This year, the previously localized DFNA3 and DFNB1 loci on chromosome 13q11, which are associated with non-syndromic deafness, have in Caucasian families been attributed to mutations in codons 24, 34, or 77 of the gene encoding the gap junction protein connexin 26 (Cx26). We have been studying several families of a village in eastern Ghana known nation-wide for an extraordinarily high prevalence of profound non-syndromic deafness. A linkage analysis performed including 29 subjects from 6 families had revealed a lod score of 4.5 at marker position D13S175 corresponding to the known DFNA3/DFNB1 loci. Sequencing of the Cx26 coding region in 21 deaf subjects from 11 families revealed in all of them a homozygous C -> T mutation in the first position of codon 143 resulting in the non-conservative amino acid exchange of an arginine to a tryptophan residue. Twelve heterozygous family members were identified, all of these have normal hearing, which was documented in nine of them by audiometric examinations.
Cx26 is a trans-membrane protein expressed in several structures lining the cochlear duct. By oligomerization into hexamers, it forms trans-membrane toroid structures which fuse to generate intercellular communication channels allowing for the cell-to-cell diffusion of small molecules such as inorganic ions. Since the R143W mutant described here causes a recessive form of disease, its presence appears not to impair the function of the wild-type molecule in heterozygous individuals.
Among the families included in our study, the disease haplotypes differed largely indicating that the mutation arose approximately sixty generations ago. This finding underlines the stability of the deaf community, and, in addition, it shows that the mutation had several centuries to spread into surrounding populations and possibly across the sea.
*) Department of Ear, Nose, and Throat, School of Medical Sciences, University of Science and Technology, Kumasi,
Ghana
 
 

Last year, studies in a population which has been long-term exposed to S. mansoni transmission revealed a susceptibility locus for schistosomiasis on chromosome 5q31-q33. As a phenotype, the quantitative trait of schistosome egg excretion was used after correction for age, sex, and water contacts. We confirmed this finding in a recently exposed group of families but found a considerably weaker gene effect. Therefore, we continued the genome-wide search in this sample to possibly identify additional susceptibility loci. A three-step strategy has been applied. So far, complete scans in 34 members of 2 families and selected typings of 62 members of 7 families yielded trends for five more candidate regions. These are presently being tested in 180 additional individuals from 21 families.
*) Department of Parasitology, University of Leiden, Leiden, The Netherlands
⁺) Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium
 
 

Parkinson’s disease (PD) is a common neurodegenerative disorder of unknown origin. Although it usually occurs sporadically, evidence has been obtained in recent years supporting an etiologic role of genetic factors. This year, a mutation of the a-synuclein gene, which is located on chromosome 4, has been identified by positional cloning as the cause for a familial form of PD in the Mediterranean which differs from the sporadic form by a rapid course and an uncommonly early onset. We have studied six families from central Europe showing evidence for an inherited PD which most closely resembles the widespread, sporadic form of the disease including a late onset at around 60 years. A genome-wide search for susceptibility genes revealed linkage with markers on chromosome 2p13. In two of the families, which originate from Northern Germany and Southern Denmark, evidence was found for a common founder. Interestingly, the penetrance of the mutation appears to be low, most likely below 40%, which is compatible with a role of this locus in the widespread, sporadic form of PD.
*) Neurologische Klinik, Klinikum Großhadern, Ludwig-Maximilians-Universität, München
**) formerly BNI
⁺) Section of Neurology, Department of Internal Medicine, University of Nebraska, Omaha, NE, USA
^#) Neurodegenerative Disorders Centre, University of British Columbia, Vancouver, Canada
^§) Dipartimento di Scienze Neurologiche, Università La Sapienza, Rome, Italy
^&) Neurologische Klinik der Universität Lübeck, Lübeck
 
 

