemical conditions we now know to be inadequate, with OXYGENAND FILARIIDS the parasites studied being metabolically stressed and essentially in various stages of dying during The first part of my talk will be devoted to the the period of in vitro maintenance. interaction between oxygen and fllariids. A dis Until fairly recently the number of studies in cussion of oxygen requirements leads to a con which attempts had been made to culture fllariids sideration of a multiplicity of important matters in vitro were discouragingly few. In 1970, I re in the biology and -parasite relationships of viewed the literature8 and found that a total of these parasites. It serves as a mechanism to in only 43 papers had been published on this subject troduce for discussion a variety of important in approximately 60 years, the first appearing in problems, for example: 1) our inadequate un 1912. Almost all were concerned with survival derstanding of the physico-chemical and nutri and maintenance in vitro; no significant growth tional factors that are required by fllariids for or development of the was reported. Most growth, molting, differentiation and reproduc of the studies were conducted with microfllariae, tion in vitro; 2) our lack of information about a few with adult worms and in only four inves the detailed nature of the tissue and fluid mi tigations were attempts made to obtain growth croecological milieu which provides the intimate of third stage larvae from the vector. habitat for the various species of fIlariids, em In the ensuing years, however, research on all phasizing the great gaps in our understanding of aspects of filariasis has increased considerably the pathophysiology of the host-parasite rela and with it a renewed interest in cultivation as tionship; and 3) the problems and possible mis an experimental tool. Some encouraging progress interpretations that may arise in biochemical, has been made and for the first time significant immunological and drug evaluation studies con growth and differentiation in culture of several ducted using currently inadequate in vitro pro species of fIlaniids have been reported. cedures. These studies are beginning to provide us with an insight into some aspects of the biological Culture systems processes of these parasites. They have also posed intriguing questions about important facets of One of the pressing needs in filariasis research the host-parasite relationship, hitherto unsus has been the development of in vitro cultivation pected. As examples, I would like to discuss some systems that would permit the normal growth information derived from work we have done and maintenance of all stages in the life cycle of and results reported from other laboratories. filarial worms. For example, the availability of Oxygen requirements of parasitic protozoa and such culture systems would provide new and im helminths have been studied for decades, par portant approaches to the study of those factors ticularly with regard to low oxygen tensions and which regulate and control developmental pro so-called conditions of anaerobiasis. It is appar cesses. Immunological studies would be en ent that exceedingly few species of parasites are hanced in many ways. Stage-specific antigens strict anaerobes. The pioneering work of Trager could be harvested and a variety ofinvestigations and his colleagues9@'°demonstrated unequivo studying the arms of the immune effector systems cally the importance of a relatively low oxygen could be critically examined under physiologi tension for the successful cultivation in vitro of cally appropriate conditions. Screening of drugs Plasmodiumfakiparum. Interestingfindingsre and the study of the mechanism of drug action garding the oxygen relationships of Entamoeba PRESIDENTIAL ADDRESS 223 histolytica also have emerged from the quanti gration of the microfilariae to the peripheral tative analytical investigations of Gillin and Dia blood. This ability of microfllanae to titrate rel mond.―Studies of the effects of the gas phase on atively small changes in p02 and to respond be schistosomes and parasitic in culture haviorally suggests that developing larval and also have been conducted but the results have adult stages may also have the capacity to do so. been difficult to interpret because information Experimental studies conducted in vitro to ex on the actual oxygen tensions present in the cul amine these phenomena should be quite reward ture system has not been provided. ing. The sensory receptors that respond to oxygen In studies conducted by Dr. Eileen Franke in are not known. It is possible that the amphids my laboratory on the cultivation of Dipetalo and phasmids may function as chemoreceptors nema viteae, we reported the development of for the gas. How the circadian rhythms of other third stage larvae from the arthropod vector to microfilarial species are entrained into the phys young adults in a