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WILLIAMS Major professor Date MAY 15, 1989 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 __h_—.____ _ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmaive Action/Equal Opportunity Institution CELLULAR AND HUMORAL IMMUNE RESPONSES IN OI‘CHCXIERCIASIS: CORRELATIONS WITH CLINICAL SIGNS AND INFECTION INTENSITY BY MOHAMED Y. ELKHALIFA A DISSERTATION Suhnitted to Michigan State University in partial fulfilment of the requirements for the degree of DQZTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY AND PUBLIC HEALTH 1989 ABSTRACT CELLULAR AND HLMORAL IMMUNE RESPONSES IN OMIHCXZERCIASIS: CORRELATIONS WITH CLINICAL SIGNS AND INFECTION INTENSITY BY MOHAMED Y. ELKHALIFA Characterizations of circulating lymphocyte subset populations, ig_ vitrg lymphocyte responsiveness, serumemediated effects on lymphocyte proliferation and anti—Onchocerca volvulus antigen antibody recognition patterns were performed on groups of onchocerciasis patients selected from over 200 examined parasitologically and clinically in Sudan and Sierra Leone. These patients manifested a very broad range of clinical signs and infection intensities; some were heavily infected and asymp- tomatic while at the other extreme were individuals with severe clini- cal signs, but fGW’ or no detectable' microfilariae (mf) in their biopsies. Patients from Sudanese foci were worse afflicted than the W. Africans. Lymphocyte subsets in peripheral blood were not ranarkably different from those in control samples. Lymphocyte proliferative responses to soluble Q. volvulus antigen (sAg) were poor in infected persons; however, mitogen and PPD responses were in the normal range in one group of patients from southwestern Sudan, but were profoundly depressed in a group from.Eestern Sudan. The latter were malnourished and in poor physical condition in a drought-stricken zone. Sera from 90 patients exhibiting a wide range of clinical signs and parasite burdens had no significant effects on i_n yigg lymphocyte responsive- ness. Proliferative responses and INF-Y production by lymphocytes were very significantly depressed in the presence of live microfilariae and worm secretions/excretions (S/E) . Inmunoblotting analyses revealed diverse antibody responses to a wide range of worm antigen bands, with up to 25 recognized by some sera. Subclass specific monoclonal probes revealed a predominance of IgG4 responses, often accounting for almost all the band spectrum seen with 196 reagents. There was, however, no obvious relationship between band number, location or intensity, and the clinical or parasitological status, except that there was an overall trend towards higher clinical severity scores in patients whose sera recognized more and more deeply staining bands. Specific patterns of band recognition with 1963 probes were not associated with Sowda patient sera, unlike previous reports. Anti IgE probes revealed fewer bands with lesser intensity, canpared to anti IgG, and they were also not peculiar to any patient subset. Overall, 9. volvulus infection and clinical consequences were not consistently associated with systemic deficits in cell mediated or humoral immunity. The demonstration of potent suppression of lympho- cyte reactivity by mf and 8/8 i_n 3E2 suggests that direct parasite intervention in host cell responses could be taking place i_n v_ivp_, perhaps at the local micro-environment level. TO NADIA WITH LOVE AND RESPECT iv ACKNWIEDGEMENI‘ It is with great appreciation that I would like to express my indebtedness to several individuals who helped me make this work possible. I am extremely grateful to Professor Jeffrey F. Williams for his enduring and skillful guidance of my Ph.D. program. The opportunities made available through him played an important role in my scholastic develognent. The scientific and intellectual exchanges we have had over the years, coupled with his consistent high standards will have a lasting hnpact. I would like to extend my sincere appreciation for the direction and encouragement of my committee menbers, Professors: James Bennett, James Jensen and waiter Esselman. Special thanks are due to Professor Charles D. Mackenzie, who helped in various parts of this project. His expertise in the immuno- pathology of onchocerciasis have helped me develop a better under- standing of the disease. Furthermore, I would like to thank Drs. Hashim W. Ghalib, Khalid ElSinary, Tracy Cotter and Rick Snith for their assistance in field and laboratory work, and BK. Jehn MCMahon for his invitation to the British Medical Research Unit at Bo, Sierra Leone . Thanks are also due to Ms. Denise Harrison for the time she so freely and willingly gave preparing this thesis and various manu- scripts. It was with untiring dedication that my wife, Nadia, and my parents Yousif Elkhalifa, and Safia Daoud gave their constant and invaluable support. Finally, I want to remember and acknowledge the willing participation of patients in the various remote areas of Africa, without whom these studies would not have been possible. Their endurance and tolerance helped us maintain the proper humanistic perspective in our research. This work was largely supported by funds from. the NIH-Sudan Medical Parasitology Project (NIH grant #AI-l6312) and was done in collaboration with the Medical Research Council and the Ministry of Health in Sudan. vi TABLE INTRODUCTION. . . . . . . LITERATURE REVIEW . . . . Geographical Distribution Life Cycle. . . . . . . Diagnosis . . . . . . . Clinical Presentation . Pathogenesis. . . . . . Pathology . . . . . . . Treatment and Management of OF CONTENTS Onchocerciasis. Immune Response in 9. volvulus infection. . RmEREmES. O O C O I O 0 ARTICLE 1 - SUPPRESSION OF HLMAN LYMPHCXIYTE RESPONSES TO SPECIFIC AND NON-SPECIFIC STIMULI IN HUMAN ONCHOCERCIASIS. ARTICLE 2 - IMMUNOGLOBULIN RESPONSES IN ObCHCXIERCIASIS - A COMPARATIVE STUDY OF IM‘iUNmLOT PROFILES. APPENDICES. 1. CLINICAL EVALUATION OF ONCHOCERCIASIS PATIENTS. 2. PATIENTS' DATA RECORD FORM. vii . 33 . 44 .105 .144 .144 .163 TABLE LIST OF TABLES ARTICLE 1 PAGE Clinical and parasitological status of Onchocerca volvulus infected subjects in four distinct foci endemic for onchocerciasis in Sudan and Sierra Leone 0 O O O O O O O O O O O O O O O O O O O O O O O O 70 Peripheral blood lymphocyte subset distribution in onchocerciasis patients and normal controls from Bahr Elghazal, Sudan, and Bo, Sierra Leone. . . . 78 Blastogenic responses of peripheral blood lymphocytes of 9. volvulus infected individuals and non-endemic area controls frcm Bahr Elghazal and sundus, Sudan. . . . . . . . . . . . . . . . . . . 80 Effects of sera from Q. volvulus-infected individuals and controls fran different geographical areas on lymphocyte blastogenic responses to phytohemagglutinin and concanavilin A. . . . . . . . . 82 viii LIST OF FIGURES ARTICLE 1 F IGURE PAGE 1 An onchocerciasis patient from the Bo area. . . . . . 72 2 Severe onchocercal dermatitis seen in a Sudanese patient 0 I O O O O O O O O O O O O O O O O O O O O O 74 3 A Sudanese patient with localized limb involve- mnt (soma) O O I O I O O O O O O O O C O C C O O O O 76 4 Effect of sera from Sudanese Q. volvulus infected and non-endanic controls on lymphocyte Blastogenic responsestomitogens................84 5 Inhibition of blastogenic responses of lymphocytes from 9. volvulus infected subjects and non-endemic controls from Sudan in the presence of 9. volvulus female Secretory/Excretory products . . . . . . . . . 86 6 Effect of Q. volvulus microfilariae Secretory/ Excretory products on lymphocyte blastogenic responsestomitogens................88 7 Effect of co-cultivation of Q. volvulus microfilariae on Sudanese 9. volvulus infected patients and controls lymphocyte blastogenic responsestomitogens................9l 8 Effect of serial dilutions of Q. volvulus female Secretory/Excretory products on lymphocyte blastogenic responses to mitogens . . . . . . . . . . 93 9 Effect of 9. volvulus Secretory/Excretory products and skin microfilariae on the production of Interferon Gamma by mitogen stimulated lymphocytes.....................95 ix FIGURE 10 11 12 ARTICLE 2 An asymptomatic Q. volvulus infected individual with microfilarial inten51ty greater than 100 Inf/mg O O I I O O O O O O O O O O O O O O O 0 Discrete papular eruption seen in this patient, confined to the upper back, giving him a clinical score of l . . . . . . . . . . . . Severe onchocercal dermatitis is seen on this patient (clinical score of 3) . . . . . . . . . . Acute severe onchocercal dermatitis (score of 3) is seen in this patient mostly in the form of acute papular eruption and excoriation marks. . . A "Sowda" patient with extensive dermatitis of the left leg while the right is spared . . . . Localized onchocercal dermatitis affecting the right arm of this patient . . . . . . . . . . . . Total immunoglobulin levels in onchocerciasis patients stUdi$O O O O O O O O O O O O I O O O 0 SDS-PAGE of 9. volvulus antigens and Secretions/ meretions O O C I O O O O O O O O C C I O O O O 0 IgG recognition pattern of Q. volvulus antigens separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose filters . . . . . IgE reactivities of the same group of patients in Figure 9A. 0 O O O O O O O O O O O O O O O O O 9. volvulus antigen recognition patterns by different IgG subclasses. . . . . . . . . . . . . Total 196 and IgG4 recognition pattern of .9. volvulus antigens. . . . . . . . . . . . . . . PAGE .116 .116 .118 .118 .120 .120 .122 .124 .126 .129 .131 .133 FIGURE 10 11 12 APPENDIX 1 An asymptomatic patient with an onchocercal nodule in the pelvic region . . . . . . . . . . A patient with localized severe onchocercal dermatitis and femoral lymphadenopathy. . . . . Severe acute onchocercal dermatitis with papular eruption, excoriation marks and pigmentory changes (score of 3) . . . . . . . . Discrete papular eruption localized to the upper back (score of l) . . . . . . . . . . . . Moderate acute changes (score of 2) . . . . . . Another Sowda patient in which the left limb is severely afflicted. . . . . . . . . . . A patient with generalized severe onchocercal demtitis. O I O O O O O O O O I O I O O O O O Marked atrophy of the skin, a consequence of chronic onchocerciasis. . . . . . . . . . . . . Severe secondary bacterial infection in a young boy with 9. volvulus infection. . . . . . . . . Classical depigmentory changes seen in both lower legs of this patient. . . . . . . . . . . Choreoretinal atrophy with focal pigmentation . Optic nerve atrophy in an onchocerciasis patient. xi PAGE .148 .148 .150 .152 .152 .154 .154 .156 .158 .160 .162 .162 INTRODUCT ION Onchocerciasis is a chronic parasitic infection caused by the filarial nematode Onchocerca volvulus. This disease afflicts some 20-40 million people world wide, and is considered a major cause of blindness in tropical Africa. Despite the morbidity and consequent economic losses, prevention, treatment and control of onchocerciasis lag behind other infectious conditions in the developing nations, where the disease is exclusively seen. Onchocerca volvulus occurs mostly in remote rural areas where the inhabitants have little politi- cal impact on the central decision making establishment. The loss of manpower because of blindness coupled with the migra— tion of people from fertile lands for fear of blindness has created severe economic difficulties in endemic areas. These factors initi- ated the implementation of a large control program in the Volta River Basin in West Africa. The program, now 10 years old, is directed towards eradication of the vector, the blackfly. This program has interrupted the transmission cycle, but at a very high price. Sus- tained financial support is needed, well over the 20 year plan pro- jected initially, in order to insure continued success. In other endemic foci, rudimentary chemotherapeutic regimens from the 1940's are the only available means to combat the disease. Treatment with both the macrofilaricidal drug, Suramin and the microfilaricidal agent ix) diethylcarbamazine (DEC) have serious, and on occasion, produce fatalities. This has limited their use by concerned medical person- nel, and results in their rejection by infected individuals, for fear of the agonizing side effects. The recent introduction of ivermectin as a macrofilaricidal drug is encouraging because fewer side effects are associated with it. However, caution is warranted until we have more experience with the drug and its side effects. At present we are confronted with a disease that carries high morbidity, though seldom fatal, for which there is no suitable treat- ment for the adult parasites. There is no convincing evidence of acquired resistance and little hope of any vaccine being developed. Prevention of the infection through vector eradication in most areas is impractical, because of the enormous expenditure needed. Explora- tion of the different immunologic mechanisms involved in the pathology and pathogenesis of Q. volvulus infection may bring about better understanding of the biology of the disease, ultimately leading to better treatment and preventive options. Experience from other infectious diseases for which we have developed better chemotherapeu- tic agents or vaccines, does not give reason for optimism in the fight against onchocerciasis. The logistical difficulties encountered in delivering "magic cures" to patients in remote endemic rural areas remains a challenge. The work contained in this thesis involved studies on both cell mediated and humoral immunity in onchocerciasis in patients in Sudan and for comparative purposes, in Sierra Leone, W. Africa. The epide- miological and biological background to _(_)_. volvulus infection in these regions is reviewed below, along with an overview of the current state 3 of knowledge of the pathogenesis and host-parasite relationship. A brief account is provided of the problems of chemotherapy and control, and of the most recent findings in the areas of cell-mediated and humoral responses to 9. volvulus. LITERATURE REVIEW Geographical Distribution Onchocerciasis occurs throughout the greater part of tropical Africa. It extends from Senegal on the west coast to Ethiopia in the east. Poci endemic for the disease are also seen as far south as Malawi in Africa, and in Central and South America. In the Middle East, the disease is confined to the Arabian peninsula. For the most part, onchocerciasis endemic foci are located in rain forest and Savannah regions. However, occurrence of the disease outside those two climatic regions is becoming recognized now as more careful epidemiological surveys are conducted. One unexpected focus for onchocerciasis is in Abu Harmad in northern Sudan. The disease was discovered in a dry arid region of the Nubian Desert in 19581. It is not unlikely that additional foci will be discovered and the disease should be suspected wherever blackflies of the Simuliurm species are found. a. Onchocerciasis in Sudan: Probably the first clinical description of the disease in Sudan was in 1908 by E‘nsor in Bahr ElChazal provincez. Later the disease was termed Jur blindness since it was contracted along the banks of River Jur3. Subsequently, additional foci were discovered in Upper D Nile4, Southern DarfurS, in Kasala Province in Eastern Sudan6'7 and in Abu Hamad in the Northern province8. Thus, the distribution of onchocerciasis in Sudan is wide, encom- passing different climatic regions, from the rain forest in the south to the desert in the north. Each geographical area has its peculiari- ties regarding the infection intensity and clinical manifestations. It is estimated that about 1 million Sudanese (5% of the popula- tion) are infected with Q. volvulus. The disease is most prevalent near seasonal fast running waters which constitute optimal breeding sites for the vector, the blackfly. The vector in the South and Southwest has been definitively identified as Simulium damnosum silo. There is a single report about infected _S_. damnosum from the Abu Hamad areall, however, subsequent workers were unable to substantiate this. No entomological studies to identify the vector along the Atbara River area were carried out. Clinically, the dermatological manifestations can be seen in all foci, but the Atbara River focus offers the most severe presentation7. It is believed that eye lesions, culminating in blindness, are more severe in the southern part of Sudan with exceptionally few cases of eye involvement or blindness in the Abu-Hamad and Atbara River foci. Factors which may contribute to differences in clinical presentations include genetic backgrounds, parasite strain variability, length of the transmission season and intensity of infection. b. Onchocerciasis in Sierra Leone: Two important benchmarks in the history of onchocerciasis were established in Sierra Leone. Qichocercal nodules”, were first described there, and the life cycle of 9. volvulusl3 was unravelled. 6 The ecological system in Sierra Leone was primarily rain forest, but because of the intense deforestation and clearance of land for farm- ing, not much of the original primary forest remains unaltered. Numerous foci of onchocerciasis exist in the Northern, Eastern, and Southern provinces. Around the Bo area, in the South, onchocerciasis is prevalent in villages along the tributaries of the Tabe and Sewa Rivers”. Detailed entomological, clinical, and epidemiological surveys of this area by the British Medical Research Council staff and the Sierra Ieonian Ministry of Health showed an overall prevalence of 68.6%”. Clinically, there are relatively mild skin and eye lesions, but there is an unusually high rate of blindness (7.5%), the explana- tion for which is not entirely clear. Pour species of the _S_. damnosum complex are implicated as vectors for the disease in the Bo area. Onchocerciasis transmission occurs year-round, with a peak during the rainy season (October to December), when 80% of the transmission takes pl acel4 . Life Cycle Qichocerca volvulus is transmitted by blackflies of the genus Simulium which act as an intermediate host. Although closely related parasites are seen in mammals, humans are the only definitive hosts for _Q. volvulus. Several species of Simulium have been shown to transmit the infection, but , S. damnosum complex members are the main vectors, especially in Africals. Others such as S. ochraceum and _S_. 