The classical transmission disequilibrium test (TDT) was proposed by Spielman and co-workers in 1993 to analyse family-based case-control studies. Recently Risch and Merikan-gas recommended the TDT as a very useful method for the detection of susceptibility alleles in a genome-wide setting. However, their analysis was based on the very special assumption that the disease-causing allele itself was present among the marker alleles tested (in the following called "optimality assumption"). We show that even minor departures from the optimality assumption may lead to very high sample sizes needed due to drastic power losses of the test. Sample sizes quickly reach more 10,000 study units, thus rendering the strategy proposed by Risch and Merikangas unrealistic. Furthermore, the prior probability for the optimality assumption to hold may safely be considered low, adding to the caution warranted when applying the Risch and Merikangas approach.
*) INSERM U436, Paris, France

The DQ genes of the MHC class II region are single copy genes in the mouse, rat, swine and rabbit. In humans and dogs, multiple DQ genes have been identified but only one DQ molecule appears to be expressed. A feature unique to ruminants is a certain variability in the number of DQ loci in the various haplotypes. In cattle, a total of three DQA and two DQB loci have been distinguished whereby the majority of haplotypes carry two functional DQA and DQB loci. Studying the polymorphism of the bovine DQA and DQB genes in African cattle, we have identified two unusual alleles, muh13 and kuhx, which are most closely related to the previously described DQA and DQB alleles, respectively, and yet differ from these to an extent that they may as well represent distinct DQ loci. RNA analyses using Northern blots and RT-PCR indicate that both genes are expressed. The complete cDNA sequences were determined through RT-PCR. Southern blot hybridisation using 3´-UTR specific probes showed that muh13 and kuhx or similar alleles also exist in cattle of European breeds. The results were confirmed by sequencing of PCR products which had been amplified from the DNA of the respective cattle using specific primers. Studying an N´Dama pedigree, we found that DQA*muh13 and DQB*kuhx belong to the same haplotype which further contained the alleles DQA1*BNI2, DQB4*1 and DRB3*8A. Whether the gene products of DQA*muh13 and DQB*kuhx combine to form a divergent DQ molecule and whether this molecule is of immunological relevance remains to be shown.
*) formerly BNI

The development of genome maps for livestock species is considered crucial for the identification of genes effecting traits of economical importance. Previous linkage maps of the bovine genome are comprised primarily of highly polymorphic microsatellite markers. However, in order to enable the identification of candidate genes by consulting the more advanced human and murine maps, the bovine marker set must include markers in a number of anchor loci. Using locus-specific primers, segments of selected anchor loci were amplified and sequenced. In the genes of renin (REN), lysosomal a-mannosidase (MANB), thyroglobulin (TG), metallothionein 2 (MT2A), BoLA-DYA (DYA) and low density lipoprotein receptor (LDLR), allelic variants were identified which could be distinguished by PCR-RFLP. The members of the International Bovine Reference Panel (IBRP) were genotyped, the typing data were submitted for linkage analysis, and thus the markers became integrated into the internationally defined bovine genome map. Locus assignments to the respective bovine chromosomes (BTA) were made as follows: DYA - BTA23, LDLR - BTA27, MANB - BTA7, MT2A - BTA18, REN - BTA16, TG - BTA14. The newly developed type I markers are now available for comparative genome studies.
*) International Livestock Research Institute, Nairobi, Kenya
 
 

The class II region of the major histocompatibility complex (MHC) is of interest because it contains many genes whose products play important roles in the immune response. At present, the MHC of cattle receives increasing attention because it was found to harbour susceptibility alleles for several diseases of economic importance. Whereas the physical structures of the human and mouse MHCs have been studied extensively using pulsed field gel electrophoresis (PFGE) and large overlapping genomic clones, the knowledge about the organization of the bovine MHC is limited to data from recombination analyses and to inferences from the gene order established in man and mouse. As a first step to physically map the region, we developed sequence tagged site (STS) markers by designing primers specific for the known bovine class II genes DQA, DQB, DRA, DRB, DMA, DMB, DYA, and DIB, and for the human LMP2 and LMP7 genes, which also map to the class II region. The primers were used to screen a library of the bovine genome represented in 21,500 yeast artificial chromosomes (YACs). A total of 24 YACs were identified which contained at least one STS marker from the class II region. As measured by PFGE, the sizes of the YACs were between 365kb and 1600kb with an average of 735kb. Their chimerism status and physical location are presently being determined by fluorescence in situ hybridization; 6 out of 17 YACs studied so far were found to be non-chimeric. Alignments based on their contents of STS markers are being confirmed by determining their end fragments and defining their overlaps in order to form contiguous arrays (contigs), which may be used, for instance, to generate a finer map, to distinguish between loci and alleles, to collect additional microsatellite markers and to further identify expressed sequences within the bovine MHC.
*) American Breeders Service Institute, DeForest, Wisconsin, USA
⁺) Institut für Anthropologie und Humangenetik, Universität Heidelberg, Heidelberg
^#) Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere, Dummerstorf
 