chemically defined medium iological rhythms of their hosts is far less well supplemented with serum.'2'4 Of critical im understood. The information that has appeared portance was the gas phase, a relatively low oxy in recent years on mammalian circadian rhythms gen tension being required in order for growth, may provide the physiological and biochemical molting and differentiation to occur. Surprising basis for the underlying host-parasite relation ly, the window for the required partial pressure ship. The location of the circadian pacemaker in of oxygen was found to be relatively narrow. De the mammalian brain driving the rhythm of mel velopment of the larval stages occurred only be atonin synthesis'6 and the integration of function tween a partial pressure of oxygen of approxi of the retina and pineal gland provide an exper mately 30 to 50 mm Hg. At higher and lower imental tool to explore this interesting problem. oxygen levels development was inhibited. Di petalonema viteae thus appears to be a microaer In vivo relationships ophilic organism, at least in its mammalian cycle. Using essentially the same medium and low It is instructive to examine the information oxygen tension, Dr. Vishnu Sneller reported at available concerning the oxygen tensions in tis last year's annual meeting of the Society that sues and body cavities that serve as habitats for adult worms of Brugia pahangi could be main various genera and species of filariids. The types tained for 3½to almost 7 months. Microfllariae of niches in mammalian hosts occupied by fi that were collected after approximately 3¾ lariids range widely: subcutaneous tissues for months of culture developed to third stage larvae , Dipetalonema viteae and in mosquitoes. Loa ba; the pulmonary cavity for Litomosoides These investigations suggest the importance of carinii, the abdominal cavity for Setaria species; quantitatively evaluating the oxygen tensions re lymphatic vessels for and quired in cultures established with fllariids of the Brugia species and blood vessels for Dirofi humans and of other experimental species. lana immitis. The oxygen tensions of all of these In most such previous studies, mixtures of air! habitats are relatively low, being in the neigh CO2 have been used with discouraging results. borhood of approximately 40 mm Hg or lower. That filariids appear to have the capacity to Although the oxygen tensions of tissues, body respond to very small differences in oxygen ten cavities and body fluids have been studied by sions present in a host is suggested by the inter many investigators, much of the recent infor esting studies of Hawking'5 on the mechanism mation is derived from studies of tumorous and of microfilarial periodicity of Wuchereria ban cancerous growths, wound healing and cardiac crofti. The nocturnal migration of the microfi vascular physiology. The sophisticated tech lariae appears to be dependent upon the differ niques utilized by investigators in these fields ence in oxygen tension that exists between such as that of colpophotography'7 and stop-mo pulmonary arterial and venous blood. Microfi tion microcinematography'8 could be of consid lariae accumulate in the pulmonary vasculature erable value in studies of the vasculanzation and when the venous-arterial difference in oxygen gaseous microenvironment in the immediate vi tension is 55 mm Hg or greater. A decrease in cinity of larval and adult stages of filanids. No the venous-arterial oxygen tension to approxi detailed studies have been made to follow the mately 44 mm Hg or below results in the mi vascular changes that occur over time in the 224 WEINSTEIN pathogenesis of filarial , particularly of Krebs cycle and adult fllariids that rely on gly the microcirculation in the immediate vicinity colysis for energy production. Many species of of developing and adult worms. In onchocerci fllariids, such as Brugia pahangi, Dipetabonema asis, young nodules may show capillary neofor viteae, Dirofilania unifonmis and Onchocenca vol mation. Ultimately, however, vascular degen vulus, are exclusively homolactate fermenters. eration occurs accompanied by fibrotic changes This aspect has been investigated in detail for of an avascular nature in the onchocercoma and adults of B. pahangi and D. viteae using isotope skin.'920 In in which obstruc and aerobic fermentation balance studies.23 These tive lymphangitis, stasis of lymph flow and thick two species observed in vitro over a 24-hr period ening of the walls of the lymph vessels occur, it continued to move actively in the absence of appears possible that oxygen tensions in the im oxygen. However, movements of L. carinii rap mediate vicinity of the filariids may be quite low. idly ceased in a medium depleted of oxygen. This Detailed study of changes in the vascular archi species, in addition to requiring oxygen for tecture in the filariases relative to local oxygen movement, also differs from