7 metallicum play a major role in transmission of the disease in Central and South Americals. Simulium are small, strongly built flies, the females of which bite preferentially on animals or birds in order to obtain a blood meal needed for oogenesisl6. The male flies do not bite, and hence do not participate in the transmission of the parasite. When the female feeds on an infected individual she ingests skin microfilariae along with the blood. Upon reaching the stomach, microfilariae penetrate the abdominal tissues and go into the thoracic muscles where they undergo maturation and metamorphosis, transforming into the infective larvae. This may take 6-10 days, depending mainly on environmental temperatures”. The infective larvae then migrate to the proboscis. When the fly bites again, it deposits these infective larvae along with saliva, which contains an anticoagulant. The larvae then prob- ably migrate in the subcutaneous tissue, and develop into mature male or female worms, usually within 12 monthsls. The female worms elicit a granulomatous inflammation around them, and with time they became encapsulated within a mesh of fibrous tissue and inflammatory cells forming what is known as the onchocercal nodule. While the females have relatively restricted mobility within the human body, the male worms are believed to travel from one nodule to the other. Fertilization of eggs occurs following mating of a male and a female worm. The result of this is the periodic release of thousands of microfilariae by the female worms9'19. Microfilariae are found in the skin and subcutaneous tissue and they can travel to the eye. Microfilariae and the inflammatory responses directed against 3 them, not the adult worms, are believed to be the major cause for the pathologic events seen in the host. The life span of adult 9. volvulus, although not known with certainty, is estimated to be in the range of 15 to 20 yearszo. The adult female is a white to tan, threadlike, worm, between 25 and 70 cm in length, and 1/3 mm in diameter21. The male, though similar in appearance, is much smaller in size, measuring up to 4 cmlin length21. The microfilariae are 220—360 u long and 5-10 u in diameter”, and their life span is estimated to be about 6 month323. Breeding of blackflies takes place in well aerated fresh running waters. The mature female deposits about 200—400 eggs at a time on aquatic plants or submerged rocks and vegetation24. Four to thirty days are needed for the larvae to hatch fram eggs, temperature being the major influencel6. The larval stage is the target for control measures against black flies. The larvae then develop into pupae within 10 days, and the pupae mature into adult flies within 2-4 days. The longevity of blackflies is not fully known, although some may live for up to 180 dayszs. It is suggested that flies infected with Q. volvulus live for a much shorter time than non-infected flies. The flight range is variable and can reach >400 mileszs, though infected flies are thought to have a much reduced flight range, possibly because the parasite is located within the thoracic muscles. The large flight range has created problems of re-invasion of onchocer- ciasis control program regions from neighboring uncontrolled areaszs. Besides being responsible for transmission of .9. volvulus, blackflies inflict damage on man and animals by their vicious biting 9 behavior. People easily become sensitized to blackfly bites and on subsequent exposure can develop both immediate and delayed hypersensi- tivity reactions”. This is thought to contribute to the skin pathology seen in onchocerciasis. Animals may experience fatal anaphylaxis or bleed to death if bitten by large numbers of blackflie527'28. Thus,- the idea of eradicating the vector as a control measure for the spread of onchoc- erciasis is appealing, since human morbidity and animal mortality caused by blackflies can also be eliminated. However, because of the nature of their breeding sites, often in small seasonal streams over wide geographical areas, control of blackflies has proven difficult and expensive . Diagnosis a. Detection of the Parasite: The most reliable method for the diagnosis of Q. volvulus is the demonstration of microfilariae in a bloodless skin snip. The simplest way is by using a needle and a sharp double edge razor to obtain the snip._ Care should be taken not to have a bloody specimen since confusion may occur if the blood contains microfilariae of 2. Erstans or _ng _l_<_)_a_. The snip can be incubated in normal saline or tissue culture medium, at ambient temperature, for l/2 hour to 4 hours. Increasing the incubation period gives a better chance for microfilar- iae to emerge from the skin snips of patients with low microfilarial densities.29 The use of a corneoscleral punch is recommended in 10 research surveys, and in large medical centers because it provides relatively uniform samples. However, because of the expense and the need for frequent sharpening it may not be suitable for use by para- medical staff in remote endemic foci. For diagnostic purposes several skin biopsies from the pelvic and subscapular areas should be obtained. A negative skin snip does not necessarily exclude infection and additional snips should be obtained and tested. The probability of obtaining a negative skin snip in patients with low microfilarial densities (less than 2.5 mf/mg) is about 50%”. In such patients repetition is recommended. In addition, DEC provocation test have been advocated to confirm clinical impressions in such patients. The test consists of administration of DEC orally or in the form of topical lotion29'3l. The patient is then observed for development of the characteristic maculopapular eruption which is part of the Mazzotti reaction, described as a side effect of DEX: therapy 32. The latter procedure, since it entails risks, should be reserved for patients who consistently prove to be skin snip negative, but having the characteristic clinical stigmata of onchocerciasis. Identification of subcutaneous onchocercal nodules constitutes an important part of the clinical and parasitological evaluation of patients. This can be achieved by visual inspection and palpation for the nodules, usually in the pelvic region. Demonstration of Q. volvulus adult worms after surgical removal of nodules constitutes definitive diagnosis of _g. volvulus infection. Recently, a non-invasive method for detection of 9. volvulus nodules using ultrasound33 has been described. However, the practical utility of such a method remains doubtful. 11 b . Immunod iagnosi s: It is obvious that detection of microfilariae, though it has a high specificity, is not a very sensitive method. False negatives are seen in prepatent individuals and in patients with low microfilarial densities, either due to low levels of infections or strong immuno- logic responses resulting in destruction of microfilariae. Scientists have thus explored more sensitive procedures, i.e. immunologic diagno- sis. Different formulations of immunological assays have been listed for detection of anti-onchocercal antibodies. These include hemogluti- nation34, complement fixation35, radioimmunoassay36, and enzyme linked immunosorbent assays37, all of which utilize _g. volvulus crude antigenic extracts for the detection of specific anti onchocercal antibodies. Obviously, one major problem of this approach is that it lacks specificity. The extensive cross reactivity between 9. volvulus antigens and other filarial antigens makes it difficult to differen- tiate between patients infected with O. volvulus from those infected with other filariae or helminths38. In addition, the mere presence of anti-onchocercal antibodies does not necessarily indicate infection. For example, exposure to the infective stage, which may not develop into an adult worm, can give rise to an antibody response. Because of this, recently the trend has been towards detection of worm antigens in serum, urine and other body fluids39'40'4l. This approach is attractive since the presence of worm antigens is indicative of active infection. Theoretically, detection of Q. volvulus antigens should have high specificity and sensitivity, especially when one utilizes monospecific antisera directed against unique worm antigens. The limited experi- 12 ence with Q. volvulus antigen detection has shown that the sensitivity is interfered with by the presence of host antibodies directed against those unique antigens, thus giving false negatives“. The use of crude adult worrm antigenic extracts'a‘2 and microfilarial excretory and secretory products43 for skin hypersensitivity reactions did not meet with much success. More work is obviously needed in the area of immunologic diagnosis to come up with an acceptable, reliable, and practical assay. Recent advances in molecular biology which culminated in the identification of _g. volvulus specific DNA probes are encouraging and may offer more sensitive and specific approaches to the diagnosis of Q. volvulus in the future44'45. Clinical Presentation Infection with _g. volvulus results in a spectrum of clinical manifestations. Many variables can affect the outcome of the disease, including host immune response, exposure rate, age and strain varia- tion. Although in most textbooks and publications the disease is often presented in its most severe form, a single visit to an endemic area would convince anyone of the concept of spectrum. Generally the clinical presentation ranges from completely asymptomatic individuals to those who have severe skin and eye manifestations. Different categories exist between these two extremes. 13 a . Dermal Changes: Skin changes are best classified into two main categories46: 1. Acute changes: (he of the early skin changes is the development of a papule, an event signifying an inflammatory reaction around dead or dying micro- filaria47'49. Papules can be discrete and their number and distribu- tion may vary. Sometime, they may coalesce together. The development of papular eruption is usually accompanied by itching, and self inflicted injuries, from excoriation, is a common finding in onchocer- ciasis. The injured skin surface may get secondarily infected, thus aggravating the initial insult to the skin. The papules may disappear completely without leaving any mark of their previous existence or they may become indurated, depigmented initially and hyperpigmented later47'48. Death of large numbers of microfilariae either naturally or secondary to chemotherapy, e.g. Mazzotti reaction32, leads to the development of large numbers of papules accompanied by edema of the skin. The acute changes seen in onchocerciasis are readily reversible. However, with time, repeated insults to the skin lead to the more characteristic chronic changes. 2. Chronic changes: These are thought to result from repeated local pathologic events in the skin evoked by dead or dying microfilariae. The chronic clinical stigmata of onchocerciasis include dermal atrophy with loss of elasticity and thinning of the epidermis“. The skin becomes wrinkled, assuming the appearance of old age in afflicted patients. 11+ Pigmeit loss, especially on the skin overlying the tibia is character- istic, giving the name of leopard skinso. In some patients marked thickening of the skin (hypertrophy) is seen prior to the development of skin atrophy. The hypertrophied skin may also be edematous and darker in color. This may be an intermediate stage between the acute changes and the more permanent chronic changes. It may result from the continuous, repeated itching which can lead to lichenification of the epidermis. In addition, the inflammatory response in the dermis is often accompanied by increased fibroblastic activityso. Thus, thickening of the skin results from thickening of both the epidermal and dermal layers of the skin. Patients differ in the degree of skin involvement. Both acute and chronic changes can be seen within the same patient, in distinct body areas49. Some patients may show predominantly acute changes while others may exhibit only chronic changes. Furthermore, some patients may show no evidence of disease, and can only be diagnosed as 9. volvulus infected during routine epidemiological surveys. An interesting form of onchocercal dermatitis is known as Sowda (Soda), the Arabic word for black pigmentation. This was first described in Yemen51, but, similar forms exist in virtually all onchocerciasis endemic foci. Sowda is defined as an asymmetric limb involvement, usually of the legs, where pronounced acute and chronic changes are seen. Most prominent changes are edema, papular eruption and hyperpigmeitation together with hypertrophy of the skinsz. This is associated with enlarged non-tender lymph nodes in the region draining the afflicted limb. Skin biopsies from such patients usually p— 13 yield no microfilariae, and the diagnosis is often made on clinical grounds. In the author's opinion the definition of Sowda may need some modification from the exclusivity of single limb involvement to a localized severe reaction in one limb not precluding the presence of other milder skin changes in other parts of the body. Our experience with Sowda cases in Sudan shows that most patients demonstrate true papular eruptions in other parts of the body, especially the back, in addition to the presence of severe localized disease in one limb. Another clinical presentation related to the skin is the hanging groin. This is a consequence of regional lymphedema and skin atrophy leading to loss of the elastic tone of the skin. Consequently the dependent structures in the femoral and inguinal regions gravitate downwardsS3 . b. Ocular Changes: Q. volvulus microfilariae can invade the human eye, sometimes in large numbers. The ocular changes seen in patients can be divided into temporary and permanent changes. These changes are pathologic consequences to the presence and/or death of microfilariae within the eye. Both anterior and posterior chamber lesions can be seen. Banc- tate keratitis ("fluffy opacities") are seen in the cornea and are caused by an acute inflammatory reaction around dead or dying microfi- lariae54'55. This usually resolves within a few weeks with no permaneit consequence. A serious and permanent form of eye damage is sclerosing keratitisSG. This starts in the limbus in the form of haziness and extends into the corneal epithelium accompanied by pigrmentary changes. Later it changes into opacification extending into 16 the central cornea. Soon extensive scarring with neovascularization is seen all over the cornea. At this stage, the transparent cornea is essentially replaced by fibrous tissue which does not permit passage of light. Blindness is inevitable. Other changes within the anterior chamber include anterior uveitis leading to development of synechiae and iris deformity, iritis and cataract. Posterior segment changes are becoming more recognized as an important cause of blindness. They vary widely from a feW'depigmented retinal spots to severe Choreoretinal changes. Initial changes secondary to acute inflammation are seen as pale edematous areasss. These are followed by focal areas of hypertrophy which precede cho- reoretinal atrophy and clumping of retinal pigmeit57. Eveitually subretinal fibrosis occurs. Reduction in vision is related to the extent of retinal lesions. When the lesions become extensive and confluent total loss of vision occurs. The optic disc and nerve are not spared57. Optic neuritis and optic atrophy are commonly seen in onchocerciasis. Many literature reports suggest that optic nerve changes can be consequent to diethylcarbamazine therapy, and that the damage is induced by microfilarial death57'58. However, it is known that DEC induced damage is an acceleration of a biological process, induced by the occasional death of microfilariae. The natural process of optic neuritis and atrophy may already be occurring, but at a slower rate. This concept remains to be investigated. 17 c. Nodules: These are firm, subcutaneous tumors composed of adult worms, entrapped and encapsulated within a fibrous stroma. Onchocercal nodules are firm in consistency, rounded and readily movable. They are often found on bony prominences of the pelvic girdle, ribs and cranium. Nodules by themselves do not cause any significant medical problem. However, their removal is clinically justifiable since it will reduce the overall worm burden, and consequently the number of microfilariae produced. Multiple worms can be found within each single nodule and there is a proportional increase in the number of worms within a nodule as the patient's age increases”. Although onchocercal nodules are commonly seen within the subcuta- neous tissue in association with the musculo-skeletal system, unusual internal locations, such as in the wall of the aorta, have been reportedsg. d. Lymph Nodes: Enlarged superficial lymph nodes which may be fibrotic are seen in onchocerciasis patients. These are usually regional lymph glands that drain an affected areas (e.g. limb). They are usually non-tender, except following treatment. e. Other Manifestations: Onchocerca volvulus microfilariae were found in many of the deep organs including the liver, kidney, spleen, pancreas, lung, peripheral nerves, and arteriessg. The significance of such "deep organ 18 invasion" is not clear, but there is little evidence that it contrib- utes to the clinical picture of the disease. It probably follows DEC therapy. Pficrofilariae can also be found in blood60, urine60'6l, sputum62, vaginal secretions63, peritoneal fluid64, and cerebrospinal fluid65'66. The latter finding, coupled with clinical observations of epilepsy and dwarfisim, have led to the belief, by some investigators, that microfilariae may invade the brain and pituitary gland67'68. These suspicions, however, remain to be substantiated scientifically. Pathogenesis Most host tissue damage is believed to be a consequence to an inflammatory response directed against the microfilariae69'46. The adult worms only elicit localized granulomatous reactions in the subcutaneous tissue resulting in onchocercal nodules. The female worm periodically releases thousands of microfilariaeg'19 which migrate to the skin and subcutaneous tissue as well as the ocular tissue. Mechan- ical destruction of host tissue by microfilariae, especially in the eye has been proposed; however, little evidence exists to that effect. Other possible mechanisms of direct tissue damage could be through the production of a collagenase-like substance secreted by the worms”- This may play a role in the pathogenesis of skin atrophy where one finds degenerating collagen bundles. The most important pathologic changes, however are due to the inflammatory responses directed 19 against dead or dying microfilariae, and these are a function of the host immune system46. Two theories currently exist as to why live microfilariae are protected from the host immune system. The first deals with the masking of“ microfilarial surface antigens by host proteins49'7l. Nficrofilariae are then recognized as self by the host immune system, and no inflarmatory response would be evoked. However, it has been shown that live microfilariae _i_n_ m have IgE irmmunoglobulins in their surfaces indicating that host proteins may not mask all the antigenic sites. Interestingly, the preferential binding of IgE to live microfilariae may play a protective role in microfilarial avoid- ance of the immune system, since _i_n_ M studies have shown that eosinophil mediated, complement enhanced killing of microfilariae is dependent on 196 and not IgE72. The other mechanism of avoidance could be through active suppression of the host immune system. Micro- filariae of Br_ugi_a produce an irmmunoinhibitory substance(s) which suppresses lymphocyte responses to antigens and mitogens as well as causing marked reduction in the production of lymphokines73. DEC and ivermectin do not kill microfilariae _ig yi_t_r_o_, even at high non-physiologic concentrations74'75. Hewever, ig_yiyg, they lead to rapid killing and disappearance of microfilariae accompanied by hnmunologic reactions (e.g. Mazzotti reaction)32'76. The mechanism of mucrofilarial killing by these two agents, though poorly understood, is thought to be immunologically mediated. Possibly their mode of action could be through inhibition of suppressor production by micro- filariae. 