 

The overall organization of the MHC is well conserved among mammals. But whereas the well studied MHCs of humans and mice span approximately 4 Mbp and have a recombination length of about 2cM, recombination analyses of the class II genes of the bovine MHC revealed two distinct clusters which are approximately 17cM apart. One cluster, designated class IIa, harbours the DQ and the DR gene families. The second one, designated class IIb, contains the bovine homologues of the human DMA, DMB, DOB, and LMP2 genes, as well as MHC-class-II related sequences designated DYA and DIB. In order to find out whether the large genetic distance is due to a recombination hotspot or to physical distance, we screened a bovine YAC library by PCR with primers specific for class IIa and class IIb sequences. We identified the two YAC clones 1A9 and 212E9, the former of which contained DQA, DQB, DRA, and DRB, the latter DMA, DMB, DIB, DYA, DOB, LMP2, and LMP7 sequences. The PCR products obtained using the YAC clones as templates were sequenced and compared with published sequences to confirm their identities. Fluorescence in situ hybridization revealed that YAC 1A9 exclusively maps to band 21 on chromosome 23 whereas YAC 212E9 exclusively maps to band 12-13 on the same chromosome. The result shows that the observed genetic distance of 17cM is consistent with the physical location of the cluster IIa and IIb sequences on chromosome 23. Thus, in contrast to the situation in humans and mice, the bovine MHC class II region physically is divided into two subregiones.
*) Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere, Dummerstorf
⁺) American Breeders Service Institute, DeForest, Wisconsin, USA
^#) Institut für Anthropologie und Humangenetik, Universität Heidelberg, Heidelberg
 
 

Evidence for renal abnormalities has been described both in patients with filariasis and in animal models of filarial infections. We investigated parameters of kidney function in the various forms of human onchocerciasis. The study included (a) a population-based, cross-sectional investigation of patients with and without onchocerciasis in a mesoendemic village near Macenta/Guinea, (b) a hospital-based, cross-sectional investigation of patients with generalized onchocerciasis, sowda and of putatively immune individuals and (c) a cohort study of onchocerciasis patients treated with ivermectin. Renal function was assessed by measuring serum creatinine and proteinuria. Measurement of total urinary protein was related to urinary creatinine because these ratios have been shown to have a high correlation to the timed 24-hour urinary protein excretions. A qualitative analysis of urine proteins was used to discriminate glomerular from tubular disorders. Kidney sizes were determined by ultrasound. No disturbances of renal morphology or function could be detected that could be attributed to onchocerciasis. Following ivermectin treatment, a slight but significant increase in the excretion of urinary proteins was detected in patients with high microfilarial densities (> 80/mg skin). Notably, protein excretion of sowda patients was comparable to that of patients with generalized onchocerciasis revealing no evidence for an immune-mediated glomerular disorder in sowda.
*) Parasitology Section
 
 