B. pahangi and D. tensions could provide important background viteae in that it accumulates acetate, acetoin and information regarding oxygen requirements of unidentified compounds in addition to lactate. filariids for growth, development and survival in Litomosoides caninii does not appear to possess vivo, particularly if coupled with in vitro inves an actively functioning tricarboxylic acid cycle tigations. How these parameters are modified by to account for its utilization of oxygen24 although chemotherapy, often with accompanying inflam some reports to the contrary have recently ap matory responses,isnotunderstood,particularlypeared.25.26Instead, it apparently requires oxygen as they may affect the viability of metabolically primarily for the oxidative decarboxylation of injured parasites. pyruvate to acetate and CO2. presumably with the production of ATP. Oxygen and mammalian cells If oxygen is not required by fllariids for energy production through a functional Krebs cycle, an Much information on the oxygen tensions re anomalous situation exists with regard to the na quired by a variety of mammalian cell types ture of their mitochondria. These organelles are growing in vitro has become available in recent highly cristated in all three species,24 such a con years. Such studies appear to have considerable dition usually implying an active TCA cycle. This relevance to cultivation attempts with particular is an interesting problem requiring resolution groups of parasites and offer a model framework since it has important implications with regard for future investigations. Some of the results ob to potential sites and mechanisms of drug action. tained from studies on the relationship of mam malian cells to oxygen tension in vitro may be Collagen synthesis summarized as follows: 1. Both neoplastic and non-neoplastic cells have It is also necessary to reconcile the seemingly equal or better plating efficiencies at low oxygen contradictory fact that although molecular oxy tensions (l%—3%02) than under a gas phase con gen presumably is not required for the move taining air (20% 02).21.22 ment of B. pahangi and D. viteae adults, it is 2. Oxygen inhibition of growth acts directly on required for the developmental processes in the the cells and not necessarily by destroying some growth and differentiation of third stage larvae essential medium component.22 of D. viteae to the adult form in vitro. Obser 3. The mechanism of oxygen toxicity is not vations of this nature suggest that oxygen, al clearly understood and is complex. An attractive though possibly not required for energy-yielding hypothesis is that reactive oxygen radicals formed pathways, may be needed for other metabolic from reductive intermediates of oxygen during processes. For example, collagen synthesis, which normal cellular metabolism damage the cells. is essential to cuticle formation, is an oxygen-dependent process in which proline-4- Oxygen and filaniid energy relationships monooxygenase converts the proline in proto collagen to hydoxyproline.27 It is of interest that Obviously, metabolic differences exist be the enzyme in adult Ascanis is partially inhibited tween mammalian cells that have a functional at oxygen concentrations exceeding l%—5%. PRESIDENTIAL ADDRESS 225

However, inhibition does not occur in the aero agents for nematodes and the use of azasteroids bic egg, suggesting the existence of isozymes.28 as metabolic probes in the study of cholesterol Whether isozymes of this hydroxylase occur in metabolism in these organisms. It will be of in the larval and adult stages of fllariids requires terest to determine the requirement for oxygen investigation. in this regard.

Ecdysteroid synthesis Oxygen toxicity

Another oxygen-requiring process that may be It is apparent that the need for oxygen by linked to fllariid development is the synthesis of nematodes is quite diverse and complex. In the ecdysteroids such as ecdysone. This steroid func case of fllariids such as D. viteae, two completely tions as a molting hormone in insects and is of different mechanisms appear to be operative. At critical importance to the developmental pro quite low oxygen concentrations the limiting as cess. Ecdysone and ecdysteroids have recently pect may be insufficient oxygen to support syn been isolated from adult Dirofilaria immitis tak thetic and metabolic processes required to sus en from the dog host29 but the origin of these tain the life of the organism. At relatively high hormones was not determined. Studies done in oxygen tensions, however, the mechanism of my laboratory on the larval cultivation of Nip oxygen inhibition very probably is due to its tox postrongylus brasiliensis under axenic condi icity. Explanations to account for the oxygen tox tions and in medium of known sterol composi icity that occurs in many species of organisms tion have provided strong evidence supporting have come from a