20 The inflammatory response directed against the microfilariae lead to destruction of microfilarae as well as surrounding host tissue. The killing is mostly by eosinophils where they first attach to the microfilarial surface and then degranulate, expelling their toxic granules into the surface of microfilariae as well as the surrounding host tissue73'103. The degree of pathologic changes seen is a function of the magnitude of the host inflammatory response. Thus, in a philosophical sense, it is better not to mount any inflammatory response. In such a case, both the host and the microfilaria remain viable and relatively healthy. Another factor that contributes to the pathologic changes seen in the skin is the intense excoriation and itching seen in Onchocerca volvulus infected individuals. This, when repeated, can lead to skin damage. The self inflicted trauma is often followed by secondary bacterial infection which causes more skin damage. The role of blackfly bites in the pathogenesis of skin lesions is becoming more recognized. Blackfly bites cause intense hypersensitivity reactions which may lead to additional disfigurement of the skin27'28. The association of blackfly bites and leopard skin has been noted”. The involvement of inmmune complexes in the pathogenesis of onchoc- ercal lesions remains unclear’ig'79'80, as does the role of autoantib- odies described recently as possible causes of the development of eye pathology8l'82. Thus, to summarize, the most important cause of pathologic changes is the host inflammatory response directed against the microfilariae. Excoriation and blackfly bites play additional roles in development 21 of skin damage. Mechanical damage caused by migrating microfilariae may cause tissue pathology in ocular tissues. Pa tholggy a. Skin: The histopathologic changes seen in onchocerciasis usually parallel the clinical picture. In asymptomatic patients microfilar- iae are seen in the upper dermis free of any inflammatory infiltrate. At times mild perivascular cuffing composed predominantly of lympho- cytes, plasma cells and macrophages is seen around small blood vessels in the upper and deep dermise583'84. With acute clinical changes, one sees prominent perivascular cuffing and the cellular infiltrate would include in addition eosinophils and mast cells, 47'77. Dermal edema with separa— some of which may be degranulating tion of collagen bundles as well as hyperkeratosis, focal parakerato- sis, and acanthosis have been described”. Microscopically the papule represents an intraepithelial microabscess containing frag- ments of microfilariae and varying numbers of lymphocytes, macro- phages and eosinophils77'85. Eosinophils, either intact or degranu- lating, are seen in close proximity to the microfilariae“. In chronic disease there is thinning of the epidermis with flattening of the rete ridges. Collagen in the upper dermis is disrupted and later becomes fragmented. This is accompanied by increased deposition of mucinous ground substancesBS. Increased fibroblasts and fibroblastic activity may be seen in initial chronic 7’7 as changes; however, in later stages fibrosis may ensue83. Microfilariae then are seen mostly in the deep dermis. Macrophages, laden with melanin pigment may also be noted in the upper dermis47'50. The clinical entity of Sowc‘la is characterized by an extensive diffuse inflammatory infiltrate involving both the upper and deep dermises. The cellular infiltrate is composed of lymphocytes, macrophages, eosinophils, arnd plasma ce11586. The latter is seen predominantly surrournding dermal appendages and blood vessels. Microfilariae are rarely seen in tissue sections taken from Sowda patientssz. Edema may also be prominent as well as dilatation of lymphatic vessels86. The epidermis shows hyperkeratosis and parakeratosis, arnd increased pigmentation of the basal layer of the epidermis is also seen86. b. The Nodule: The onchocercal nodule is essentially a granulomatous reaction directed against adult worms, mainly females. The cellular infil- trate in the nodule is composed of mononuclear cells and eosinophils and with time, a thick fibrous capsule develops and the worm becomes entangled within fibrous septae47. Death of adult worms within nodules is accompanied by caseous necrosis and the center of the nodule may become liquified. An intriguing question is how the adult worm derives its nutrients within the nodule and whether the nodule constitutes a barrier that offers protection to the worm from systematically administered chemotherapeutic agents. It was initially postulated that the worms derive their nutrient supply by causing microhemor- 23 rhages through erosion of blood vessels in the vicinity87. Smith et al.88 elegantly demonstrated the fine capillary network surrounding worms within nodules. They fournd no evidence of extravasation of blood or erosion of blood vessels. Neuvascularization was evident in nodules, suggesting that the worm may be producing an angiogenesis stimulating factor”. It is apparent then that the worm gets its nutrients directly from the vascular network surrounding it, likely by passive diffusion. The same mechanism can be responsible for helping the worm excrete its waste products. This intimate contact with the host blood makes the worm vulnerable to chemotherapeutic agents. Chemotherapeutic agents such as chloroquine arnd ivermectin can readily be found in nodular tissue and worms (Williams, J.F., personal communication). At the same time secreted worm products and shed antigens may have access to the systemic circulation of the host . Pathologic changes in the eye are generally similar to those seen in the skin. These changes are again thought to be a consequence of microfilarial death either naturally or by chemotherapy. Unlike the skin, there is a scarcity in our knowledge about eye pathology and the few scattered reports on eye pathology deal mostly in end stage disease. TWO pathologic events can be seen in the cornea. The first is punctate keratitis, where there is focal collection of lymphocytes arnd eosinophils around dead or dying microfilariae89. This localized inflatmatory reaction usually resolves without any sequelae within 3 weeks. The second corneal change is sclerosing keratitis. Here 24 there is development of fibrovascular pannus between the Bowman's membrane and the corneal epithelium which may become hyperplas- tic47'89. The translucent cornea becomes opacified. Sclerosing keratitis, when advanced, can lead to blirriness. Inflammation of the uveal tract is also seen in onchocerciasis. It can occur as part of the natural disease or following microfilar- icidal therapy. It is characterized by the presence of inflammatory cells in the iris and ciliary body accompanied by hyperpigmentation due to increased activity of the iris pigment epitheliumgo. Later depigmentation is seen as well as hyalinization of small arteries and arteriolesgo. A spectrum of Choreoretinal changes can be seen. Focally, one may see collections of inflammatory cells, eosinophils, and plasma cells where the underlying epithelium becomes atrophicgl. Pigmentary changes in the form of pigment migration and clumping may also be seen. In advanced disease, profound Choreoretinal atrophy with loss of photoreceptors, bipolar cells and ganglion cells is evident89. The latter stages are often accompanied by optic atrophy and severe restriction in vision or total blirndness is the usual outcome. d. Lymph Nodes: Lymphadenopathy is commonly seen in onchocerciasis patients. TWO types of pathologic changes can be seen in lymph nodes. The first is mostly seen in patients with long standing chronic features and include various degrees of fibrosis. This usually begins at the capsular area and gradually extends into the interior of the lymph node92. The fibrosis is accompanied by atrophy of the germinal 25 centers and in some series, up to 80% of the lymphoid tissue is replaced by tough scar tissue92. This gives lymph nodes their characteristic discrete rubbery feeling. Microfilariae are commonly seen in such lymph nodes (in 75% of patients), located mainly within the fibrous capsule or interstitial connective tissue. The second histopathologic feature is an enlarged soft homogenous lymph node which microscopically shows marked follicular hyperplasia, and enlarged germinal centers. This is commonly seen in the acutely reactive patient, and is characteristic of Sowda patients”. Microfilariae are rarely seen in such nodes. The pathologic events occurring in lymph nodes, though not well understood, are again thought to be precipitated by inflammatory responses, directed against dead or dying microfilariae. The consequence of this, in the long term, is probably fibrous replace- ment of the lymphoid tissue. This process is mostly seen in inguinal lymph nodes; however axiliary arnd occipital lymph nodes can show similar pathologic changes. The fibrosis can eventually lead to impediment of lymph drainage which may lead to hanging groin53 arnd elephantiasis of the genitalia94 and limbsgs, seen occasionally in onchocerciasis . e. Deep Organs: There is only a single case report documenting the presence of Onchocerca volvulus microfilariae in deep organs at autopsy in a patient who died after Diethylcarbamazine therapy59. Sections from 26 the liver, kidney, pancreas and lungs all showed 9. volvulus microfilariae. This is most likely secorndary to the effect of DEC in mobilizing microfilar iae . Treatment and Management of Onchocerciasis a. Symptomatic Treatment and Treatment of Superimposed Secondary Bacterial Infection96: Severe pruritus caused by the disease can be relieved by the use of antihistamines. This kind of treatment can reduce self—inflicted injuries. Secorndary pyogenic infections of the skin seen in onchoc- erciasis can be prevented by adequate treatment of infected lesions by antibiotics . b. Specific Antiparasitic Drug Therapy: At present, specific drugs available for the treatment of onchoc- erciasis are not satisfactory”. They are often associated with adverse effects, some of which are severe and can be fatalge. Ironically, these may be the result of parasite death caused by the effective anthelmintic drug. An important sequel to treatment is the possible deterioration of vision which may ultimately lead to blind- ne5358'99. Anti—onchocercal drugs in general, and microfilaricidal drugs in particular are thought to accelerate the natural processes of para- site death74'96. Until recently, the anti—onchocercal drugs available had many undesirable side effects, which made them unsuit- able for mass treatment in any chemotherapy based control measure. 27 The recent introduction of ivermectin, with its milder side effects has renewed the hope of combating the disease by specific chemother- apy. Only 3 antiparasitic drugs are available now for clinical use in the treatment of onchocerciasis. These are diethylcarbamazine (DES) and ivermectin which are microfilaricidal drugs, and suramin which is a macrofilaricidal drug with some microfilaricidal action. 1. Diethylcarbamazine (DEC): DEC was discovered in 1947100. It has considerable activity in killing the microfilariae of Onchocerca volvulus but it has no action against the adult wonmlm. DEC is given orally and within a few days can lead to elimination of more than 80% of the microfilarial load, and patients often turn from positive to negative on skin snip 102. tests However, the microfilariae start to reappear again within 2-3 months, since the adult worm is still alive and capable of producing new microfilar iaelm" 102. The microfilaricidal mode of action of DEC in onchocerciasis is poorly understood. In lymphatic filariasis, DEX: leads to mobiliza- tion and rapid disappearance of circulating microfilariae from the blood to the liver and spleenml'loz. There, the microfilariae are phagocytised by the reticuloendothelial system calls. In onchocer- ciasis, where microfilariae are found predominantly in the skin arnd subcutaneous tissue, DEC leads to mobilization of microfilariae from the dermis to the epidermis, blood stream and urine60'74. _I_t_n_ _v_it_r_g_ studies have shown convincingly that DEC by itself does not kill microfilariae”. _Ig vivo DEC may promote cell mediated attack on the 28 parasites77' 103 . Addition of DEC to immune serum and white cells leads to increased cell adherence and killing of the microfilar- iae104. However, the concentrations of DEC used in these experiments were well above those which are pharmacologically active i_n y_iy_g. DEC is a rather safe drug when given to normal individuals. The side effects seen in onchocerciasis are directly related to its microfilaricidal action. In the characteristic Mazzotti reaction seen with DEC treatment”, the rapid killing of microfilariae in the skin, subcutaneous tissue and the eyes, results in severe allergic- like reactions. Intense itching occurs shortly after drug adminis- tration, accompanied with watering of the eyes. Hours later a strornger reaction takes place in the form of urticaria, fine maculo- papular rash, and edema of the skin, especially over the buttocks, thighs, and genitalia, together with swelling arnd tenderness of inguinal lymph nodes. Pyrexia, headache, muscle pains and joint aches, arnd tachycardia are all cormon. If onchocercal eye involve- ment is present it may become worse58'99. Death of microfilariae in the anterior segment can give rise to punctate keratitis, which is reversible; however, serious, irreversible posterior segment lesions demonstrable by fluorescein angiography can occur58. Different forms of topical application of DEC were tried in an attempt to minimize the side effects of treatment105'106'107- Lotions, creams and eye drops were used, but, unfortunately, the reactions observed in these experiments were worse than oral adminis- trationloe'loa. In spite of all these problems, DEX? remains a potent, commonly used microfilaricidal drug. It is cheap arnd affordable. However, 29 the occurrence of the Mazzotti reaction makes it less attractive and many of the patients abarndon therapy because of the reaction. 2. Suramin: Suramin is the only available approved macrofilaricidal drug. It is effective in killing adult 9. volvulus. Snramin was first discovered in 1920109. It was then used for the treatment of African trypanosomiasis. It was first used in the treatment of onchocer- ciasis in 1947. Snramin is poorly absorbed from the gastrointestinal tract and should only be given intravenously. Intramuscular and/or subcuta- neous injection cause intense local irritation, severe pain and possible sloughing and abscess formation at the site of injection. It strongly binds to plasma proteins and is slowly metabolized leading to a lorng half life (30 days)llo. The drug, bound to plasma proteins, is taken up by the reticuloendothelial system cells and proximal convoluted tubules of the kidney. The latter process might be responsible for the frequently observed renal damage in patients treated with suraminlog'uo. Suramin is a toxic drug. Reactions are more common in malnour— ished people, especially if they have hypoproteinemia. Since excre- tion of drug is slow, toxicity may be cumulativeuo. Toxic reactions during therapy are classified by Hawking109 into: I. Immediate reactions: These include nausea, vomiting, collapse, shock, sweating arnd loss of consciousness. These are best II. III. IV. 30 avoided by slowly injecting the drug and starting treatment with a small dose. Late reactions: These occur at 3-24 hours. Signs include pyrexia, photophobia, uveitis, lacrimation, abdominal disten- sion and cutaneous hypoesthesia, and are thought to be due to the nicotinic effect of the drug. Delayed reactions: These are the most conmon. The most frequent is albuminuria and cellular casts in urine, due to kidney tubular damage. Because of this serious toxic effect, examination of renal function, at least in the form of a urine-general exam, is essential before administering each weekly dose. Other less conmon components in the delayed late reaction category include exfoliative dermatitis, stomatitis and rarely jaundice. Allergic reactions: Some of these are thought to be due to the microfilaricidal action of the drug, similar to those seen with DEC. They include urticaria and pruritus, accompanied by vesicular rashes. Other reactions, thought to be primarily due to the macrofilaricidal action of the drug include tenderness and swelling around nodules containirng the adult worms. With all these unwanted side effects of Suramin, the drug remains the only available compound that offers cure for onchocerciasis. Almost 40 years have elapsed since it was first used in onchocer- ciasis, but we still lack knowledge about the pharmacokinetics, tissue distribution and effective blood levels needed for its macro- filaric idal action. 31 3. Ivermectin: This is a semisynthetic macrocyclic lactone produced by the actinomycetae, Streptomyces avermitilis. It is extremely active against a wide variety of parasitic nematodeslll'llz. Although it has no detrimental effects on adult filariae in general, it proved to be active against _O. volvulus microfilariaell3. When administered orally in a single small dose of 100-200 ug/kg body weight, it can lead to killing of microfilariae and patients can remain microfilar- iae-negative after treatment for up to 9 monthsll3. It is claimed that the side effects of the drug are less severe than those of DEC, manifested in the form of a milder "Mazzotti" reactionll4. However, recently, moderate to severe reactions were noted in patients from Sierra Leonells. In addition, a possible association between ivermectin therapy and coagulation disorders was suggested in a small number of Sndanese patientsll6. Further tasting of the drug is needed in order to exclude potentially serious side effects and evaluate efficacy on a larger scale, and in different geographical locations. The microfilaricidal action of the drug is not well understood. It has been proposed that it acts by potentiation of gamma- aminobutyric acid (GABA) action thus reducing muscle membrane poten- tial of the wormll7. In manmals, CABA is a neurotransmitter found only in the central nervous system, while in nematodes GABA is a neuromuscular transmitter. Hence, ivermectin may lead to inhibiton of excitatory and inhibitory post-synaptic potentials and paralyis. This action, however, does not seem to apply to Q. volvulus microfilariae i_n vitro, since the drug, at physiological concentra- 32 tions does not have readily detectable effects on the motility of microfilariae. Recently, microfilarial emergence from skin snips following ivermectin therapy was assessedlla. No difference in emergence was found between biopsies taken from treated or untreated patients. Thus, the hypothesis of an ivermectin-paralyzing effect on microfilariae is weakened further. Ivermectin has stimulated a lot of enthusiasm since clinical trials started, and the overwhelming success of ivermectin therapy in West Africa prompted the World Health Organization to encourage cotmunity based treatment programs. It is expected that large scale use of the drug in the treatment of onchocerciasis will be commence in many endemic areas, with the hope that the use of ivermectin may aid in the interruption of the trans- mission cycle121 . c. Nodulectomy: Surgical removal of onchocercal nodules from patients is a justifiable procedure since it reduces the worm burden in the body. Nodulectomies do not lead to complete cure because nodules may be deeply buried in the muscles and behind big jointsll9, where they are inaccessible for surgical removal. In endemic areas, for the proce- dure to have a measurable effect, new nodules have to be removed yearly. In South America, removal of head nodules is routinely practiced because it is thought to reduce the number of microfilariae invading the eyes. 33. d. Prophylaxis: The subject of prophylaxis in onchocerciasis has not received much attention. Studies with Litomosoides carinii, Loa loa, and Brggia “$1132 have provided some indirect evidence that prophylactic use of DEX: can prevent new infections. DEC was shown to be effective in preventing loaisislzo, but this might not be the case for onchocer- ciasis. Ivermectin was found to be effective in preventing infection of cattle by g. gibsoni. There was hope that it could serve as a prophylactic drug against 9. volvulus infection, but this has not been borne out by recent experiments in chimpanzees. Immune Response in O. volvulus Infection Immunological studies of Q. volvulus infection are often hampered by the scarcity of parasite material. The lack of a suitable labora- tory animal model makes it necessary to obtain the parasites from infected irndividuals residing in remote, often inaccessible endemic foci. Studies of cell mediated immune responses of patients have proven difficult, since most of the work has to be done on site, where field conditions dictate what can and cannot be performed. Simple tasks, such as 31 yi_t_r_g lymphocyte cultures become a major challenge for problems of contamination and the lack of appropriate equipment. Despite all logistical difficulties, investigation of immune responses to 9. volvulus infection is progressing at a good pace, and our knowledge about the biology and immunology of the disease has substan- tially irncreased during the last 10 years. 