Delayed-type hypersensitivity (DTH) reactions are considered to reflect antigen-specific responses of type 1 helper T lymphocytes. With regard to onchocerciasis, previous reports described positive DTH reactions to Onchocerca volvulus antigens in patients with sowda and stressed the absence of such responses in patients with generalized disease but did not include putatively immune individuals. Corresponding in vitro investigations of T-cell responses to O. volvulus antigens yielded stronger reactivity with cells from patients with sowda than with those from patients with generalized disease. Cells from putatively immune individuals were also found to respond to O. volvulus antigens, which was interpreted as evidence for an active and specific immune reaction. However, the type of T helper cell activity dominating in putative immunity was not yet clearly defined.
We studied DTH reactions to 50mg of a crude O. volvulus extract and to 1-5mg each of some recombinant antigens. After testing for adverse effects in three healthy European volunteers, we recruited in south-east Guinea i) 40 patients with generalized disease, ii) 14 patients with sowda, iii) 20 putative immunes from the same villages, and iii) 16 non-endemic controls from an area free of onchocerciasis in west Guinea. Concomitant intestinal nematode infections were found in 40-50% of individuals of all groups. Not more than 20% of the subjects of any group reacted to any of the recombinant antigens, substantial reactivity was found with the crude antigen extract only. Highest reactivity was obtained in sowda patients (80%), followed by the groups of non-endemic controls (75%) and putative immunes (60%), whereas patients with generalized disease reacted poorly (15%). Overall, the results confirmed the reactivity patterns which had been described before and which were expected from in vitro studies.
The high reactivity in non-endemic controls appears to most likely be due to cross-reactivity with other nematodes, a finding well known from serology. It raises questions about the role of interactions in the immune responses to concomitant nematode infections and suggests that the lack of response in generalized onchocerciasis may not be due to a failure of activation but rather to immunosuppression. Finally, the presence of DTH reactions in putative immunity and their absence in generalized disease argue in favour of type 1 helper cells as factors involved in protection.
*) Parasitology Section
 
 

The prevalence of E. histolytica infection in Turkey is not known. Previously, we have examined the population of two villages in Southeast Anatolia, these studies now were extended to another three villages. The primary objective of our study is to estimate the prevalence of E. histolytica and E. dispar infections in a rural area in Eastern Turkey - using stool microscopy, PCR and classification of amoeba in fecal samples by isoenzyme determination. By stool microscopy, E. histolytica/E. dispar was detected in 138/493 persons. Results from stool microscopy and PCR were divergent, nevertheless the prevalence of E. histolytica infection in the study area can be estimated: Stool microscopy was performed by skilled observers and accordingly it can be expected that the rate of false-positive results is low (some false-negative results, however, are indicated by the fact that PCR from fecal samples was positive in 11/140 samples that were microscopically negative). Culture in Robinson´s medium was positive in 35 out of 60 cases (58%), similar percentages have been found in other studies. Only 3/35 (8.6%) of the positive cultures were found to be E. histolytica by hexokinase determination and riboprinting-PCR. Accordingly it can be calculated that 8.6% of the 28% microscopically positive samples were E. histolytica, this would amount to a prevalence in the population of 2.4%. This conclusion seems to be justified because a different rate of positive cultures between E. dispar and E. histolytica has never been described. PCR directly from fecal samples was positive in only 17 out of 70 cases that were microscopically positive. These false-negative results were probably due to the fact that the samples were stored for several months and frozen at less than -20° C during air transportation, causing the degradation of DNA by nucleases present in the fecal samples. However, when differentiation by PCR was possible, only E. dispar was detected. It can therefore be concluded that the prevalence of E. histolytica is close to zero. The alternative hypothesis that PCR directly from fecal samples is (even) less sensitive for E. histolytica than for E. dispar, seems unlikely because the results correspond to our results from isoenzyme determination and riboprinting-PCR from cultures and because the same sensitivities were found in vitro. Thus the prevalence of E. histolytica in this rural population in Eastern Turkey appears to be very low. Similarly low prevalences have been found on the Seychelles (2.6%), where classification was done by isoenzymes, as well as in Brazil (3%) and in Bangladesh (1%), where classification was done by an antigen stool ELISA.
°) Parasitoloy Section
*) Dicle University, Diyarbakir, Turkey
⁺) London School of Hygiene and Tropical Medicine, London, UK
^#) Institut für Tropenmedizin, Universität Tübingen

In early stage of HIV-1-infection an increased number of CD4+ T (red) proliferate in the lymph node. The edge of a germinal centre showing many B cells in cycle (Ki-67+, blue nuclei). Among them proliferating CD4+ T cells (double positive) are present.