variety of sources. Some of the concept that nematodes are capable of syn the hypotheses that have been proposed suggest thesizing ecdysteroid-like compounds from cho that: 1) oxygen inhibits metabolism by oxidizing lesterolin their diet.3°It is important to recognize SH groups of enzymes; 2) oxygen causes the per that in insects synthesis of ecdysteroids from oxidation of lipids and the resulting lipid per cholesterol is dependent upon the presence of oxides inhibit enzyme function and affect mem molecular oxygen and presumably this would also brane integrity; and 3) oxygen radicals formed hold true for nematodes. during normal metabolism damage cells. Much work is now required not only to de Although one or a combination of these factors termine the presence and function of these cc may be operative in fllariids, virtually no exper dysteroids in fllariids and other parasitic nema imental data exist to clarify the matter. Much todes, but also their mode and specific site of work remains to be done to determine the pres synthesis. As has been found to be the case with ence and types of oxygen intermediates that may insect species, considerable differences in the cc arise during metabolism. Hydrogen peroxide has dysteroid biochemistry of nematodes should be been reported to be toxic to L. carinii.35 How anticipated. Of considerable interest in this re ever, it is not known whether scavenger enzymes gard has been our finding that azasteroids such as catalase, superoxide dismutase and glu (25-azacholestane and 25-azacoprostane) are ex tathione peroxidase are formed to remove re tremely potent inhibitors of nematode devel active oxygen intermediates. Such investigations opment, disrupting growth, molting and repro could be of considerable importance in clarifying duction,31-33comparable to that which occurs to the toxic nature of oxygen for fllariids. Oxidant nematodes grown in the absence of cholesterol. stress on Plasmodium falciparum, as discussed It is possible that these compounds act as inhib by Scheibel and Adler36 and Allison and Eugui,37 itors interfering with the incorporation of cho may seriously injure the parasite. lesterol into membranes. For example, the ad Another aspect of the possible interaction of dition of azasteroids to axenic cultures of the fi.lariids with reactive oxygen intermediates aris larval stages of N. brasiliensis results in severe es not from their endogenous metabolic synthesis repression of development, abnormal molting and of these compounds but rather from the gener degeneration of the intestinal cells. However, ation of the oxidants by the cells of the immune azasteroids may also be functioning as inhibitors response. Of interest in this regard are the recent of ecdysteroid synthesis comparable to their ac findings of Kazura and Meshnick38 concerning tion in insects.34 These findings suggest new tar the disparity in the susceptibility of the different gets for the development of chemotherapeutic stages in the life cycle of to 226 WEINSTEIN killing by neutrophils and eosinophils. Their ob from retinol was considered to be potentially in servations indicate that newborn larvae were volved in glycoprotein synthesis. more susceptible to being killed by oxidants than It is possible, however, that fl-carotene and were muscle larvae or adults. It was hypothesized retinol may also have another function in filar that adults and muscle larvae may be more re iids which is worthy of investigation. fl-carotene, sistant to killing by leukocytes than newborn lar a purported anticancer agent, has been believed vae because they contain scavenger enzymes en to have antioxidant action of a radical-trapping abling them to resist oxygen-mediated damage. type. More recently, in vitro experiments have Comparable studies of this type with filariids are demonstrated that it belongs to a previously un needed to determine whether the oxidative in known class of biological antioxidants.45 It ex termediates of neutrophils and eosinophils, which hibits good radical-trapping antioxidant behav are known to severely damage the cuticle of var ior only at partial pressures of oxygen significantly ious stages of the parasites, may be nuffifled to less than 150 torr, the pressure of oxygen in nor any degree by the presence of oxygen scavengers mal air. The low oxygen pressures in which it in the . Forsyth et al.39have hypothesized acts as an antioxidant are found in most tissues that albumin which is adsorbed in vivo to mi under physiological conditions, which would in crofllariae of Onchocerca gibsoni may be acting dude the various habitats of fllariids. At higher as a local inhibitor of eosinophil peroxidase-me oxygen pressures fl-carotene loses its antioxidant diated oxidative damage. Bovine serum albumin activity and shows an autocatalytic pro-oxidant is a known scavenger of hypochiorous acid which effect, particularly at relatively high concentra is