34 Most of the pathologic consequences seen in onchocerciasis are believed to be secorndary to inflammatory reactions against microfilar- iae. Such inflammatory reactions are controlled by the immune system and require participation of both humoral and cellular elements to occur. Even the mode of action of the microfilaricidal drugs in current use is thought to be immunologically mediated. Thus, it is of importance to have a good understanding of the different immunologic mechanisms involved in onchocerciasis and its relationship to the pathogenesis of the disease. It is often difficult to dissect immune responses to _O_. volvulus in individuals living in endemic foci, since concurrent parasitic and non-parasitic infections are commonly seen in the majority of patients. Therefore, it is imperative to include appropriate controls to minimize these variables, in any immunologic studies of patients. a. Antigens of Onchocerca volvulus: Several approaches to obtain 9. volvulus antigenic preparation have been utilized. By far the most common is the preparation of crude somatic antigen extracts from whole worms obtained by surgical removal of modules. 9. volvulus worms may either be dissected or collagenase digested from host tissue122 and then homogenized and extracted with buffer. The antigenic preparation obtained this way contains complex water soluble somatic and surface antigens, as well as contaminating host proteins. Another approach is the preparation of secretions/excretions of adult worms and microfilariae. This is achieved through En yi_t_r_9_ maintenance of live intact adult 9. volvulus or microfilariae in various culture medial-22. The culture supernatant 35 should contain actively secreted, excreted or shed wonm antigens. One of the problems encountered in this approach is that the preparation often contains host proteins. Also it is difficult to maintain the worms in serum-free media, for their survival time is greatly reduced, and consequently only a small amount of antigenic preparation can be obtained. Of course, the advantage of this approach is that the secreted/excreted antigens are likely to be the ones produced i3 yiyg by live worms and which are usually encountered by the host immune system. A third approach is the use of live microfilariae for _i_r_n_ giggg assay where the microfilarial surface acts as multiple antigenic epitopes which can be recognized by antibodies and cellle4'123. Antigenic extracts from 9. volvulus worms and microfilariae have been used in ig_yi££g_cell mediated responses as well as in testing of immediate and delayed twpersensitivity reactionslzs. They have also been used extensively for immunoblotting recognition patterns by sera from Q. volvulus infected individuals. Although the antigenic properties and the immunomodulatory effects of worm.secretion and excretions were recognized in other filarial and teflmunth infections73'124'126, they have received little attention in ornchocerciasis. To date, few reports exist on the utility of _O_. volvulus microfilariae and adult worms secretions/excretions and in most instances the goal has been immunodiagnosislv. This lack of investigation of the immunologic role of secretions/excretions, is largely due to the extreme difficulty in obtaining live worms in numbers large enough to produce sufficient amounts of these products for research use . 3 6 b. Cell Mediated Immunity: The data on cell mediated immunity in onchocerciasis are at best confusing. The earlier notions of generalized defects in cellular immunity similar to those seen in lymphatic filariasis are still being cited. ENidence for such defects stems from findings of depressed skin reactivity to the non-related antigens tuberculin and lepromin as well as to 9. volvulus crude antigenlzs'lza. The idea of generalized immunodepression was further supported by the in; 3.1.13.0. work of Merino and Brandlzg, where they found decreased reactivity to concanavalin A and by Greene et al. They demonstrated depressed reactivity to the bacterial antigen SKSD in a small number of patients arnd controlsl3o. This has lead to acceptance of the concept of generalized immunosuppression among researchers, the clinical relevance of which was suggested by the appearance of a single report of an association between onchocerciasis and the devel- opment of lepromatous leprosyl31. Hague and colleagues examined pooled sera from ornchocerciasis patients and found that the addition of such sera suppressed lymphocyte responses to mitogensl32. They postulated that serum inhibitory factors are responsible for "this state of generalized immunodepression in onchocerciasis". Evidence to the contrary, i.e. lack of generalized immunosuppres- sion is becoming more recognized in recent literature. Onchocerciasis patients living in endemic areas are not more susceptible to other infectious diseases when compared to non-infected endemic controls from the same ethnic and socioeconomic background. Bartlett et al. 125 have shown that _(_)_. volvulus infected subjects have normal delayed skin reactivity to the non-related antigen of Necator americanus. 37 Brycesonl33 and Greene130 found normal lymphocyte blastogenic responses to mitogens in onchocerciasis patients. Lack of generalized irmmunosuppression is reported by Brattig et al.134 where they were able to demonstrate normal peripheral blood lymphocyte phenotypes in onchocerciasis patients. In fact, the latter authors found an increase in NK activity among reactive (Sowda) patients. Studies from Venezuela in which the effect of individual patient's sera on lympho- cyte blastogenic responses to mitogens was studied, showed mixed patterns where some sera augmented blastogenic responses while others suppressed it135. No recent reports, however, clearly negate the idea of general- ized immunodepression in onchocerciasis. In the first by Ward and co-workersl36 they found intact i_n; y_i_t_n£ lymphocyte blastogenic responses in Guatemalan onchocerciasis patients to mitogens and the non-related antigen PPD, when compared to endemic controls. The normal lymphocyte proliferative responses were accompanied by normal production of interleukin-2 (IL-2) and Gamma interferon (IFN-y). Unexpectedly, they also found good lymphocyte blastogenic responses to cruie Q. volvulus antigen among their patient population. However, they noted decreased IL-2 production by cells from Q. volvulus infected subjects when compared to non-infected, endemic controls. The second report comes from Gallin and her colleaguesl37 where they found normal lymphocyte responses to mitogens and the bacterial antigen streptolysin 0 among Liberian ornchocerciasis patients. They also found low reactivity to crude Q. volvulus soluble antigen among patients and endemic area controls. The E v_it_£g reactivity to 9. W could be greatly increased in both groups by addition of 38 IL-2. In the above two studies, careful planning to include endemic and non-endemic controls was taken, as well as the utilization of pertinent clinical information to include or exclude participants in their studies. Such characteristics, which were mostly ignored by previous investigators, have proven useful in analysis of results. However, in all of the studies sited here only crude Q. volvulus antigenic extracts were used. These are composed of numerous antigens some of which may not be readily available for recognition by the host immune system before the worm death. Thus, to summarize, our knowledge of cell mediated immunity does not consistently support the idea of generalized immunosuppression. Intact responsiveness to mitogens and to non—Q. volvulus antigens is present. Clinically, 9. volvulus patients do not exhibit signs or symptoms of immunodeficiency when compared to their respective endemic controls. Specific hyporesponsiveness to _Q. volvulus antigen is suggested in some studies by decreased lymphocyte responses to Q. volvulus antigen and decreased production of IL-2. c. Antibody Deperndent Cellular Responses: Sera from g. volvulus infected individuals were shown to promote leukocyte adhesion to and killing of microfilariae of Q. volvulus and other animal onchocercal specie5123'l38. Not all 9. volvulus infected patients' sera have such capacity, and most of the activity is seen in sera obtained from patients with active acute skin arnd eye lesions. The cell type involved in the adhesion phenomenon is the eosi- nophi1104'138; in the presence of sera it adheres to and degranulates onto the surface of live microfilariae. The adhesion is enhanced by 39 the presence of complement. Purified eosinophils can adhere to microfilariae _ig y__i_tLo_, but they are unable to degranulate and kill them, irndicating that participation of other cell types and their secreted products is needed for killing to occur. The in; yi_t_rg finding of eosinophil adhesion is in accordance to what is seen i_n xiv—o, where eosinophils arnd their toxic products are seen in close proximity to dying microfilariae in tissue section72. Diethylcarbama- zine was shown to enhance adhesion of granulocytes to microfilariae ig’ yj££g74'139, however the concentrations used in those experiments were much higher than the physiologic therapeutic plasma level. Eosinoph— ilia is a common feature in patients with onchocerciasis, sometimes reaching 70% of all leukocytele4. Administration of DEC to patients leads to rapid disappearance of eosinophils from peripheral blood to the tissues where they were considered to participate in killing of microfilariae74. All of these findings suggest an important role for eosinophils in the destruction of microfilariae, and they provide no support for the notion that neutrophils are the major players in the killing of ‘microfilariae123. The paucity of neutrophils in the inflammatory infiltrate around microfilariae seen on histologic sections of the skin suggests that their role in killing of microfi- lariae arnd hence in the pathogenesis of this disease is rather lim- ited. Antibody dependent cellular cytotoxicity is most likely promoted by antibodies of the IgG class rather than IgE, since in histological sections, 196 can be seen on the surface of dying microfilariae, while 1913 is seen on apparently health microfilariae, without any asociated 40 cellular reaction72. No work has been done to determine the subclass of IgG responsible for microfilarial killirng. d. Humoral Immune Response Until recently, most of the studies on humoral immune response in onchocerciasis were directed towards diagrnosis. It has been mentioned in the "Diagnosis" section that several immunologic procedures were employed in pursuit of improving the specificity and sensitivity of these tests, but the failure to identify an Onchocerca volvulus specific antigen has severely curtailed efforts for development of a reliable and practical immunologic test. Recently, with the introduc- tion of more sophisticated techniques, it has been shown that low molecular weight g. volvulus antigens may offer the greatest diagnostic specificity of the disease. These low m antigens gener- ally lack cross reactivity with other filarial antigensl4o. Of these antigens, two seem to offer the most promising potential; A 33 KD and its supposedly ZlKD breakdown product141. These two antigens were shown to react specifically with sera from Q. volvulus infected individuals with a specificity of 100% and sensitivity above 90%. However, more work is needed to refine the techrniques for practical diagrnostic use arnd more sera from patients with wider clinical presen- tations have to be tested to confirm their usefulness. Detection of circulating Q. volvulus specific antigen(s) is an attractive approach, since it can discriminate between patients harboring the infection from those who were exposed, but not infected or those who are cured from the infection, but still serologically positive. The two pieces of work dealing with this issue did not show 41 greater advantage over the old fashioned method of demonstrating the parasite in skin biopsies. The sensitivity ranged from 31% to 75% in one report‘10 and increased to about 80% in the other after an _g. volvulus specific monoclonal antibody was used39. Nonetheless, antigen detection remains an attractive option which can be used to detect parasite products in various biological fluids, Thus minimiz- ing the invasiveness of skin sampling and blood drawing both of which are sometimes opposed by patients. High levels of total immunoglobulins, notably those of the IgG and IgE classes are usually seen in sera from onchocerciasis patients 142'143’144. Serum levels of immunoglobulins do not usually correlate with the clinical status of patients, perhaps because of the frequent occurrance of other concomittant parasitic infections among onchocer- ciasis patients. Recently however, it has been shown that signifi- cantly higher levels of IgG and 1913 are seen in patients with local- ized onchocerciasis (Sowda) , when compared to otlners with the general- ized fonnl34. Studies aimed at detection of Q. volvulus specific antibody responses have shown that parasite specific 1913 can be seen in asso- ciation with the infection143 with up to 70% of patients manifesting high titresl44. The highest levels of specific IgE anti 9. volvulus antibodies seem to reflect clinical severity of the disease 145- Unlike delayed intradermal skin testing where patients' reactivities differ, immediate hypersensitivity reactions to onchocercal antigens are invariably seen in onchocerciasis patient5122'127. These findings coupled with findings in lymphatic filariasis 146'147'148 bolstered beliefs about the immunobiological significance of 1913 in the path- 42 ogenesis of the disease. In 1982, Weiss and colleagues149 showed intense IgE reactivities to more than 50 g. volvulus antigenic bards using Wastern immunoblotting techniques. The above authors, however, used extremely concentrated patients' sera (dilution 1:2) which raises questions as to the specificity of their procedure. Since then no one has attempted to specifically look at IgE reactivities using the Western blotting technique. However, recently Cabrera et al.,]‘50 looking at IgG3 reactivity of Sowda patients to Q. volvulus antigens, also examined other immunoglobulin classes reactivities, including IgE. Although they did not comment on IgE recognition pattern to separated 9. volvulus antigens the results of their immunoblots clearly show minimal reactivity with IgE. The same authors have shown very restricted patterns of IgE recognition using Onchocerca gibsoni as a source of antigen151. Reproducible patterns of Q. volvulus antigen recognition by other immunoglobulin classes using Western blotting have been demonstrated by a number of investigatorsl41'150'151'152'153. Differential recognition of worm antigens by antibodies of the IgG immunoglobulin class and its relation to the clinical presentation of patients attracmd much attention recently. The emerging pattern is that patients with severe disease terd to recognize more antigenic bands with greater intensity152'153. However, this is a generalization, since on occasion, asymptomatic patients and those with mild disease may recognize a similar or greater number of antigenic bands with strornger intensitieslsz'153. This diversity ard lack of consistent antigen recognition patterns in relation to the clinical signs is probably due to tlne dynamics of the disease, where the immunologic 43 status of patients may change, without an immediate change in the clinical picture. Patients may revert from a reactive state to non-reactive or vice versa. Most of the immunological studies in onchocerciasis, including those corncerned with immune responses, are cross-sectional ones. Better understanding of immunologic responses to the parasite should come from longitudinal studies, where the clinical ard immunologic changes can be closely mnonitored. In tlne most recent studies by Cabrera et al.150 the role of IgG immunoglobulin subclasses in onchocerciasis was studied. The authors looked at IgG3 recognition patterns of Q. volvulus antigens by sera from Sowda patients and those with generalized onchocerciasis. They claimed that IgG3 antibodies present in sera obtained from Sowda patients all specifically recognize 72 KD and 9 RD 9_. volvulus antigenic bands, while ornchocerciasis patients with other forms of the disease failed to recognize either band. They proposed that this 1963 recognition could serve as a diagnostic test for Sowda patients who are often negative parasitologically. However, their data were unusual in that these two proteins were not recognized when blots were processed with anti-total 196. 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Experimental chemotherapy of filariasis. III. Effect of 1-diethylcarbamyl-4-methy1piperazine hydrochloride against naturally acquired filarial infection in cotton rats and dogs. J. Lab. Clin. Med. 32: 1314-1329. Hawking, F. 1981. Chemotherapy of filariasis. Antibiotics Chemother. 30: 135-162. Hawking, F. 1978. Diethylcarbamazine: a review of the litera- ture with special reference to its pharmacodynamics, toxicity and use in the therapy of onchocerciasis and other filarial infec- tions. World Health Organization Document. WHO/ODCHO/78-142. 