Recent studies have shown that the nasopharyngeal tonsils (adenoids) contain HIV-producing cells during HIV-infection. In the present study we have examined 14 specimens that included 7 adenoid and 7 palatine tonsils. Clinical data were also available. The blood CD4+ T cell counts were between 200 to 900/ml and the histories indicated that the patients were asymptomatic, other than the presenting symptom that the tonsils were sufficiently enlarged to cause obstructive symptoms or raise the potential of malignancy. For morphological and molecular pathological analysis classical histology and immunohistochemistry (CD1a, CD4, CD8, CD68, lysozyme, p55) were performed. For detection of HIV-1 productively infected cells in situ hybridization using a 35S-labeled RNA probe was performed. Most of the cells with intracellular HIV-1 protein were small but multinucleated. The majority of these syncytia could be double labelled for HIV-1 RNA and a dendritic cell marker S-100. In the palatine tonsil, the infected cells were not found in the stratified squamous epithelium that is adjacent to the pharynx. Instead, S-100+ infected syncytia were localized to the surface of tonsil crypts. This mucosa is termed lymphoepithelium because large numbers of lymphocytes, primarily B but also CD4+ T cells, are abundant within a ramifying epithelial network. This mucosa contains antigen-transporting so-called M cells that lie above regions where S-100+ dendritic cells are juxtaposed with CD4+ lymphocytes. Infected cells were found in lymphoepithelium and not respiratory epithelium of nasopharyngeal tonsils or adenoids. We propose that lymphoepithelia, the histological term that describes the specialized regions where antigens access mucosa-associated lymphoid tissue, are sites where HIV-1 replication can be enhanced in syncytia derived from dendritic cells.
This study is a German-American collaboration. The American investigators are R. Steinman^#), M. Pope^#), S. Frankel⁺), B. Wenig*), C. Hansen^), D. Heffner*), A. Nelson⁺)

Supported in part by BMBF and the Körber-Foundation
^#) Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, USA
⁺) Armed Forces Institute of Pathology and the Division of Retrovirology, Washington, DC, USA
*) Department of HIV Vaccine Development, Walter Reed Army Institute of Research, Rockville, Maryland, USA
^) Georgetown University Medical Center, Washington, DC, USA
 
 

B-lymphocyte dysfunction has long been known to occur in HIV-1-infected individuals at early and late stages of the disease. It has been suggested that naive immunoglobulins encoded by the VH3 gene family interact aberrantly with human immunodeficiency virus type 1 (HIV-1) gp120 via a superantigenic epitope, causing initial expansion and eventual depletion of VH3-expressing B cells. However, this possibility has not been prospectively assessed during an AIDS virus infection. We determined VH family usage in rhesus monkeys during primary infection with chimeric viruses expressing HIV-1 evelopes on a simian immunodeficiency virus (SIVmac) backbone (SHIVs). Four SHIVs with different envelopes and pathogenicities were studied. VH family usage was prospectively assessed in peripheral blood mononuclear cells and lymph node cells of these monkeys by a semiquantitative PCR technique. Changes of the B-dependent zone of the lymph node were monitored with immunohistochemistry. A profound early depletion and subsequent re-expansion of circulating B cells was observed following infection with all four SHIVs. B cell expansion within the lymph nodes could contribute to the elevation of circulating B cells since the lymph nodes showed follicular hyperplasia, enhanced B cell proliferation and elevated numbers of B cells in the medullary sinuses. No single VH family was consistently altered. In particular, the average representation of VH3-bearing B lymphocytes did not change. This observation suggests that the envelope glycoprotein of HIV-1 does not selectively expand or deplete the VH3 repertoire of primate B cells during acute AIDS virus infection, contrary to predictions of the gp120 superantigen hypothesis.
Supported in part by BMBF and the Körber-Foundation
This study is a German-American collaboration
^#) Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center Boston, MA, USA
*) Dana Faber Cancer Institute, Boston, MA, USA
 
 