generated by the reaction of eosinophil per tions. oxidase with H202 and Cl- and which is capable The results obtained by Burton and Ingold45 of killing newborn T. spiralis larvae. have demonstrated that fl-carotene has the po tential to play an important role in protecting Carotenoids and vitamin A lipid from peroxidation in vivo. The chain breaking action of fl-carotene complements that Other chemical factors in the tissue environ of vitamin E since fl-carotene is effective at low ment of fllariids may also serve to protect them oxygen concentrations and vitamin E is effective from oxygen damage. Vitamins, such as vitamin at high oxygen concentrations. As pointed out E and ascorbic acid, are important antioxidants previously, fllariid habitats have low partial pres in vivo and their effects on larval development sures of oxygen and it may, therefore, be antic using in vitro cultures should be assessed. In this ipated that retinoids would exhibit antioxidant regard, Stürchleret al.4°have reported that the properties in fllaniids such as 0. volvulus, Brugia retinol (vitamin A alcohol) concentration in tis pahangi and Foleyella. Studiesto examine this sues of 0. volvulus is very high, averaging eight relationship should be of considerable interest. times higher than the skin of infected individu Another important function of retinoids, that als. Carotene and retinols have a multiplicity of involved in the regulation ofgene expression, has functions and it will be of interest to investigate recently been reported. Retinoic acid, as a nat these in fllariids. urally-occurring metabolite of vitamin A, has Carotenoids, although not extensively studied, been known to modify the proliferative rate and have been reported to be present in a variety of differentiation of a variety of normal and trans parasitic helminths: in the larval cestode Schis formed human cells. Recent studies46'47 suggest tocephalus solidus,4' adult trypanorhynch that retinoic acid and serum retinol-binding pro cestodes42and in Foleyellafurcata, a filariid par tein can directly regulategene expression of mouse asite of chameleons.43 Comley and Jaffe,@ stim macrophages and human prómyelocytic leuke ulated by the observations on the high concen mia (HL-60) cells and specifically induce the syn tration of carotene and vitamin A in 0. volvulus, thesis of tissue transglutaminase. Such regulation have studied the biochemistry of these com of gene expression as a property of retinoic acid pounds in Brugia pahangi. The adult fllariid took and its binding protein may have important im up and incorporated fl-carotene and retinol and plications with regard to the growth and differ the in vitro conversion of fl-carotene to retinol entiation of 0. volvulus and other fllariids which was demonstrated. The retinyl phosphate formed concentrate the vitamin to a marked degree. In PRESIDENTIALADDRESS 227 vitro culture should provide a valuable tool to version of the vitamin to adenosylcobalamin assess their direct effect on the actual growth and which participates in an isomerization reaction differentiation of larval and adult stages. in the conversion of succinyl CoA to methyl Vitamin A deficiency, unfortunately, is an all malonyl CoA. Vitamin B,2 can also be converted too-common occurrence among human popu to the metabolically active coenzyme, methyl lations in areas endemic for filariasis. Laboratory cobalamin, which acts as a cofactor for methi studies on the association of vitamin A and filari onine synthetase, in the conversion of homocys asis obviously should be integrated with clinical teine to methionine. In the elegant work of Jaffe investigations in the field. Sturchler et al.4°posed and his colleagues on the folate metabolism of the pertinent question of whether onchocerciasis fllariids, they were not able to demonstrate the depletion of retinol in host tissues might be a presence of methionine synthetase in Brugia pa contributory factor in the pathogenesis of corneal hangi.48This strongly suggeststhat methylcobal opacities, localized dermatitis (sowda) and other amin, if present, is either inactive or it does not symptoms occurring in both clinical onchocer exist in this parasite. On the basis that we have ciasis and hypovitaminosis A. not been able to demonstrate the presence of cobalamin in this species, I believe the latter in COBALAMINS AND FILARIIDS terpretation to be correct. Interestingly, we have searched for the presence of methiomne synthe Please forgive what may appear to be some tase and methylcobalamin in a few other genera what exaggerated speculations on the possible of parasitic helminths. Although we were able to fl-carotene-vitamin A relationship in filariids. My demonstrate the adenosylcobalamin form of the interest in the unusually high concentration of coenzyme in these organisms we have been un these compounds in these nematodes stems from successful in finding any suggestion