103. 104 . 105. 106. 107. 108. 109. 110. 111. 112. 113 . 114. 115. 52 Gibson, D.W., D.H. Connor, H.L. Brown, H. Fuglsang, J. Anderson, B.O.L. Duke, and A.A. Buck. 1976. Onchocercal: Ultrastructural studies of microfilariae and host tissues before and after treat- ment with diethylcarbamazine (Hetrazan). Em. J. Trop. Meg. Hyg. 25: 74-87. Mackenzie, C.D. 1980. Bosinophil leukocytes in filarial infec- tions. Trans. R_y. Soc. Troy. Med. Hyg. 74: 51-58. Langham, M.B., Z.D. Traub, and R. Richardson. 1978. A transepid- ermal chemotherapy of onchocerciasis. Tropenmed. Parasit. 29: 156-162. Taylor, H.R., M.B. Langham, B.M. deStahl, L.N. Figueroa, and F. Beltran. 1980. Chemotherapy of onchocerciasis: A controlled trial of topical diethylcarbamazine (DEC) in Guatemala. Tro- penmed. Parasit. 31: 357-364. Hutchinson, D.G.A., H.A. ElSheikh, B.R. Jones, J. Anderson, H. Fuglsang, and C.D. Mackenzie. 1979. Adverse reactions to cuta- neous diethylcarbamazine in onchocerciasis. Lancet. 2: 46. Anderson, J., and H. Fuglsang. 1978. Further studies on the treatment of ocular onchocerciasis with diethylcarbamazine and suramin. Trans. Roy. Soc. Trg. Med. Hyg. 62: 450-457. Hawking, F. 1978. Suramin: with special reference to onchocer- ciasis. Adv. Pharmacol. Chemotherapy. 15: 289-322. Hawking, F. 1978. Suramin: A review of the literature with special reference to its pharmacodynamics, toxicity and use in the therapy of onchocerciasis. World Health Organization Document. WHO/OICHO/7 8: 143. Campbell, W.C. 1981. Efficacy of avermectins against filarial parasites: A short review. Vet. Res. Comm. 5: 251-262. Aziz, M.A. 1986. Ivermectin vs. onchocerciasis. Parasitology Today. 2: 233-235. Aziz, M.A., S. Diallo, I.M. Diop, M. Laviviere, and M. Porta. 1982. Efficacy and tolerance of ivermectin in human onchocer- ciasis. Lancet. 2: 171-173. Awadzi, K., K.W. Dadzie, H. Schulz-Key, D.R.W. Haddock, H.M. Gilles, and M.A. Aziz. 1985. The chemotherapy of onchocerciasis. X. An assessment of four single dose regimen of MK-933 (ivermec- tin) in human onchocerciasis. fl. Trop. Med. Parasitol. 97: 63-78. Whitworth, J.A.G., G.H. Maude, and D.W. Taylor. 1988. Cammmity based treatment with ivermectin. Lancet. 2: 97-98. 116. 117. 118. 119. 120. 121 . 122. 123. 124. 125. 126. 53 Homeida, M.M.A., I.A. Bagi, H.W. Ghalib, H. Elsheikh, A. Ismail, M.A. Yousif, S. Sulaiman, H.M. Ali, J.L. Bennett, ard J.E. Wil- liams. 1988. Prolongation of prothrombin time with ivermectin. Lancet. 1: 1346-1347. Kass, I.B., A.O.W. Stretton, and C.C. Wang. 1984. The effects of avermectin and drugs related to acetylcholine and 4 aminobutyric acid on neurotransmission in Ascaris suum. M01. Biochem. Parasit. 13: 213-225. Mossinger, J., H. Schulz-Key, and K. Dietz. 1988. Emergence of Onchocerca volvulus microfilariae from skin snips before and after treatment of patients with ivermectin. Trop. Med. Parasit. 39: 313-316. Duke, B.O.L. 1970. Onchocerciasis: Deep worm bundles close to hip joints. Trans. Ry. Soc. Trop. Med. Hyg. 64: 791-792. Nutman, T.B., K.D. Miller, M. Mulligan, G.N. Reinhardt, B.J. Currie, C. Steel, and E.A. Ottesen. 1988. Diethylcarbamazine prophylaxis for human loiasis. Results of a double blind study. J. E. J. _I‘gd. 319: 752-756. Qipp, E.W., M.J. Bernardo, A.E. Kiszewski, R.C. Collins, H.R. Taylor, M.A. Aziz, and B.M. Greene. 1986. The effect of ivermec- tin on the transmission of Onchocerca volvulus. Science. 231: 740-742. Schulz-Key, H., E. Albiez, and D. Buttner. 1977. Isolation of living adult Onchocerca volvulus from nodules. Tropenmed. Parasit. 28: 428-430. Greene, B.M., H.R. Taylor, ad M. Aikawa. 1981. Cellular killing of microfilariae of Onchocerca volvulus: Fosinophil ad neutrophil-mediated immune serum dependent destruction. J. hmmmol. 127: 1611-1615. Kaushal, N.A., R. Hussain, T.E. Nash, ard E.A. Ottesen. 1982. Identification ard characterization of excretory/secretory prod- ucts of Brugia malaLi, adult filarial parasites. J. Immunol. 129: 338-343. Bartlett, A., J. Turk, J.L. Ngu, C.D. Mackenzie, H. Fuglsang, and J. Arderson. 1978. Variation in delayed hypersensitivity in dermal onchocerciasis. Trans. Roy. Soc. Tag. Med. Hyg. 72: 372-376. Foo, B., M. Nowak, B. Copeman, and M. McCabe. 1983. A low molecular weight immmosuppressive factor produced by Onchocerca gibsoni. Vet. Immunol. Immunopath. 4: 445-451. 127. 128. 129. 130. 131. 132. 133. 134 . 135. 136. 137. 138. 54 Ngu, J.L., P.M. Ndumbe, V. Titanji, and R. Leke. 1981. A diag- nostic skin test for Onchocerca volvulus infection. Tropenmed. Parasit. 31: 165-170. Rougemont, A., M. Boisson-Pontal, P. Pontal, ard F. Gridel. 1977. T‘uberculin skin tests and B.C.G. vaccination in hyperen- demic area of onchocerciasis. Lancet. I: 309. Merino, F., and A. Brand. 1977. Immunological studies in onchoc- erciasis patients. Tropenmed. Parasit. 28: 229-234. Greene, B., M. Fanning, and J. Ellner. 1983. Non-specific suppression of antigen-induced lymphocyte blastogenesis in Onchoc- erca volvulus infection in man. Clin. E_:x_p. Immunol. 52: 259-265. Prost, A., M. Nebout, ard A. Rougemont. 1979. Iepromatous leprosy and onchocerciasis. Brit. Med. J. 1: 589-590. Hague, A., A. Capron, A. Qaaissi, L. Kouemeni, J. Lejeune, B. Bonnel, and R. Pierce. 1983. Immune unresponsiveness and its possible relation to filarial disease. Contr. Microbiol. Immunol. Bryceson, A.D.M. 1977. What happens when microfilariae die? Trans. Roy. Soc. Trop. Med. Hyg. 70: 397-401. Brattig, N., F. Tischendorf, E. Albiez, D. Buttner, ard J. Berger. 1987. Distribution pattern of peripheral lymphocyte subsets in localized and generalized form of onchocerciasis. Clin. Immunol. Immunopath. 44:149. Rarez-Rojas, G.E., M. Valderrama, C. Anderson, I. Machado, N. Bianco, ard L. Yarzabal. 1983. Immunidad mediada por celulas en la onchocercosis modificaciones por factore sericos. Filariasis Humans _e_n_ _e_l Territorio Fed. Amazonas. 2: 93-99. Ward, D.J., T.B. Nutman, G. Zea-Flores, C. Portocarrero, A. Lujan, ad E.A. Ottesen. 1988. Onchocerciasis and immunity in humans: enhanced T-Cell responsiveness to parasite antigen in putatively immune individuals. J. Infect. Dis. 157: 536-543. Gallin, M., K. deords, J.J. Ellner, K.D. Ertman, A.T. White, H. Newlad, R.H. Taylor, and B.M. Greene. 1988. Cell-mediated immune responses in human infection with Onchocerca volvulus. J. Immunol. 140: 1999-2007. Williams, J.E., H.W. Ghalib, C.D. Mackenzie, M.Y. ElKhalifa, J. Ayuya, and M.A. Kron. 1987. Cell adherence to microfilariae of Onchocerca volvulus: A comparative study. Ciba Fourdation Monograph. 127: 46-76. 139. 140. 141 . 142. 143 . 144 . 145. 146. 147. 148. 149. 35 King, G.H., B.M. Greene, and P.J. Spangicolo. 1983. Diethylcar- bamazine citrate: An antifilarial drug stimulates human granulo- cyte adherence. Antimicrob. Agents Chemo. 24: 453-456. Lobos, B., and N. Weiss. 1986. Identification of non-cross- reacting antigens of Onchocerca volvulus with lymphatic filariasis serum pools. Parasitology. 93: 389-399. Lucius, R., H. Schulz-Key, D. Buttner, A. Kern, B. Kaltmann, J. Prod-Hon, F. Seeber, R.D. Walter, K.C. Saxena, ard H. Diesfeld. 1988. Characterization of an immunodaminant Onchocerca volvulus antigen with patient sera and a monoclonal antibody. J. §x_p. _JMed. 167: 1505-1510. Buck, A., I.P. Anderson, K. Kawata, and K. Hitchcock. 1969. Onchocerciasis: Some new epidemiological ad clinical findings. Ad. J. Trop. E. dyg. 18: 217. Weiss, N., F. Speiser, ard R. Hussein. 1981. IgE antibodies in human onchocerciasis. Application of a newly developed radioal- lergosorbent test (RAST). Acta. Tropica. 38: 353-362. Somorin, O., D.C. Ajugwo, and D.C. Heiner. 1977. Immunoglobulin E in adult Americans and Nigerians. 3. J. T‘rop. _JMed. Hyg. 26: 872-876. Kowemeni, L., A. Hague, and A. Capron. 1982. Detection of IgE antibodies in onchocerciasis. Possibility of using allergens from Dipetalonema viteae extracts that cross-react with allergenic determinants in crude extracts of Onchocerca volvulus. Clin. _p. Immunol. 50: 541-548. Hussain, R., R.C. Hamilton, V. Kumaraswami, N. Franklin Adkinson, ad E.A. Ottesen. 1981. IgE responses in human filariasis. I. Qiantitation of filarial-specific IgE. J. Immunol. 127: 1623-1629. Hussain, R., ad E.A. Ottesen. 1983. IgE responses in human filariasis. II. Quantitative characterization of filaria- specific IgE. J. Immunol. 131: 1516-1521. Hussain, R., and E.A. Ottesen. 1985. 1913 responses in human filariasis. III. Specificities of IgE ad IgG antibodies campared by immunoblot analysis. J. Immunol. 135: 1415-1420. Weiss, N., M. Gualzata, T. Wyss, ad B. Betschart. 1982. Detec- tion of IgE binding Onchocerca volvulus antigens after electropho- retic transfer and immuno-enzyme reaction. Acta. Tropica. 39: 150. 151 . 152. 153 . 154 . 155 . 56 Cabrera, Z., D.W. Buttner, and R.M.E. Parkhouse. 1988. Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper—reactive oncho-dermatitis (Sowda). Clin. Efl. Immunol. 74: 223-229. Cabrera, Z., M.D. Cooper ard R.M.E. Parkhouse. 1986. Differen- tial recognition patterns of human immunoglobulin classes to antigens of Onchocerca gibsoni. Trgn. Med. Parasit. 37: 113-116. Lucius, R., J. Prod-Hon, A. Kern, G. Hebrard, and H.J. Diesfeld. 1987. Antibody responses in forest and savana onchocerciasis in Ivory Coast. Trpp. Med. Parasit. 38: 194-200. Lucius, R., D.W. Buttner, C. Kirsten, and H.J. Diesfeld. 1986. A study on antigen recognition by onchocerciasis patients with different fornms of disease. Parasitology. 92: 569-580. Ottesen, E.A., V. Kumaraswami, R. Paranjape, R.W. Poindexter, ad S.P. Tripathy. 1981. Naturally occurring blocking antibodies modulate immediate hypersensitivity responses in human filariasis. J. Immunol. 127: 2014-2020. Hussain, R., and E.A. Ottesen. 1986. IgE responses in human filariasis. IV. Parallel antigen recognition by IgE and IgG4 subclass antibodies. J. Immunol. 136: 1859-1863. SUPPRESSION OF HUMAN LYMPHCEYTE RESPONSES TO SPI‘IHFIC AND NON-SPECIFIC STIMULI IN HUMAN OMIHCIIEKZIASIS Mohamed Y. ElKhalifa and Jeffrey F. Williams Department of Microbiology ad Public Health, Michigan State University, Fast Iansing, MI 48824 57 38 ABSTRACT Characterizations of circulating lymphocyte subset populations, _id y_i_t£d lymphocyte responsiveness, and the serum-mediated effects on lymphocyte proliferation were performed on groups of onchocerciasis patients selected from over 170 examined parasitologically and clini- cally in Sudan and Sierra Lecne. These patients manifested a very broad range of clinical signs ard showed widely divergent parasite intensities; some were heavily infected and asymptomatic while at the other extreme were individuals with severe clinical signs, but few or no detectable microfilariae (mf) in their biopsies. Relationships between parasite burden and clinical severity varied; in Sierra Leone severity increased with detectable mf numbers, but the reverse was true in one group in Sndan. Patients from Sudanese foci were, on the whole, more badly afflicted than the W. Africans. Lymphocyte subsets in peripheral blood were not remarkably different from those in samples from erdemic area and non erdemic area controls, except in regard to a significant increase in NK cells in infected W. Africans. Lymphocyte proliferative responses to soluble Onchocerca volvulus antigen (sAg) were poor in infected persons, however, mitogen and PPD responses were maintained in the normal range in one group of patients from southwes- tern Sudan, but were profoundly depressed in a group from Eastern Sudan. The latter were malnourished and in poor physical cordition in a drought-stricken zone. Sera from 90 Sudanese and W. African patients exhibiting a wide range of clinical signs and parasite burdens had no significant effects on responsiveness 1.9. m of lymphocytes frum either normal or infected subjects. However, proliferative responses and IFN-y production by lymphocytes were very significantly depressed 59 in the presence of live microfilariae of d. volvulus or secre- tions/excretions (S/E) from microfilariae or from females, but not male adult parasites. Lymphocyte responses were maintained near normal when exogenous IL-2 was added to the cultures. The results indicate that overall 9. volvulus infection and clinical consequences are not consistently associated with systemic deficits in immune cell popula- tions or responsiveness, or circulating suppressive factors; however, lowered lymphocyte responses can be a feature when health problems are perhaps exacerbated by prolonged malnourishment. Specific lymphocyte responsiveness to d. volvulus sAg was poorly manifested in all patients examined. The demonstration of potent suppression of lymphocyte reactivity by mf and S/E _id y_i_t_:_rd suggests that direct parasite intervention in host cell responses could be taking place Bl £132: perhaps at the local micro-environment level, mediated by effects on IL-2 production. A hypothesis is presented corncerning the consequences of anti-microfilarial therapy _1_r_n v_iy_g, and its potential effect on disruption of local immunosuppression. 6O INTRODIIITION Modulation of host immune responses is considered a major factor in the disease course followed by patients infected with the filarial nematode Onchocerca volvulusl'z. Clinical manifestations may range frorm a camplex of massive acute and chronic inflammatory changes in the skin and eyes of those infected, to mild pruritus or even asymptomatic infections at the other extreme. Acute exacerbations are generally associated with chemotherapy3, and, increasingly, death of microfilar- ial forms in tissues is becuming recognized as a significant event in the pathogenesis of onchocercal lesions in skin and ocular tissues 4'5. In spite of the clinicopathological and parasitological evidence to support this interpretation of onchocerciasis ad its natural history, immunological determinants and correlates of disease stages have proven very difficult to identify. By analogy with findings in chronic lymphatic filariasis6'7'8, specific and non-specific immunosup- pressive mechanisms have been sought in onchocerciasis patients, but the evidence is conflicting about their contributions to the outcume of the host-parasite interaction. Both within ad between patient groups there is a great deal of heterogeneity with regard to lymphocyte responses to Q. volvulus antigens, mitogens ad bacterial antigenic stimulig'lo'll, and to the presence of circulating suppressors or enhancers in the sera of infected subjects12'13. Variables such as age, chemotherapeutic history, and geographic strain of parasite have recently been shown to have important bearing on immunological response patternsl4'ls, and the need to include endemic area "normals" in such studies has been emphasizedzs. 61 In developing a camparative account of onchocerciasis in several distinct geographic foci we have had the opportunity to examine the influence of patients' sera and parasite products and antigens on the .12 y_i_t_rd lymphocyte responses of controls and of patients with differing clinical manifestations and d. volvulus infection intensities in a variety of epidemiological contexts. Our results confirm the occurrence of i_n div—0 suppressive changes in some patient subgroups, ad demonstrate the production of inhibitory factors for human lympho- cyte proliferation by live adult females and microfilariae of d. volvulus Bl v_it_r_o. The effects of these inhibitors could be largely overcame by addition of exogenous IL-2, raising the possibility that direct intervention by the parasite into the immunoregulatory pathways of the host's immune system may be a component of suppression mecha- nisms i_n vivo. 62 MATERIALS AND METHODS Study Areas. Patients from four different geographical areas endemic for onchocerciasis were studied. Three are in Sudan, and each shows some slightly different characteristics in the prevailing form of the disease: Abu-Hamad in the Northl6; Surdus in the Fast4'17, ad Bahr El Ghazal in the Southwest”. An additional group of patients was examined and sampled in Bo, Sierra Leone, West Africa 19. Patients and Control Selection. 174 patients were examined: 109 from Sudan and 65 from Sierra Leone. From each focus individuals representing the fullest possible range of clinical manifestations and parasite burdens were included. Subject ages ranged from 12-70 years, (mnean, 32.8), with 120 males and 54 females. Two groups of non- infected controls were included: the endemic area controls were 13 (eight males and five females) irdividuals living in onchocerciasis erdemic areas, but clinically ad parasitologically not identifiable as onchocerciasis patients. The normal (non-erdemic area) controls were 32 (22 mnales ard 10 females) individuals living in Khartoum, Sudan, where the disease is not established. These people were similar in ethnic make-up ard in their socioeconomic backgrourd to the Sudanese infected patients. Ages of both control groups ranged from 18 to 63 years (mean, 33.8). Clinical Examination. All participants were given a complete physical examination with special attention given to the palpation of _O_. volvulus nodules, ad to the evaluation of skin and eye changes consistent with d. volvulus infection. The clinical history taken included details of any antifilarial treatment received and of surgical removal of nodules. Details of the examination procedure ard the ()3 assessment of lesions are described elsewhere4'5. As a result of physical findings, individuals were classified primarily on the basis of the type, distribution and severity of skin changes, using a point scale of 0-3, with 3 being most affected. For the purposes of compari- son 3 groups of symptomatic patients emerged from this scheme; those with mild disease (score of l) , moderately affected people (score of 2) ad those severely afflicted (score of 3). Asymptomatic patients with no signs or symptoms of onchocerciasis, but with microfilariae in their tissue biopsies were given a score of 0. Parasitological Examination: To determine the microfilarial intensity of d. volvulus infection in each individual, bilateral skin biopsies (snips) were taken from the iliac crest using a Walser cor- neoscleral punch. Skin snips were then incubated in tissue culture medium RPMI 1640 (Sigma Chemical Co., St. Louis, M0) for 4 hours. Emerging microfilariae were counted and the wet weight of the snips was determined. Microfilarial intensity was expressed as the number of microfilariae per milligram of skin (mf/mg). Urine, stool and blood samples from patients in Abu-Hamad and Bo were examined for other parasites by conventional techniques. Lymphocyte Subset Phenotyping of Patients and Controls. Anti-1eu 2a, 1eu 3a, 1eu 4, let 11b, and 1eu 12 mouse monoclonal antibodies were purchased from Becton Dickinson (Mountain View, CA). These probes recognize, respectively, human lymphocyte surface antigens, CD8, CD4, CD3, CDl6, ad CD19. Colloidal gold-conjugated goat anti-mouse antib- ody (Geometric Data, Wayne, PA) was used as detector for the primary mouse monoclonal antibody bound on lymphocyte surfaces. 