The use of combination with two nucleosid analogues and an HIV-1 protease inhibitor (triple drug therapy [TDrT]) in HIV disease yields a dramatic reduction of viral RNA in the plasma and a significant increase in circulating CD4+ T cells. The use of TDrT early in disease holds the potential of preventing virus-induced damage to the immune system. We monitored the short-term (28 days and three months) effect of TDrT on 12 patients with 14 - 75 months histories of minimal HIV-1-disease, e.g., CD4+ T cell counts constantly >500 ml and little or no lymphadenopathy. Thus, it was the first time that the extent of viral replication and immunoarchitectural changes in unenlarged lymph nodes early in disease could be determined. We found HIV-1 RNA+ CD4+ T cells and viral RNA in association with follicular dendritic cells (FDC) both in enlarged and non-enlarged lymph nodes. The number of CD4+ T cells was in the normal range both in germinal centers and the T-dependent zone but CD8+ cells were markedly elevated. The FDC network was well preserved. In contrast to HIV negative lymph nodes, CD4+ T cell proliferation was active, with marked increases in the number of Ki-67+CD4+CD45R0+ cells. After 28 days and three months of therapy, productively infected T cells decreased dramatically and often were not apparent. The labeling of the FDC network for HIV-1 RNA also decreased, but not for gag protein p24. We conclude that HIV-1 replicates and accumulates in lymph nodes before severe damage of the immune system occurs, that in this stage of disease de novo production of T cells occurs in the lymphoid tissue, and that the infection is sensitive to TDrT not only in plasma but also in the lymph nodes.
Supported in part by BMBF and the Körber-Foundation

*) Medical Department, University Hospital Eppendorf, Hamburg
^#) Department of Surgery, University Hospital Eppendorf, Hamburg
^Y) Carl Zeiss Company, Jena
°) Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, USA

Host tissue penetration by parasitic nematodes may be mediated by both, mechanical processes and proteolytic enzymes released by the parasites. Analysis of excretory-secretory (ES) products of Onchocerca volvulus microfilariae and adult stages in various protease detection assays demonstrated that they contain several distinct proteolytic enzymes. The ES products of the microfilariae comprised one low and two high molecular weight proteolytic bands that degraded gelatin in substrate gels while ES products of males contained several high molecular weight proteases in the range of *100 kDa degrading gelatin. Further enzymatic analysis revealed that the low molecular weight protein in microfilarial ES products but not the high molecular weight proteolytic components found in microfilarial and male ES products was able to cleave soluble elastin incorporated in substrate gels. In our experiments we demonstrated that 1 ng porcine elastase degraded soluble elastin while an amount of 10 mg Clostridium histolyticum collagenase was required for elastin cleavage. The definite evidence for the occurrence of an elastase-type protease could only be procured by the degradation of insoluble, fibrillar elastin which was not affected by collagenase. Insoluble elastin was cleaved by microfilarial ES and female extract but not by male ES products demonstrating the expression of secreted elastase by O. volvulus microfilariae. Elastase-type proteases are supposed to be involved in the wrinkling and thickening of the skin and the development of "hanging groin" observed in chronically infected onchocerciasis patients. ES proteases of both developmental stages degraded the extracellular matrix proteins fibronectin, laminin and collagen type IV, but only microfilariae are able to cleave elastin fibrils. The optimal protease activity for each of the proteases was found to be at a neutral pH. Inhibitor studies demonstrated their classification as serine and metalloproteases.
Supported in part by BMBF
*)Provisional Research Station of the Institute in Macenta, Guinea
 
 

The majority of O. volvulus-infected persons shows signs of cellular anergy including deficient T cell proliferative responses. The underlying mechanisms of the hyporesponsiveness are poorly defined. Histological and in vitro evidence elaborated by other investigators and by our group indicate a central role of the monocyte/macrophage lineage as effector cells in O. volvulus infection. Monocytes/macrophages and dendritic cells represent the major antigen presenting cells. We were therefore interested to investigate the effect of O. volvulus somatic and excretory-secretory products of adult and microfilarial stages on monocytes and macrophages. The modulatory effects were shown to be time dependent. After six hours of exposure of the monocytes to O. volvulus products low levels of TNF-a were detected in the cell culture supernatants, and after 24 hours the IL-2 receptor (a chain) was upregulated. Exposure of the cells for two to three days resulted in a pronounced production of IL-10 and a pronounced reduction of HLA-DR, B7.1 and B7.2 expression. The downregulation of these receptors which are engaged in the antigen-presentation was partially abolished by anti-IL-10 and less by anti-TGF-b antibodies. The expression of CD40, the adhesins LFA-3, ICAM-1 and VLA-4 but not LFA-1 (b-chain) and VLA-5 was less reduced. Extracts of adult filariae and microfilariae affected monocytes in mononuclear cell suspensions as well as purified monocytes and in vitro generated macrophages thus demonstrating a T cell-independent monocyte response to O. volvulus products. Various components of the O. volvulus products seem to mediate the monocyte modulatory effect which are currently analysed. The results indicate that the monocyte may be a major target cell for immunomodulatory parasite molecule leading to deactivation of its pivotal function in afferent and efferent immune mechanisms.