of the pres my previous experience in unexpectedly having ence of methylcobalamin or methionine synthe found extraordinarily high concentrations of vi tase.49 Perhaps the ready availability to parasites tamin B,2 in a considerable variety of parasitic of preformed host methionine accounts for this. helminths. Unraveling aspects of cobalamin In any event, these results suggest the possibility physiology, biochemistry, evolutionary distri that in the evolution of fllariids genetic deletions bution in helminths and its role in the host-par have occurred eliminating this arm of cobalamin asite relationship has occupied much of the time metabolism. of my laboratory during the past years. Interest ingly enough, unlike the situation in most eu karyotic , vitamin B,2 and its cobalamin GENETICS OF THE FILARIASES derivatives do not occur in any of the fllariids Parasite genetics we have assayed. These findings correlate well with the fact that fllariids are primarily lactate Such studies emphasize our ignorance about fermenters. The large number of parasitic hel the very important field of the evolution and minths which contain cobalamins form propio genetics of parasitic helminths, particularly fi nate and do so through a cobalamin coenzyme lands. Although a considerable amount of work dependent step. This has been found to be true has been done by systematists to delineate the especially for the more “primitive―groups of evolutionary patterns of fllariids based on mor parasitic nematodes such as strongylids and as phology and life history studies, little is known carids. The more highly evolved tissue and vas about the genetics of these organisms. This mat cular parasites such as Angiostrongylus and fi ter is of great significance to our understanding lariids have apparently lost the ability to take up of the epidemiology and disease relationships in and utilize cobalamins and presumably have de filariasis. In this regard, the genetics of the mam leted the enzymatic pathway from succinate to malian host and the genetics of the arthropod propionate which requires the B,2 coenzyme, ad vector inexorably interlock with the genetics of enosylcobalamin. the parasite to form a dynamic interacting trinity. Vitamin B,2 is an interesting molecule in that These relationships unquestionably are ex it can function in two completely different coen tremely difficult to approach and analyze exper zyme forms. I have already mentioned the con imentally but the rewards could be significant in 228 WEINSTEIN our understanding of the complexity of the fi human 0. volvulus from Guatemala, Venezuela lariases. and Africa. Part of the problem seemingly relates Epidemiological data abound suggesting the to technical difficulties of chromosome analysis. genetic modulation of the filariases in nature. For Resolution of this problem could help interpret example, in onchocerciasis it is well-known that the clinical, vector transmission and epidemio the clinical expression of disease differs consid logical differences that are manifested in oncho erably between the Sudan-savannah zone and the cerciasis in different areas of the world. West African forest. In the Cameroon, experi It is apparent that a number of approaches are mental data indicate that there appear to be two required to help resolve the matter of identifi main Onchocerca/Simulium complexes, each cation of species and strains of fllariids. Appli distinct from the other and associated with a cation of the techniques of molecular biology, different clinical pattern. Findings such as these isozyme analysis and immunology undoubtedly have important implications for research and will be of great value. Monoclonal pro control efforts. cedures and the use of DNA probes are providing Because of the considerable technical prob important new tools, but will require great care lems involved, the genetics of filarial worms re in developing specificity and sensitivity. mains obscure. Only a few experimental studies have been conducted, these essentially being Genetics of vectorial capacity simple crossing attempts. Hybridization exper iments were done between B. malayi, B. pahangi Although the genetics of vectorial capacity of and B. patei and crosses were obtained with var fllaniids is undoubtedly the best understood as ious combinations of the three species.5°Hybrid pect of genetic modulation in the filariases, the males failed to produce spermatozoa but females field is still at a fairly primitive level of devel were fertile and produced microfllariae when opment and offers considerable opportunities for crossed with males of the parental species. The studies basic to understanding the host-parasite strains of parasites used in these studies had been relationship. It is of vital importance to any con in the laboratory for different periods of time and sideration of attempts for the control of filariasis were from different geographic