64 The test procedure was similar to that described by the manufac- turer. Briefly, from. each individual a buffy' coat suspension was obtained from EDI‘A-treated blood. Aliquots of buffy coats were first incubated 30 minutes with one of the primary monoclonal antibodies, then with the second, conjugated antibody for another 30 minutes. Cells were washed with phosphate buffered saline after each incubation. Finally, a thin film was made, fixed in methanol and counter-stained with modified Wright's stain. Enumeration of lymphocyte subtypes was done microscopically by counting the number of positive cells per 200 lymphocytes. Cells considered positive had at least four gold par- ticles on their surfaces. Preparation of O. volvulus Adult WOnm Secretions/Excretions (S/E) ad Soluble Antigen (SAL). Intact nodules were surgically excised from patients urder local anesthesia. To obtain live female parasites, nodules were digested in collagenase using the method described by Schulz-Keyzo. Trimmed excised nodules were incubated in culture medium RPMI 1640 containing 10% pooled normal human serum, penicillin, 100 U/ml, streptomycin, 100 ug/ml (Gibco Laboratories, Grad Islad, NY) ad collagenase type IV (Sigma Chemical Co.) at a concentration of 2 mg/ml. Digestion was carried out at 37°C for 12 hours after which female parasites were recovered by gentle dissection. d. volvulus mnales were obtained either. by collagenase digestion or from gently minced nodular tissue. Isolated adults were thoroughly washed in serumpfree RPMI 1640 with antibiotics. All 9. volvulus female and male worms were~maintained individually at 37°C in RPMI 1640 supplemented with antibiotics for 24-48 hours. For active males, the culture period was exterded up to 96 hours. Culture 65 supernatants were dialyzed at 4°C against three changes of 1,000 volumes of RPMI 1640 using Spectrapor membrane tubing (Spectrum Medical Industries Inc., Los Angeles, CA), with a molecular weight cut off of 10,000 Daltons. Supernatants were then filter-sterilized, aliquoted ad stored at -70°C. The protein concentration in supernatants was measured using the Lowry method21. Isolated d. volvulus female worms were homogenized in phosphate buffered saline, pH 7.2 and extracted overnight at 4°C. Particulate material was pelleted by centrifugation at 5,000 x g/30 minutes and the supernatant was further clarified at 100,000 x g/l hour. The final supernatant was dialyzed against RPMI 1640 and processed as for d. volvulus S/E products. Preparation of O. volvulus Skin and Nodular Microfilariae (mf) ad Their Secretions/Excretions. Live skin mf were obtained by incubating skin biopsies obtained from d. volvulus patients in RIMI 1640 ad antibiotics for 4 hours. Nodular mf were obtained by teasing excised tissues containing 2. volvulus female worms. The tissues were then incubated in RPMI 1640 with antibiotics for 1 hour. Emerging microfi- lariae from skin or nodular tissues were purified using the agar gel techniquezz. Preparation of _O. volvulus mf S/E. Microfilariae obtained either from biopsies or nodular tissues were maintained at a concentration of 1 x 103 mf/ml in RPMI 1640 plus antibiotics at 37°C for 48 hours. Mf culture supernatants were then processed as for those of d. volvulus adult worm S/E. Lymphocyte Stimulation. Peripheral blood was drawn from patients ad controls into heparinized vacutainers (Becton Dickinson, Ruther- 66 ford, NJ). Peripheral mononuclear cell (PMbC) separation was achieved by centrifugation on Ficol-Hypaque density gradients (Pharmacia, Piscataway, NJ)23. Unless otherwise specified, washed PMMZ were cultured in triplicate at a concentration of 2 x 105 cells/well in U bottom 96 well microtitre plates (Corning Glass Works, Corning, NY), in 0.2 ml of RPMI 1640 supplemented with antibiotics and 10% human serum. Lymphocyte responses to mitogens. To measure lymphocyte blasto- genic responses, EMMY. were cultured with either phytohemagglutinin (PHA) or concanavalin A (Con A) (Sigma Chemical Co.) at concentrations of 5 ad 10 ug/ml, respectively. All cultures were maintained at 37°C in a humidified atmosphere containing 5% C02, 18 hours prior to harvesting, cells were pulsed with 1 uCi of H3 thymidine (New Englard Nuclear, Boston, MA) per well (specific activity of 6.7 mnC/mol) . Cells were then harvested onto glass fibre filters using a Bellco harvester (Bellco Glass Inc., Vineland, NJ). The incorporated radioactivity in counts per minute (cpm) , was read in a Beta Scintillation Counter. Lymphocyte responses to O. volvulus sAg and a non-related antigen. PMIC were cultured in the presence of d. volvulus sAg or purified protein derivative (PPD), each at a concentration of 20 ug/ml. These cultures were maintained for a total of 144 hours ad harvested 18 hours after addition of l u Ci of H3 thymidine. Effects of O. volvulus adult worms S/E ad mf S/E on lymphocyte blastogenic responses to mitogens. PMIC were cultured with either PHA or Con A, in the presence or absence of S/E from d. volvulus females, males or mf. Similar experiments were carried out in which 9. volvulus S/E was replaced by either 9. volvulus sAg or PPD. d. volvulus S/E, d. volvulus sAg ad PPD were all used at concentrations of 20 ug/ml. 67 Serial dilutions of d. volvulus female S/E were also tested. Cultures were maintained for 72 hours, and harvested 18 hours after pulsing. The percentage inhibition caused by addition of d. volvulus derived materials was calculated using the following formula: cpm (mitogen + O.v. S/E) - cont. cpm Percent inhibition = l- x 100 cpm (mitogen alone) - cont. cpm Effect of cocultivation of live skin and nodular O. volvulus mf on lymphocyte blastogenic responses to mitogens. mm were cultured with either PHA or Con A in the presence or absence of live mf. 30 skin mf/well and concentrations of 30, 50 and 120 nodular mf/well were tested. Cultures were harvested at 72 hours. Effect of Interleukin-2 addition. To assess the effect of exogenous Interleukin-2 (IL-2) on the blastogenic response of PMIC cultured in the presence of mitogens ad serial dilutions of d. volvulus S/E, IL-2 (Cellular Products Inc., Buffalo, NY) was added to the test cultures at a concentration of 20 units per ml. Effects of patient sera on lymphocyte blastogenic responses to mitgen . PMM: obtained from a normal donor were cultured with 20% heat inactivated individual patient or normal control serum in the presence of either PHA, 5 ug/ml or Con A, 10 ug/ml. Cultures were harvested at 72 hours. Gamma Interferon (IFN-y) Production. The effect of d. volvulus female worms S/E and Q. volvulus live mf on the production of IFN-y, was measured by culturing 1 x 106 PMbC/ml with PHA in the presence or 68 absence of & volvulus female S/E, 20 ug/ml, or d. volvulus skin mf, 150 mf/ml. Cultures were incubated at 37°C for 24 hours and the resultant culture supernatants were stored at -l96°C or -70°C. To some cultures IL-2 was added at the beginning of the incubation at a concen- tration of 20 units per ml. Concentrations of IFN-Y in the superna- tants were measured in international units per ml (IU/ml) using an IMRX solid phase monoclonal antibody-based radio-immunoassay (Centocor Inc., Malvern , PA) . Statistical analysis. All statistical analyses were based on the unpaired Student's 't' test. 69 RESULTS Clinical ad Parasitological Results. The parasitological findings on the skin snip examinations of the four subgroups of d. volvulus infected individuals are shown in Table 1. Infection intensity was highest in Bo, Sierra Leone, whereas markedly lower numbers of parasites were evident in biopsies from patients from Abu-Hamad and Sundus, Sudan. Physical examinations revealed striking differences in disease manifestations, both between and within patient populations. In Sundus there was generally an inverse relationship between parasite intensity and the severity of clinical disease, but in Bo, Sierra Leone, there was a trend toward increasing severity with greater mf intensity (Table 1). The severely affected patients in Bo had the highest mf counts; in Surdus those severely affected had the lowest mf counts. None of the patients included in the study had a history of antifilarial chemotherapy; however, almost 50% of Bo patients had experienced surgical removal of onchocercal nodules within the past two years. Overall, the clinical manifestations of onchocerciasis were milder in B0, ad only 14 (22%) of the patients fell into the categories of moderate to severe onchocerciasis (Figure 1), compared to 71 (65%) of the Sudanese (Figure 2). In Abu-Hamad, infection intensities were low, generally in the 1-2 mf/snip range, even though clinical manifestations were present and often severe. Four 9. volvulus-infected individuals in Sudan had predominantly an asymmetric severe limb involvement (Figure 3), but none of the irdividuals seen in Sierra Leone showed this pattern. The overall clinical presentations and microfilarial 70 5:99.” ammomfi tcm 3.225 :mmEon 95:25.9 .525 0: mm; 99: act 68?: he was poet-+34. :_ 36:95 _mtmEQoE 399.... 65 99:38 RNA .0 $23 2539a m 5.. m.m v + t 50.0 NF on v m mwmmmfi :Em Swim Nd- v + : o.m mm 0.00 or N ommowfi :2... $9822 I o + o «in 9. mm mm P ommmwfi 5.... 2:2 I o .+ a mm m 0.: mm o 393% 5 29m 02 9.5:. .oz 9&2: .oz 9.5.: .oz 9.5:. .oz Boom 8333 8.8.... fins .81: 8..."... sons awaken 0 525 :35 :33 0:3.— Eim a _ am .323 58 62.21-32 .2655 do .838 6:03 Stew DE... :85 :_ $868855 :2 2885 .8. 8:56 52 :_ 983% 8892 93329. 8803020 .0 3.9m _mo_mo.2_mm:mo t:m _mo_:=o . m..m<._. 71 Figure 1. An onchocerciasis patient from the Bo area. Note the mild fine papular erruption seen on the upper back. The clinical presen- tation of this patient is seen in the majority of Bo patients. 73 Figure 2. Severe onchocercal dermatitis seen in a SJdanese patient. Note areas of hypo and hyperpigmentation, papular erruption and excoriation marks . 71+ Figure 3 . (Sowda) . 75 A Sudanese patient with localized limb involvement 76 77 intensities were not influenced by age or sex, however, in the Sundus group, younger patients had more pronounced acute changes4. No other filarial infections were diagnosed by examination of blood from patients in Bo, aindus and Abu-Hamad, but one of the eight individuals from Bahr El Ghazal had Mansonella perstans. Malaria was most prevalent in patients from Sundus; despite being sampled in the dry season, 30% were blood film positive for either _13. falciparum or g. vivax or both. In contrast, only one individual from Bo, Sierra Leone was positive for g. falciparum infection. Intestinal helminthiasis was prevalent among individuals examined in Bo, where 52% of onchocerciasis patients and 42% of the endemic area controls had intestinal nematode infection detected in a single fecal sample. Patients from Abu-Harmad were essentially free of parasitic infections other than 9. volvulus. Endemic area controls were asymptomatic and had no demonstrable Q. volvulus microfilariae in skin biopsies. However, they showed similar prevalence rates for other endemic parasitic infections (e.g. malaria, intestinal helminthiasis) . Non—endemic area control subjects had no remarkable characteristics on physical examination, negative skin biopsies and were free of intercurrent parasitic infections. Lymphocyte Sibset Phenotypes. Pefipheral lymphocyte subset phenotyping was done on 30 g. volvulus patients and 8 endemic area controls from Bo, Sierra Leone, and on 8 Q. volvulus patients from Bahr El Ghazal and 17 non-eidemic area controls from Khartoum, Sudan. The only significant differences seen (P = < 0.03) was an increase in natural killer (NK) cells in infected individuals from Sierra Leone when compared to endemic area controls (Table 2). The NK cells were not counted in samples from Sudanese subjects. No significant differ— 78 .9523 _m~m:m_m Emm :_ 52v goo .0: was $935 2.8 x2; Amodvé w_o:.:oo ucm 9:223 om :mmEmn 355:6 Ema—Ema 2326.55,. oz v.0 H MK; we H tom ox H 0.9 new H v.5 mum H mdw An TE m_o=:oo _mEcoz .02 mo H mm; m. P F H Now we H 5.9» adv H mdo _.m H mxm 8-5 .3556 :55 .mEmzmn. 3 H :3 9.: H 3 mm H New 3 H a. z. 3.. H 9% mm H No. 3-5 $9.80 $5 o_Emn:m .NeHm. «OH: EH :8 N309. 3: H05 .83.? ans om .mEozmn. 659 80:50 .80. =8 p .30. 2.8 .89 2.8 850. «=3 x: 33235 b. .02»: h .82. «=3 m = mans. .m:om.. Seem .om Em :mczm ..m~m:m_w 33 E9. £938 :25? Em 28:3 $888855 :_ :ozaezmfi 68% 260352 803 .9239. 79 ence in T-helper to T—suppressor ratio was seen between patients and controls and the absolute counts of both phenotypes were similar in the two groups. Lymphocyte Responses to Mitogens. Lymphocytes from eight 9. volvulus infected individuals from Bahr El Giazal responded normally to PHA, Con A and PPD. There were no significant differences in lympho- cyte blastogenic responses to PHA, Con A and PPD between the 15 non- endemic area controls and these 9. volvulus infected individuals (Table 3). 9. volvulus soluble antigen caused only slight stimulation of peripheral blood lymphocytes of Q. volvulus infected subjects and controls; however, there was no significant difference in the blasto- genic responses between the groups (Table 3) . The Q. volvulus infected patients showed infection intensities from 2.0 to 56.0 mf/mg; four had moderate (score of 2) and four had severe generalized (score of 3) dermal lesions. Although those eight 9. volvulus infected subjects were originally from Bahr El Giazal, they had been residing in Khartoum for at least one year. When similar experiments were carried out on twelve Q. volvulus infected subjects from Sundus the results were different. These patients were generally in poorer physical condition than those from Bahr El Gdazal; they had been residing in a remote area and subsisting on an inadequate plane of nutrition. Their mf intensities ranged from 0.5 to 46 mf/mg, and the clinical scores assigned were as follows: seven had a score of 1; three had a score of 2; and two had a score of 3. These individuals showed much reduced responsiveness to PHA and PPD campared to controls, and they had the same very slight response to the presence of Q. volvulus sAg as did patients from Bahr El Ghazal. 8O 0:00 .02 02.. .000: 0.0; _.0;\m._00 .9 x m .0 000.0 .00. 0.0::00 02.00000. :0... 0:0 9:0..00 .0 00:05 N 0:. :0. 000... 0:0; :00..:0 0:0 800...: .0 00:20: .:0.0...o_. .ow H E00 :09: 00 00000.90 0. 0.00.. ommd mo fim vmoé Pom; H0000 H03... Heme... . H0000 0% 5.0 08.9 80.9 movd 53.0 H o 5.00 H 03.9 H mohm H mama: Den. mmmd Kmd F DZ 52 H mmodm H 90.8 < :00 80.9 55. P 80.9 30.5 H 5060 H w 56 H m 5.03 H m 5.0.1 0:00:00 0:0 5550005000050 0. 0005000. 0.500.003 0309.052 :0 000.0 50.50.0000 .5550 50.. 0.0.500 0:0 0.000.205 00.00.500.050. .0 50.. 0.00 .0 0.00..m >.w4m._._mw>mw ZO_ww._ 255 .0. .0. .5 .o. 4<2¢OZ no [om \ o 0 n W S d aJu \. 8. 0. U m a X m nomw 9 [com 85 Figure 5. Inhibition of blastogenic responses of lymphocytes from Q. volvulus infected subjects and non-endanic controls from Sudan in the presence of _g. volvulus female Secretory/Excretory products. Concentrations of 9. volvulus S/E, O.v. sAg and PPD were 20 ug/ml. 22:2 (1 $0 § 00000 mg; ‘1 d l 33 o o F ‘ l l I I l a? a? a? a? a? o o o o o co :0 v N sesuodsel 915004de 10 uomqguul +PPD < c O 0 o' + 0 PHA PPD + .2 g». 0)- 0 II- + Is ntro endemic co % Non- infected - 0. volvulus 87 Figure 6. Effect of Q. volvulus microfilariae Secretory/Excretory products on lymphocyte blastogenic responses to mitogens. i y—i W. “H 100% ' I I BE a? l a? a? o o o o co co 0 N SGSUOdSGJ eMooqduMl go uogquuul l a? O Nodular mi S/E E0 \ x0) Nodular mf S/E skin mf S/E @ConA - PHA 89 volvulus mf with PMMZ, also caused inhibition of lymphocyte responses. When 30 skin mf were added to each well there was almost canplete inhibition of PHA and Con A responses (Figure 7), but when the mf from nodules were used, higher numbers were needed to cause significant inhibition. Nodular mf at 50 mf per well had no effect. Inhibitory effects were not different when either _g. volvulus infected individuals or non—endanic normal controls were the donors of PMMZ. Cell viability in the presence of all S/E preparations was main- tained at greater than 90% for up to 6 days. The inhibitory effect of 9. volvulus female S/E was concentration dependent, (Figure 8) declining to undetectable levels at 1.2 ug/ml. ‘Ihe activity of S/E from either source was not lost on dialysis (M.W. cut—off 10,000) and was stable on prolonged storage at -70°C and on exposure to 56°C for 30 minutes. Addition of exogenous IL-2 (20 iu/ml) partially overcame the g. volvulus S/E inhibitory effects at 20 ug/ml, while at lower S/E concentrations, the inhibitory effect was not detectable in the pres- ence of IL-2 (Figure 8). line amount of IFN-Y produced from mitogen stimulated cells was substantially reduced when PMMZ were cultured with either 9. volvulus female inhibitory S/E or 9. volvulus skin mf. In the presence of 20 u/ml IL-2, IFN-Y production was improved, but not to control levels in either case (Figure 9). 90 Figure 7. Effect of co—cultivation of g. volvulus microfilariae on Sudanese '9. volvulus infected patients and controls lymphocyte blastogenic responses to mitogens. I0 I\\\ \\ \\\\\\\\\\\\\\\\\\\\\\\\\\\ r a? a? a2 a? O O O O Q V N sesuodseJ eMooquuAI go uomngul 50 skin mf Nodular mf Nodular mf skino mt Nodular mi Nodular mi 120 30 @ConA - PHA Figure 8. Effects of serial dilutions of 9. volvulus female Secretory/Excretory products on lymphocyte blastogenic responses to mitogens. Addition of interleukin-2 at 20 U/ml partially overcame the inhibition. 93 0-... .0 05000.0 Em 00320.. .0 w 05 c. 800...... WW. .0 805...... I 5.000.. 5.00.0.0 5.010 5.000. 5.0000 _ N \ \ is .u. [.000 m \ 0. U 0.. 200.. I \ 0 d U. 0 ...\ 00 m \ . .. 0 S 10 lo \ 000 m S ...\.8. Figure 9. Effect of Q. volvulus Secretory/Excretory products and skin microfilariae on the production of Interferon Gama by mitogen stimulated lymphocytes . 95 0-0_.:=.;.0 .<:0 2:00; .200 kD) were seen, with up to 25 bands recognized by some sera with IgG specific probes. Subclass specific monoclonal probes revealed a predominance of 1964 responses, often accounting for almost all the band spectrum seen with 196 reagents. Many individuals had little or no evidence of reactivity in other subclasses. There was, however, no obvious relationship between band number, location or intensity, and clinical or parasito- logical status, except that there was overall more reactivity with Sudanese rather than Sierra Leonean sera. Within Sudanese sera subsets there was a trend towards higher clinical severity scores in patients whose sera recognized more and more deeply staining bands. Specific patterns of band recognition with 1963 probes were not associated with Sowda patient sera, unlike previous reports. The most intense IgGB reactivity was observed in an asymptomatic Sudanese patient. Anti IgE probes revealed fewer bands with lesser intensity, compared to anti 107 196, and they were also not peculiar to any patient subset. Antibodies were detected to up to 10 bands in secreted/excreted (S/E) antigens frcm adult females of Onchocerca volvulus maintained _in vitro, but these corresponded in position to bands in soluble worm extracts. Altogether 15 polypeptid S/E bands were labelled with 355 methionine under these conditions, but the use of these materials does not seem to offer advantages in exploring antibody responses to 9. volvulus and their procurement is difficult and costly. 