areas. Compa by the replacement of vector populations with rable investigations were done more recently by filaria-refractory populations. Filariid develop Cross et al.5' which in the main confirmed and ment in the insect vector is invariably intracel extended these studies. The possibility was sug lular and a single host cell type is associated with gested that in certain areas of Indonesia, B. ma each filariid species. Apparently, only ticks pro layi and B. pahangi are hybridizing naturally in vide more than one cell type in an organism for cats and may be transmitted to humans. The the development of a filarial species. In insect interesting observation was made that it is pos vectors, however, these are exclusively either the sible such hybrids conceivably could develop to thoracic muscle cells, the cells of the malpighian adults without producing microfllariae, leading tubules or the fat body cells. Presumably, the to occult filarial infections. large size of these cells, adequate to accommo Genetic studies should be firmly rooted in date a developing , governs the restriction chromosomal analysis. However, chromosomal to these three host cell types. The mechanism of cytological studies have been done for only a few recognition and penetration of a specific host cell species of filarial worms. Analyses of the karyo type by a migrating microfilaria in an insect is types of B. pahangi and B. malayi revealed that not at all understood but apparently it is not a both species are karyotypically indistinguishable fortuitous event. It is possible that the microfi from each other confirming their close taxonom lana recognizes and responds to specific cues and ic relationship.52 However, chromosomal studies to a particular cell surface membrane compo of Guatemalan strains of 0. volvulus from hu nent, but I am unaware of any attempts to study mans and 0. gutturosa from bovines indicate this matter. that they are karyotypically distinctive.53 Bio Susceptibility of mosquitoes to with chemical isozyme patterns and morphological fllariids is inherited, being controlled by simple data also support these conclusions. However, sex-linked alleles in some groups, and a polygenic some confusion exists among different investi condition in others. Refractoriness to infection gators concerning the chromosomal patterns of has been found to be dominant to susceptibility PRESIDENTIALADDRESS 229 but incomplete penetrance occurs.54'55 Different nutritional requirements and development in genes control the development of the various culture, particularly if antibiotics were used. Oth filariid species in the different host cell types.56 er issues in the relationship of fllariids and their Since arthropod development is fairly rigidly symbionts may also be of significance. It would controlled by various hormones such as cody be of interest, for example, to determine the an sone and juvenile hormone it was anticipated tigenicity of the symbionts and whether their that manipulation of host hormones might affect presence in fllariids may prove to be confusing filarial development. So far, this has not proved to immunological studies. Conceivably, they may to be the case but this aspect has not been ex also interfere with DNA and RNA analytical plored in depth. studies. It is likely that mosquito refractory genes di This entire matter has become highly complex rectly affect host cell types in which the fllariids and controversial and is sufficiently important develop but the mechanism remains ob to require further detailed investigation. At issue scure.56'57How mosquito gene regulation affects is the understanding of whether the symbionts parasite intracellular differentiation at the bio significantly affect the reproductive biology of chemical and molecular level is one of the im mosquitoes, the genetics of the vectorial capacity portant challenges for future investigators. If it of fllariids, as well as other aspects of fllariid becomes possible with a high degree of success biology. to culture the arthropod phase of the filanal cycle in vitro using infected mosquito cell explants, it Geneticsof the mammalian host may provide a tool with which to examine such factors experimentally. Specific information concerning the genetic The role of intracellular microorganisms that modulation of Maria! infections in mammals is exist in mosquito vectors as well as in many still scanty. Interest in this aspect for the lym species of fllariids is quite perplexing. The precise phatic fllariases63 derives primarily from corre nature of these organisms is unknown since they lating parasite prevalence data in particular lo have not been isolated and studied in culture. It calized human populations with clinical has been proposed that in mosquitoes the rick expressions of infection such as the asympto ettsia-like organism, Wohibachiapipientis, is the matic state or symptomatic conditions such as basis for cytoplasmic incompatibility.58 It has fevers, chronic lymphatic obstruction and trop also been suggested that the mosquito symbiont ical eosinophiuia. In onchocerciasis in Africa, a provides some necessary component for the de difference exists in the severe ocular complica velopment of filarial parasites in mosquitoes.59 tions of the disease in infected forest patients The nature of the symbionts in fllariids is also compared with those in the savannah. In addi unknown, being considered rickettsia-like or tion, paradoxically, in the forest the number of Chlamydia-like.60'@2Although the precise route nodules per individual is vastly more than in the of transmission of the symbiont in fllariids is savannah.