108 INTRODIETION Recent explorations of the relationships between the clinical manifestations of human filarial infections and patterns of inmune responses have begun to identify potential correlates of pathogenesis 1. In lymphatic filariasis, where categorisation of patient subsets has been based on well established parasitological and clinical crite— ria, distinct characteristics of immunoblot profiles have emerged in each group that provide insights into mechanisms of diseasez. In human onchocerciasis immunoglobulin and antibody responses have not been as well examined so far, and the picture is less clear. This may be due in part to the spectral nature of pathologic responses and the complex- ities of evaluation of a wide range of changes in dermal and ocular tissues. It is also likely to be due in part to the geographic varia- tion of the disease in endemic foci3, perhaps attributable to recently identified genetically distinct "strains" within 9. volvulus 4. Nevertheless, recent reports suggest associations between antibody responses in immunoglobulin subclasses and clinical signs that are of central importance to understanding the nature of onchocercal lesions, and the functions of immunoglobulin subclasses in disease causations. We have been involved in the epidemiological characterization of onchocerciasis in a number of distinct foci in Sudan where, even within the country, the disease assumes very different forms in each of the provincial foci6. We have developed a comprehensive clinical scoring system based on the severity of acute and chronic changes that has proven valuable in classification of lesions and their changes over time in relation to parasitologic status7. We report here on immunoblot 109 profiles in clinically distinct patient subgroups, from savannah and desert regions in Sudan, and some comparative aspects with regard to similarly examined samples from the forest foci of Sierra Leone8. 110 MATERIALS AND METHODS Study Areas. Patients from four different geographical areas endemic for onchocerciasis were studied. Three are in Sudan, and each shows some slightly different characteristics in the prevailing form of the disease: Abu-Hamad in the Northg; Sundus in the East7, and Bahr El Gnazal in the southwestlo. An additional group of patients was examined and sampled in Bo, Sierra Leone, West Africa8. Individuals Studied. A total of 90 serum samples were collected from groups of 10-20 patients each in Abu flamed, Bahr El Gnazal, Sundus, and Bo (Sierra Leone). The selection of patients for immuno- blot studies was made to ensure representation of the range of prevail- ing clinical and parasitological features in each focus. Control patients samples included those from individuals in the endemic areas who were parasitologically negative, and others from Central Sudan, who were resident in a non-endemic region. Clinical Examination. All participants were given a complete physical examination with special attention given to the palpation of _Q. volvulus nodules, and to the evaluation of skin and eye changes consistent with _g. volvulus infection. The history included details of any antifilarial treatment received and of surgical removal of nodules. Details of the examination procedure and the assessment of lesions are described elsewhere7. As a result of physical findings, individuals were classified primarily on the basis of the type, distribution, chronicity, and severity of skin changes, using a point scale of 0-3, with 3 being most affected. For the purposes of comparison 3 groups of symptonatic patients emerged from this scheme; those with mild disease (score of l), moderately affected people (score of 2) and those sev- 111 erely afflicted (score of 3). Asymptomatic patients with no signs or symptoms of onchocerciasis, but with microfilariae in their tissue biopsies were given a score of O. Parasitological Examination. To determine the microfilarial intensity of 9. volvulus infection in each individual, bilateral skin biopsies (snips) were taken from the iliac crest using a Walser cor- neoscleral punch. Skin snips were then incubated in tissue culture medium RPMI 1640 (Sigma Chemical Co., St. Louis, M0) for 4 hours. Emerging microfilariae were counted and the wet weight of the snips was determined. Microfilarial intensity was expressed as the number of microfilariae per milligram of skin (mf/mg). Urine, stool and blood samples fron patients in Abu Hamad and Bo were examined for other parasites by conventional techniques. Preparation of O. volvulus Soluble Antigen (sAg) and Secre- tions/Excretions (S/E). Intact nodules were surgically excised from patients under local anesthesia. To obtain live female parasites, nodules were digested in collagenase using the method described by Schulz-Keyll. Trimmed excised nodules were incubated in culture medium RPMI 1640 containing 10% pooled normal human sera, penicillin, 100 u/rml, streptomycin 100 ug/ml (Gibco Laboratories, Grand Island, NY) and collagenase type IV (Sigma Chemical Co.) at a concentration of 2 mg/ml. Digestion was carried out at 37°C for 12 hours after which fenale parasites were recovered by gentle dissection. Isolated adult females were thoroughly washed in serum-free RPMI 1640 with antibiotics. Q. volvulus female worms were maintained individually at 37°C in RPMI 1640 supplemented with antibiotics for 48 hours. Culture superna— tants were dialyzed at 4°C against three changes of 1,000 volumes of 112 phosphate buffered saline, pH 7.2 using Spectrapor membrane tubing (Spectrum Medical Industries Inc., Los Angeles, CA), with a molecular weight cut off of 10,000 Daltons. Supernatants were then filter- sterilized, aliquoted and stored at -70°C. The protein concentration in supernatants was measured using the Lowry methodlz. Additionally, to obtain radiolabeled S/E, live 9. volvulus female worms were cultured in RPMI 1640 supplemented with antibiotics in the presence of S35 methionine (DuPont, Boston, MA), specific activity 1086 Ci/mmol, at a final concentration of 80 uCi/ml, for 24 hours. Proteins in the culture supernatants were then precipitated by 5% trichloroacetic acid (TCA). For sAg preparation, isolated _g. volvulus fenale worms were homogenized in phosphate buffered saline, pH 7.2 and extracted over- night at 4°C. Particulate material was pelleted by centrifugation at 5,000 x g/30 minutes and the supernatant was further clarified at 100,000 x g/l hour. Immunoblotting Procedure Sodiun dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) of _g. volvulus sAg, and S/E was performed according to the method of Lammeli et al.13, using a 7.5-15% gradient gel. The stacking gel was 3%. The separated proteins were electrophoretically transferred onto nitrocellulose sheets according to the method of Towbin et al.14. The portion of the nitrocellulose membrane containing molecular wt. standards and Q. volvulus sAg was then stained with amido black. The rest of the membrane was cut longitudinally into 0.5 cm strips. 113 The strips were then reacted with individual patient's sera (90 sera for Q. volvulus sAg and 10 Sera for S/E) diluted 1:200 following an overnight blocking with 1% BSA and 0.5% tween 20. After washing with Tris buffer, pH 7.2, the strips were incubated with anti-human IgG, IgGl, IgGZ, IgGB, 1964 or IgE mouse monoclonal antibodies (ICN Immunobiologicals, Lisle, IL, USA), diluted 1:2000. Following washing, the strips were incubated with horse radish peroxidase enzyme conju- gated goat antimouse antibody at a dilution of 1:2000. These concen- trations were arrived at by bracketing those recommended by the manu- facturer in preliminary tests. Additionally, peroxidase conjugated, affinity purified polyclonal, antihuman IgE (ICN Immuiobiologicals) was used. The substrate solution consisted of diaminobenzidine (0.5 mg/ml) and hydrogen peroxide (0.15%) in phosphate buffered saline, pH 7.2. The reaction was stopped after 15 minutes by washing with cold water. Visualization of different protein bards ard estimation of bard intensity was determined visually. Analysis of S35 Labeled O. volvulus S/E: s35 methionine labeled S/E, precipitated by TCA were solubilized in SIS sample buffer ard separated by SDS PAGE using the protocol described above. Autoradiographs of dried gels were exposed for 7 days. Immunoglobulin Qlantitation: Total 196, IgM, and IgA levels were determined in patient's sera using commercially available radial immunodiffusion plates (Kallestad, Austin, Texas). Total 1913 levels were measured by an enzyme immunoassay (Abbot Laboratories, North Chicago, IL). 1 l 4 RESULTS The parasitological and clinical features of patients in each region were markedly different. Details of the regional characteris- tics have been described elsewhere, (ElKhalifa et al., submitted) but the patient samples used in this study covered the range, in each area, from infected but clinically unaffected to severely afflicted with onchocercal lesions but with no denonstrable parasites in skin biopsies (Figs. 1-4). There were six affected with the localized asymmetric form of the disease in Sindus patient groups (Figs. 5-6), although this syndrome was seen in all Sudanese regions. Altogether 90 samples from infected individuals including 7 with Sowda syndrome, were examined in the immunoblot system. Most patient sera showed high concentrations of total IgG, ard extremely high 1913 titers (Fig. 7). Separation of Q. volvulus antigen extracts on SDS-PAGE produced a reproducible pattern of about 25 bards with molecular weights ranging fron above 200 kD to 12 kD (Fig. 8 Bl). The patterns of 196 responses of the patients varied within the sub— groups, both with regard to the numbers and positions of bards recog- nized and their relative intensity. Sera from normal non-endemic area controls tended to show IgG reactivity with a bad in the 50 kD region, ard occasionally there were diffuse responses at higher NW levels which were poorly resolved. Reactivity to bards representing virtually the whole spectrun of Mw's visible on the SIB-PAGE profiles was present within each patient sample subgroup. Although, in some instances a trerd towards increase 196 reactivity with increased clincal scores was noted in some groups, but for the most part this was lacking in other groups (Fig. 9b) . Bards with m lower than 50kd were consistently more 115 Figure 1. An asymptomatic Q. volvulus infected irdividual with microfilarial intensity greater than 100 mf/mg. Note the presence of an onchocercal nodule while the adjacent skin is essentially free of any acute or chronic changes except for a single small papule. Figure 2. Discrete papular eruption seen in this patient, confined to the upper back, giving him a clinical score of l. The microfilar- ial intensity in the skin of this patielt was 20 mf/mg. 116 117 Figure 3. Severe onchocercal dermatitis is seen in this patient (clinical score of 3) . Note the macropapular eruption with lichenfi- cation of the skin. Hypo and hyperpigmented, indurated papules are apparent. Some of these papules are excoriated, others are healing, while few have secordary bacterial infection. Figure 4. Acute severe onchocercal dermatitis (score of 3) is seen in this patient mostly in the form of acute papular eruption and excoriation marks. Note areas of hypo ard hyperpigmentation (left upper back) . 118 119 Figure 5. A "Sowda" patient with extensive dermatitis of the left leg while the right leg is spared. Note the hyperpigmented maculo- papular eruption as well as the edema of skin in the left leg. It is worth noting that examination of this patient, one year after taking this photograph, showed remarkable improvement of the dermatitis in her afflicted limb, without any anti-onchocercal therapy. Figure 6. Localized onchocercal dermatitis affecting the right arm of this patient. Note the maculopapular eruption as well as the thickening and hypertrophy of the skin. The features seen here fits the category of Sowda syndrome, despite the unusual upper arm loca- tion. 120 Figure 7. Total Immunoglobulin levels in onchocerciasis patients studied. Total level of serum 196 is elevated (mean 3202 _+_ 1288 mg/dL). More notable is the extreme elevation of 193 (5668 i”. 5739 iu/ml). These high 198 levels are not associated with any intense recognition of Onchocerca volvulus soluble antigens by western blotting technique, as shown in Figure 10. 122 _E\:_ E .950 § 439: E 0:00 I NJ in! so. \ 0.0 . 000; 000.N 000.0 000.v 000.0 000.0 Figure 8. SDS-PAGE of 9. volvulus antigens and secre- t ions/excret ions . A. An autoradiograph of SDS-PAGE, S35 methionine labeled, TCA precipitated secretions/excretions of Q. volvulus female wonms, showing at least 15 distinct protein bands with various molecular weights. B.l SIB-PAGE of adult Onchocerca volvulus PBS soluble extracts stained with Coumassie blue. 8.2 SDS-PAGE of adult Onchocerca volvulus secre- tions/excretions stained with Coumassie blue. Molecular weight markers are seen on the right. 124 ,. . 5ft.“ '3 . a ’r Figure 9. IgG recognition pattern of 9. volvulus antigens separated by SDS-PAGE ard electrophoretically transferred onto nitrocellulose filters. A. Sera from patients with different clinical presentations as well as controls were used. Lane 1, Non-erdemic control; Lanes 2 and 3, endemic controls; Lane 4, asymptomatic; Lanes 5 and 6, mild disease (score of 1); Lane 7, moderate (score of 2); Lane 8, severe disease (score of 3); Lanes 9 and 10, Sowda patients. Note the increase in the number and intensity of the bards recognized with the increase in the severity of the disease. The endemic ard non-erdemic controls show non specific reactivity at the level of 50 KD as well as high MW bards. B. 196 reactivity in another set of Q. volvulus patients with a spectrum of clinical manifestations. Lane 1, normal individual; Lanes 2 and 3, patients with a score of 3; Lanes 4 ard 5, patients with a score of 2; Lane 6, a patient with a score of 1; Lane 7, an asymptomatic patient . It is apparent here that the IgG reactivity of those patients does not necessarily correlate with their clinical presentation. 126 emuemm N 127 sharply separated. Most prominent among bards commonly recognized were those at W 43 kD, 33 kD and 21 kD (Fig. 9a and b) where 80% of patients, but none of the erdemic controls reacted to them. Reactivities with anti IgE probes were generally more limited in scope and intensity (Fig 10). These reactions were not enhanced by altering the protocol to inclLde a secord (enzyme-labelled) antibody directed against the unlabelled class specific anti IgE probes. Gener- ally less number of bands spanning the m of 12-220 kD were detected, but with no obvious association with particular patient subgroups or their infection or disease status. 01 further analysis of IgG responses with immunoglobulin subclass- specific probes the predominance of IgG4 in the antibody reactivity of sera from patients in all categories was most striking. In many cases bards recognized by 1961, 1962, ard 1963 were barely visible (Fig. 11), whereas reactions with the 1964 probe covered the entire range of those seen with the IgG class-specific secord antibody. Repeated absorption of patient's sera with IgG4 specific antibody failed to enhance reac- tions with other subclass specific probes (data not shown). The IgG4 responses, mirrored those of total IgG regardless of the clinical or prasitological status (Fig. 12), revealing the same marked heteroge- neity as with 196 reactivity. No clear evidence emerged of a relation- ship between the degree of infection or clinical severity ard blot profiles with any of the anti IgG reagents; however, there was an overall trerd towards greater intensity in blots from Sudanese patients from all foci when compared to sera from forest zone patients in Sierra Leone. There was also a terdency towards more bards with a higher 128 Figure 10. IgE reactivities of the same group of patients in Figure 9A. Although some of those patients have total IgE levels in excess of 18,000 iu/ml, there is an apparent overall reduction in the number ard intensity of bands recognized by those patients. Neither the clinical status, nor the level of parasite burdens were related to the recognition patterns . 129i. 130 Figure 11. Q. volvulus antigen recognition patterns by different IgG subclasses. IgGl (l); IgGZ (2); 1963 (3); IgG4 (4). A. IgG subclass recognition pattern by a serum from an asympto- matic infected patient. B. IgG subclass recognition pattern by a Sowda patient. Note that most of g. volvulus antigens are recognized by IgG4 with minimal contribution from the other IgG subclasses. No differ- ences as to the recognition by 1963 or any other IgG subclass can be identified in those two patients representing extremes of clinical presentations. LL 131 132 Figure 12. Total IgG and IgG4 recognition pattern of 9. volvulus antigens. A. Total IgG recognition pattern by a group of patients with different clinical manifestations. B. IgG4 recognition by the same group of patients, reflecting almost identical pattern of the antigenic bands recognized. In most of our patients, the reactivities seen with total IgG can be attrib- uted to IgG4. 134 intensity of staining with sera of patients with moderate to severe disease rather than with sera from asymptomatic irdividuals. Reactivity of IgG antibodies was pursued with particular attention to IgG3 responses in cases showing the asymmetry ard lymphadenopathy associated with the Sowda syndrome. In several patients with severe Sowda or early Sowda-like afflictions blot patterns were not distin- guishable from those of other patients in their respective subgroups (Fig. 11). ENen where IgG3 positivity was present, reactions of the sane sera with the anti IgG4 probe were much more intense and directed to a wider range of antigens. Several of the more pronounced IgG3 response patterns, especially to lower molecular weight bards, were seen with sera of patients who were asymptomatic. SOS-PAGE of Q. volvulus S/E showed the presence of 10 protein bards with varying molecular weights (Fig. 832). All of the S/E protein bands had correspording bards of the same we in the Q. volvulus sAg SDS-PAGE preparation. Experiments with S35 labelled methionine incorporation demonstrated that up to 15 labelled polypeptides were produced by adult females maintained i_n y_i£_r_o_ (Fig 8A). When 10 patient sera were tested against these S/E antigens, 12 bards were recognized. Again, different sera recognized different spectra of antigenic bards and there was no clear influence of the clinical presentation on the band recognition pattern. All of the protein bards appeared to be present in antigelic extracts of the entire worms, so that there did not appear to be any advantage to using the S/E fluids, especially since they were much more difficult to collect in quantity. 