@ Obviously, nongenetic factors may unclear, like that of the symbionts in mosquitoes play the vital role in these conditions. But the it appears to be transovarial. Whether the mi question, nevertheless, persists as to whether fa croorganisms infecting mosquitoes may also in milial predisposition occurs and whether genetic fect their fllariid parasites, or vice versa, has not correlates can be identified. been determined. Clear-cut examples of the involvement of Of particular significance is the role that these mammalian host genetic factors have been dem intracellular organisms may play in the biology onstrated in nematode infections in experimen of their respective hosts. The possibility exists tal animals. For example, the major histocom that they may be pathogenic in fllariids. How patibility complex (MHC; H-2 linked genes) ever, consideration should also be given to the influences the response of mice to infection with possibility that a metabolic and nutritional re Trichinella spiralis. In studies evaluating poten lationship may exist between the symbionts and tial genetic influences on susceptibility to ban their fllariid hosts comparable to that described croftian filariasis among Polynesians, significant for the symbionts present in some kinetoplastids familial clustering of patients with filariasis was and in reduviids. This would have important found and the clustering was most compatible implications for any studies on the biochemistry, with genetic transmission of disease susceptibi! 230 WEINSTEIN ity. However, there was no evidence for associ regularly receive a good deal of attention by many ation between individuals' HLA antigen speci groups. Instead, I have focused on a few matters ficities and either susceptibility to filarial infection that I believe are equally fundamental to our or predisposition to any particular clinical man comprehension of the filariases and which re ifestation of this disease.63 In a similar study with quire study. two ethnic groups, Sri Lankans and Southern Despite the importance of basic research to Indians, it was found that a heterogeneity in HLA provide an underpinning for control efforts for antigen frequency existed between patients with the filariases, I wish to close with the proscription elephantiasis and controls.65 However, the Bl 5 provided by Dr. Philip Russell68 in his presiden antigen was found to be strongly associated with tial address entitled “ExcellenceIn Research Is the development of elephantiasis in both ethnic Not Enough.―Let us assume eventual success. groups. To explain the differences obtained be The development of a promising vaccine or drug tween the two studies, it was suggested that the only signals the beginning of the long, arduous HLA antigen is probably only a marker for some and expensive task of field and clinical trials. For other disease-associated genes on chromosome example, funds to underwrite such efforts must 6 that is in linkage disequilibrium with the HLA be sought in competition with other equally antigen, in this case Bi 5. The strength of this pressing programs also struggling for priority. linkage may vary from population to population. Careful planning in the endemic area of choice It will be interesting to determine whether the must be accomplished to provide an infrastruc association ofBl 5 with elephantiasis is reflected ture of dedicated, well-trained local personnel in differences in immune response to the para capable of conducting a long range program con site. sonant with national public health goals and bud The association between blood groups and the gets. These are some of the challenges for the filariases remains controversial, to some degree future. I have no doubt but that many of the because of the differences in methodology and young members of our Society as well as others statistics that have been applied. In a recent study worldwide will participate in the work of the conducted in India,66 no association was dem exciting years that lie ahead. The reward is good onstrated between blood groups and infection science, the sense of kindredness that shapes the with W. bancrofti. 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