135 DISCUSSION Patients infected with Q. volvulus in endemic areas seem to react in widely disparate ways to the presence of the parasite. In the absence of accurate longitudinal assessment of clinical and pathologi- cal changes, it is difficult to construct a picture of the course of the disease. In our groups some individuals were both heavily infected and severely affected, whereas others were heavily infected and asymp- tnmatic; yet others were clinically afflicted with many changes consis- tent with onchocerciasis, but without demonstrable parasites in their skin tissues. The relationship between infection intensity and disease was different between groups, with a positive correlation between these two factors established in the forest zone patients from Bo, ard the reverse being true for the patients examined in Surdus7. In the face of this diversity, we were unable to identify any clear traits with respect to bard specificity, scope of reactivity, or intensity that could be implicated in pathologic differentiations of subgroups. There was a tendency for those from the forest zone to recognize fewer bands ard with less intensity than sera from Savannah regions in Sudan, but this was not marked. Previous workers have noted such differences between groups of patient samples collected from savanna and forest zones of W. Africa3. Brattig, et al.1 also examined patients with the asymmetrically localized hyper-reactive form of the disease, referred to loosely as "Sowda" syndrome. They reported that such individuals have higher titers of IgG antibodies in Elisa tests. Our qualitative immmmoblot profiles, although showing a trerd towards greater IgG reactivity of Symptomatic patients' sera, did not reflect this characteristic consis- 136 tently. On occasion, strong IgG responses similar to those described 31 by Lucius were noted by asymptomatic patients. 196 profiles of Sowda patients ard others were marked by one extraordinarily consistent trait: the large contribution of antibodies of the IgG4 subclass to the reactivity seen. In many individuals it appeared that most of the antibodies they had made, regardless of their clinical or parasitologi- cal status were of this type. Observations such as these have been made recently in other helminthiases in man,27'23, and 1964 responses form one of the hallmarks of antibodies detected in human lymphatic filar- iasisz. There, the hypothesis has been constructed that IgG4 may function in a protective or blocking role, ameliorating reactions potentially generated by the high levels of IgE antibodies to m, or Wuchereria antigen in these patientsz'ls. Concentrations of 198 are elevated in human onchocerciasis,16'22 but the blot profiles in our studies showed a rather restricted pattern of reactivities compared to those seen in lymphatic filarial infections“. The parallel antigenic recognition by IgE ard IgG4 reported in lymphatic filariasis20 was not observed in our study. Moreover, the IgE reactions in our patient groups showed no discernable trerds related to clinical signs or parasite burdens. The relatively low number of bards detected in our IgE specific blots, regardless of whether or not direct enzyme-labelled probes were used or an IgE specific enzyme-labelled secord antibody was involved, contrasts sharply with the results reported by Weiss et al.18. They found up to 50 bands detectable with anti IgE in their onchocerciasis serum pools from sub-Saharan regions of W. Africa. It should be mentioned here that the authors' experimental protocol involved high concentrations of patients' sera (sera were diluted 1:2) , 137 while we used sera diluted 1:200. It is possible that the majority of parasite specific IgE are present, but at low concentrations and with high dilution they are essentially titrated out. Our results, however, reflect similar findings to those of Nutman et al.30, where high levels of non-specific 1913 were prevalent among their loiasis patients popula- tion. We were unable to demonstrate any association between IgG3 reacti- vity to any bands and the presence of Sowda-like lesions. Recently Cabrera et al.5 have implicated IgG3 in the pathogenesis of this syndrome, showing that high levels of IgG3 antibodies were exclusively associated with Sowda patients and were directed preferentially against a 72 kD MW antigen ard a low PW antigen in the 9 k0 region, the latter being most specific. In our immunoblotting system, the lowest identi- fiable Mw Q. volvulus antigenic bard was in the 12 KD region, similar to other published reports31. Thus, we are unable to comment on the recognition of the 9 KD band ard its relation to Sowda. This discre- pancy might be due to our use of PBS soluble worm antigenic prepara- tions rather than the detergent solubilized extracts used by the above authors. As for the 72 KD bard, sera from Sowda patients in our study were not differentiable by this means. In the Cabrera et al. report, antibody recognition profiles by Sowda patients' serum pool, both the 9KD ard the 72 KD bands were strongly recognized by IgG3, but neither bard was recognized in the total IgG blots on the same patients' serum; no explanation for this difference was offered. In our series, IgG3 antibody reactivities were weak and when present they were seen equally in individuals with no dermal clinical changes suggestive of onchocer- ciasis. The reasons for these differences remain to be established, 138 but for the moment generalizations about the involvement of IgG3 responses in this syndrome do not seem justified. Parasite genetic factors, patterns of exposure and host genetic traits may all play a role in these phenomena. Some of our patients were infected with Mansonella Erstans, ard those in W. Africa had a higher rate of infection with gastrointestinal helminths than did the Sudanese groups, but these intercurrent para- sitic problems did not detectably influence the antibody profile. Possibly some of the antibodies demonstrated in blots may have been against antigens shared with gastrointestinal nematodes because filar- iae are known to have many antigens in common with major human hel- minthslg. Interestingly, some of the most consistent and best resolved areas of reactivity on blots were in the lower molecular weight regions where antigens of greatest 9. volvulus specificity have been identifiedzo'n. Of special interest to us were the 33 KD ard 21 KD antigelic bards, the recognition of which has recently been shown to be of greatest speci- ficity and sensitivity (>90%) in the diagnosis of 9. volvulus infection21. Although these two bards were recognized by the majority of our patients ard by none of the erdemic controls, their utility as a specific diagnostic test for 9. volvulus infection in our patients series is not evident because of the lower sensitivity (80%) of detec- tion by this means. The prospects for discriminating between subsets of patients with specific diagnostic assay configurations seem dirm in the face of the heterogeneity displayed here. Ehrther progress on this ad other aspects of the irmmulology of the host-parasite relationships 139 in this infection probably will require a focus on pmrified or rDNA- derived defined antigens for more meaningful interpretation. Our analysis of S/E showed that the antigenic bands seen are similar to those present in 9. volvulus soluble antigelic extracts, at least in terms of their molecular weights. These antigens are clearly synthesized by the worm as shown by the 353 methionine incorporation experiments. Although there is evidence from other helminths of advantages in using secreted or excreted antigens in serological studie524'25'26, this was not apparent in our limited analysis. In view of the extreme difficulty of procuring live wonms for this purpose ard the scanty harvest of material for study it does not appear to offer worthwhile advantages. 140 ACKNOWLEDGMENTS This work was supported by grants from NIH Grant # AI-l6312, Fogarty International Fellowship #1FOSTWO3637 and the Wolfson Fourda- tion. Particular thanks are due to the many cooperating patients and medical staff in Sudan provincial villages and at the MRC Center, Bo Sierra Leone who made this study possible. 10. ll. 12. REFEREMIES Brattig, N.W., F.W. Tischerdorf, B.J. Albiez, D.W. Buttner, ard J. Berger. 1987. Distribution pattern of peripheral lymphocyte subsets in localized and generalized forrm of onchocerciasis. Clin. Immunol. ard Irmmunopathology. 44:149. Hussain, R., ard E.A. Ottesen. 1985. IgE responses in human filariasis III. Specificities of IgE and IgG antibodies compared by immunoblot analysis. ._J_. Immunol. 135:1415. Lucius, R., J. Prod Hon, A. Kern, G. Hebrando, and H.J. Diesfeld. 1987. Antibody responses in forest and savana onchocerciasis in Ivory Coast. Try. Med. Parasit. 38:184. Cianchi, R., M. Karam, M. Henry, F. Villani, S. Kumlien, and L. Bulini. 1985. Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa. Acta Trop. 42:341. Cabrera, Z., D.W. Buttner, ard R.M.E. Parkhouse. 1988. Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper reactive oncho-dermatitis (Sowda). Clin. BE. Immunol. 74:223. Mackenzie, C.D., and J.F. Williams. 1985. Variation in the clinical presentation of onchocerciasis ard their relationship to host parasite interactions. Sudan Med. g. 21(supplement):“. Gialib, H., C. Mackenzie, J. Williams, H. E1511eikh, M. ElKhalifa, and M. Kron. 1987. Severe onchocercal dermatitis in the Ethiop- ian border region of Sudan. Ann. Trop. Med. Parasitol. 81:405. McMahon, J.E., J.B. Davies, M.D. White, J.M. Goddard, P.A. Beech- Garwood, and B.R. Kirkwood. 1986. Onchocerciasis in Sierra Leone. I. Studies on the prevalence and transmission at (baiima village. Trans. Roy. Soc. Trop. Med. m. 80:802. Williams, J., A. Abu Yousif, M. Ballard, R. Awad, M. ElTayeb, and M. Rasheed. 1985. Onchocerciasis in Sudan: The Abu Harmad focus. Trans. R_y. Soc. Troi. Med. Hyg. 79:464. Mackenzie, C., J. Williams, J. O'Day, I. (halal, H. Flockhart, ard B. Sisley. 1987. Onchocerciasis in Southwestern Sudan: Parasito- logical ard clinical characteristics. Ar_m_. g. _Tr_g- 1183- 529,- 36:371. Schulz-key, H., E. Albiez, and D. Buttner. 1977. Isolation of living adult Onchocerca volvulus from nodules. TroEnmed. Parasit. 28:428. Lowry, 0., J. Rosenbrough, A. Farr, and J. Randall. 1951. Protein measurement with the folin phenol reagent. _J. Biol. 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Wyss, ard B. Betschant. 1982. Detec- tion of IgE binding Onchocerca volvulus antigens after electropho- retic transfer ard Immunoenzyme reaction. Acta Tropica. 39:373. Ambroise-Thomas, P. 1974. Immunological diagnosis of human filariasis: present possibilities and limitations. Acta Tropica. 38:108. _- Lobos, E., ard N. Weiss. 1986. Identification of non-cross- reacting antigens of Onchocerca volvulus with lymphatic filariasis serum pools. Parsitol. 93:389. Lucius, R., H. Schulz-key, D.W. Buttner, A. Kern, B. Kaltmann, J. Prod Hon, F. Seeba, R.D. Walter, K.C. Saxena, and H. Diesfeld. 1988. Characterization of an immunodominant Onchocerca volvulus antigen with patients sera and a monoclonal antibody. 3. _p. @. 167:1505. Somorin, A.C., D.C. Heiner, ard R.E. Ajugwo. 1977. Immunoglobu- lin E in Nigerian onchocerciasis. 31.“: _J_. Tr p. Maid. Hyg. 26:872. Hofstetter, M., R.W. Poindexter, E. Ruiz-Tiben, ard E.A. Ottesen. 1982. Modulation of the host response in human Schistosomiasis III. 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Parallel recognition of filarial antigens by IgE and IgG4 antibodies. New Eng. Reg. Allergy Proc. 9:51. Nutman, T.B., W. Reese, R.W. Poindexter, and E.A. Ottesen. .1988. Immulologic correlates of the hyperresponsive syndrome of lolaSls. g. Infect. Dis. 157:544. Lucius, R., D.W. Buttner, C.A. Kirsten, and H.J. Diesfeld. 1986. A study on antigen recognition by onchocerciasis patients with different forms of disease. Parasitology. 92:569. APPENDIX 1 CLINICAL EVALUATION OF ONCHOCERCIASIS PATIENTS Clinical evaluation of patients included detailed past and present medical history. Special attention was paid to past history of skin problems. History related to previous diagnosis of onchocerciasis and whether treatment was given was also inquired about. Occupation, life style ard possible exposure to black fly bites was also investigated. General physical examination was then performed on all partici- pants. This included examination of head and neck, cardiovascular and respiratory systems, abdomen and limbs. The presence or absence of onchocercal nodules was determined by visual inspection and palpation. Enlargement of superficial lymph nodes was also noted. Special attention was given to examination of the skin ard the eyes. Assessment of Skin Lesions: The skin of all individuals studied was carefully examined for visible changes ard a detailed record of results was kept. Skin changes were divided into acute ard chronic lesions: 144 a. Acute skin changes: 1. Pruritus and itching marks 2. Papules, either discrete or containing microabscess 3 . Skin edema b. Chronic skin changes: 1. indurated papules which may be hyper or hypopigmented. 2. Hyperkeratosis and lichenification of skin 3. loss of turgor (atrophy) 4. Confluent depigmentation. Each acute ard chronic change was quantitated using a scoring system. The body is divided into head and neck, arms, torso, pelvic girdle and legs. The absence of acute changes was given a score of 0. Presence of an acute lesion in one or two body areas was given a l. The presence of a lesion in more than two was given a score of 2. Tutal scores for acute changes, chronic changes and the overall combined score for skin lesion severity can then be quantitated. Accordingly, 4 groups of patients can be identified. These are: a. Asymptomatic patients b. Patients with mild skin disease c. Patients with moderate skin disease d. Patients with severe skin disease Ocular Changes: Ocular examination included assessment of visual acuity using an illiterate E. chart. Anterior segment changes i.e. punctate kerati- tis, uveitis, iritis, and sclerosing keratitis were looked for and in some patients a slit lamp examination was performed. Posterior segment changes (chorioretinal and optic nerve changes) were deter- mined using an ophthalmoscope. Figure 1. An asymptomatic patient with an onchocercal nodule in the pelvic region. m. A patient with localized severe onchocercal dermatitis and femoral lymphadenopathy. This patient fits the classical description of Sowda . 148 149 Egg—3: Severe acute onchocercal dermatitis with papular erruption, excoriation marks ard pigmentory changes (score of 3) . 150 151 Figure 4. Discrete papular erruption localized to the upper back (score of 1). Figure 5. Moderate acute changes (score of 2). Excoriated papules, and early pigmentory changes can be seen. 152 Figure 6. Another Sowda patient in which the left limb is severely afflicted. Figure 7. A patient with generalized severe onchocercal denmatitis. Note the indurated hypo ard hyper pigmented papules. Some of the skin changes seen on this patient are similar to those seen in the Sowda patient in Figure 6. 154 155 Figure 8. Marked atrophy of the skin, a consequence of chronic onchocerciasis. 156 157 Figure 9. Severe secondary bacterial infection in a young boy with Q. volvulus infection. This is not an uncommon consequence of pruritus, itching, ard excoriation. 158 Figure 10. Classical depigmentory changes seen in both lower legs of this patient. 160 161 Figure 11. Choreoretinal atrophy with focal pigmentation. These eye changes are not uncommon in onchocerciasis. Figure 12. (ptic nerve atrophy in an onchocerciasis patient. 162 APPENDIX 2 PATIENTS' DATA RECORD FORM 163 164- MICHIGAN STATE UNIVERSITY MEDICAL PARASITOLUGY RESEARCH PROJECT mans: DATE: mo. _I‘L _“_. AGE: sex: a ' 1 . OCCUPATION: ““"' L__1 .__J PRESENT RESIDENCE: PREVIOUS RESIDENCE: x u HISTORY or TRAVEL? ; ' r : WHERE? wmmu? _. Y N Y N COKPLAINTS : SCRATCNINC ‘ 1— W OTHERS I 1 SPECIFY : L_1 L__1 FOR HOW LONG? Y N Y N ANY SEASONAL VARIATIONS? ALL YEAR AROUND D I l SURIER l l D WINTER .1 1 I IS IT RELATED TO BLACK FLY BITE? : l 1 I Y DOES SCRATCNING INCREASE BY SUI-EATING? i l DOES SCRATCH-TING PREVENT YOU FROM SLEEPING? SIT! OF SCRATCNING: HEAD D NEON UPPER LINES BACK D D 30110ch [:1 (mom :1 HIPS D D raomxx [::] ABDOHEN mamas [::] FEET DID YOU NOTICE ANY NODULE 0R SWELLING ON YOUR BODY? LEGS D WHERE? DUDE x N ANY FAMILY xrnnsx wrrm THE SAME CONDITION? 1 i [::] PAGE I 165 Y HAS YOUR VISION CHANGED SINCE You EXPERIENCED IRIS CONDITION? 1 1 1.1 HAVE YOU HAD TREATMENT FOR II? 1:1 1:1 Y N . 11“ YES. WHAT DRVC? SURANIN 1—1 m now MANY INJECTIONS? VNEN? u—d ’— Y _N DEC 1‘" 1 ‘7 now HANT 'I‘MHJ'I'I‘S? __.___-_ WHEN? Y N OTHERS 1 1 1 1 SPECIFY: Y N DID You DEVELOP ANY HYPERSENSITIVITY REACTION? 1 1 1:1 TO “NAT EXTENT? Y N ANY ACCOMPANYING IMPAIRMENT or VISION? 1 1 1:] Y N HAVE YOU BEEN DIAGNOSED AS HAVING SCHISTOSOHIASIS? 1::] 1 1 WHEN? Y N DID You RECEIVE TREATMENT? D 1 1 VNEN? Y N IS THE COLOR OF YOUR URINE ABNORMAL? 1::] 1 1 IF YES. SPECIFY: IS YOUR sroor. NORMAL? [:1 1:1 IF YES, IS THERE: DIARRHBA I 1 mucus D 31.00:: D OTHERS 1:1 SPECIFY: __ Y N HAVIYOUHADHALARIA? 1 1 1:1 mm PULSE: 3.9.: PAL“ 1 1 1:1 CYANOSED:1 1 1:] sum: ANOREXIC 1: UNDERwEICNI 1: WWW 1:1 OVERVEICNI D 03331»: 1 1 NORMAL 1:1 comEN'IS ON ABNORMALITY: PAGE 2 166 NECK: THYROID: NORMAL _7 COMMENTS ON ABNORMALITY: CHEST: NORMAL i_I COMMENTS ON ABNORMALITY: HEART: NORMAL 1 COMMENTS ON ABNORMALITY: l--J ,— ARTERIES: NORMAL 1.! COMMENTS ON ABNORMALITY: VEINS: NORMAL COMMENTS ON ABNORMALITY: ABDOMEN : NORMAL [:1 1] COMMENTS ON ABNORMALITY: HUS-SKEL: NORMAL 1:1 COMMENTS ON ABNORMALITY: NAILS : COLOR: PALE 1 1 PINK 1 1 CYANOTIC 1: OTHER 1 I SHAPE/LESIONS: VNL 1 I CLUBBING 1:1 OTNER 1 I LYMPR NODES : CERVICAL: NORMAL I_" CONMENTS ON ABNORMALITY: R. AXILLARY: NORMAL D COMMENTS ON ABNORMALITY: L. AXILLARY: NORMAL I I COWS ON ABNORMALITY: R. INGUINAL: NORMAL I I COWS ON ABNORMALITY: L. INGUINAL: NORMAL D CW8 ON ABNORMALITY: R. POPLITEAL: NORMAL D COMMENTS ON ABNORMALITY: L. POPLITEAL: NORMAL D CW ON ADNORMALII'T: GROIN EXAM NORMAL I I COMMENTS ON ABNORMALI'IY: PAGE 3 167 NO. :zzr: 'IIII‘IIDVI ‘ muz - I.|||I Ill u‘lll' I-.I‘I| Il‘ll'l"- mHmCH= zo~H:;Oxh< RIGHT TBIGN LEFT THIGH RIGHT FOREARM LEFT FOREARM SKIN LESIONS BODY PARTS CHEST AID PAGE 4 1(38 SCORING: ACUTE 11 CHRONIC I P Rx 1 CP 1 PPRx 1 ATR PRU NYK OED I PC 1 i . I I IOTALS NODULES, NUNDERS SHOULDER TRUNK NONE HEAD :IECR L R ANT POST [31:11:11] [:1 DEC} [:1 OTHER C] DE] O NO. PROVISIONAL DIAGNOSIS? HIP E] R COCCYX DU /\ . .. K ./'1. W}. VISUAL ACUITY CODE: 0 I 2 3 r. 5 6 7 8 9 6/6 6/9 6/ 18 6/36 6/60 COUNT FINGERS PERCEPTION NO PERCEP. UNABLE TO NOT AT 3 METERS OF LIGHT OR 0? LIGNT UNDERSTAND DO“! HAND MOVEMENT TEST. UNCOOPERATIVE RIGHT LEFT VISUALACUITY: 0123456789 123656789 ocuun R L R L L_L R i M1 in AC: ‘1 -_I Pupil: Opt. disc: Liv. If: Trach: Irina: Rec. ves: Dead at: Pig. disp: Pig. epith: Coin lea: Lens: C.E.a.: ’1- 0? cor: Vitreous: Rec. doc: Scl. kcr: IOP: PAGE 5 1659 99x >5 VISUAL ACUITY: W SKIN SNIP: SITE 1 2 3 l. SKIN SCRAP INC: RESULTS: NUMBER RIGHT - HEIGHT NO. BLINDNESS: CAUSE: LOCATION: PHOTO: LEFT mm NEIL": 1 WT] 1 [TH 1J1 LJJ [IE] UTFLHUEI IHIWHIH IJHHITU [11111]!!! HIHHTHHHHLlJ—I SKIN BIOPSY: TAKEN 1:1 NOT TAKEN D m: MT. PRESENT ONGNO. V. HUGE. B. LOA LOA TET. P. OTHER 3 hawa 2 2 2 2 2 F‘D-‘H'd" NO. ESTIMATE 0 O 0 0 O FROM WHERE: DIFFERENTIAL L M ED N NBC REC PCB ND PAGE 6 170 NO. SERUM: TOTAL PROTEIN VITA ICE ELISA ICA l/C ICM ADHER. A8. IGG MALARIA AB. ICD OTHER BLOOD TRANSFORMATION ASSAY USING PATIENT'S SERUM: INMIBITION ACTIVATION NO EFFECT # DOMUNOBLOTTING RESULTS: LYMPNOCYTE STIMULATION ASSAY: USING FILARIAL AGS: USING PHYTOHAD‘IOACGLUTININ 1:1 CON A I I OTNERS: CELL TYPINC OF PERIPHERAL BLOOD LYMPIDCY‘I'ES: T - HELPER: I - SUPPRESSOR: B - CELL: TOTAL LYMPNOCYTES: RATIO OF T-NELPER TO T-SUPPRESSOR: COMMENTS: CELL TYPING OF SKIN BIOPSY: ‘t'-HELPER: 1' - SUPPRESSOR: B-CELL TOTAL ‘1' AND B CELLS: RATIO OF T—NELPER TO 'I-SUPPRESSOR: NO. OF LANGERNANS CELLS: COMMENTS: PAGE 7 171 URINE: COLOR: SEDIMENT: BIOCNB‘IICAL ABNORMALITY (SPECIFY): SPECIFIC GRAVITY: NO. STEROID LEVELS: PARASITES: FECES: PARASITES: ADDITIONAL COMMENTS: PAGE 8 ‘11111111111111“