Supported in part by BMBF

*) Forschungszentrum Borstel

Microfilariae represent the pathological agent in chronic infection with Onchocerca volvulus. Because of the potential role of microfilarial antigens in the immune regulation and pathogenesis of onchocerciasis we have initiated studies to characterize the functional role of microfilarial antigens. Subclass specific antibody responses to microfilariae extract (MfE) and excretory-secretory (ES) products of microfilariae, males and females, were assessed by ELISA and Western blot analyses using sera from cases with generalized onchocerciasis with high (>90 mf/mg skin) or low (<7 mf/mg skin) microfilarial load, hyperreactive onchocerciasis (sowda), prepatent individuals, endemic controls, Mansonella perstans-infected persons, filaria-negative individuals and European controls. Generalized onchocerciasis and sowda patients showed high levels of MfE-reactive IgG1 and IgG4 isotype antibodies. Predominantly the occurrence of high IgG4 response against MfE and ES products of males discriminated between microfilaria and non-microfilaria carriers. Sera of prepatent persons comprised IgG1 and IgG3 but not IgG4 antibodies against MfE and ES products of males. The distribution pattern of isotype responses towards MfE and ES products was similar for the various serum pools. Only sowda sera showed a pronounced IgE antibody response against Mf ES products. In contrast to Mansonella perstans-infected persons and filaria-negative individuals, sera of endemic controls comprised IgE antibodies reactive with ES products of females. The ongoing analysis of isotypic reactivities of O. volvulus-exposed persons in immunoblots shows marked differences in the pattern of proteins of MfE in comparison to male and female extracts.

Supported in part by BMBF

High levels of circulating IgE, eosinophilic granulocytes and prominent tissue mastocytosis, concomitant with in vitro induction of IL-5 and IL-4, represent a Th2-type response in filarial infections and are indicative for allergic reactions. This allergenic potential - hallmark in Onchocerca volvulus infection - contrasts the rarity of immediate hyperreactivity manifestations observed in onchocerciasis. Immediate type of hypersensitivity reactions, however, are characteristic for the sowda form of onchocerciasis and for inflammatory reactions follo-wing anthelminthic drug exposure (Mazzotti reaction). We investigated the in vitro histamine release of peripheral basophils in twelve patients with a generalized form, six patients with a sowda form and five putatively immune persons who were exposed to O. volvulus without expressing any parasitological and clinical signs of onchocerciasis. We could confirm earlier results demonstrating significant basophil degranulation by exposure of these cells to low concentrations of allergenic compounds of the O. volvulus extract (10-100 ng/ml protein extract). While non-endemic Europeans did not react to the exposure of O. volvulus allergens or anti-IgE antibodies, we found in the generalized group low rates of histamine release but high rates in sowda patients and putative immunes. O. volvulus-specific IgE activities as well as total IgE serum levels were also highest in sowda patients. Correlation was stated between specific IgE levels and histamine release. Lack of basophil degranula-tion was observed after stimulation of the cells with the recombinant O. volvulus proteins S1 and OvGST1 indicating that both proteins do not represent allergens. While mycobacterial tuberculin did not induce basophil degranulation, an extract of Ascaris lumbricoides induced a strong allergenic response which is suggestive for cross-reacting allergens. The high allergenic potential detected in sowda is in line with numerous in vivo and in vitro data indicating the hyperresponsiveness of this polar form of onchocerciasis. The strong allergic re-sponse observed in putative immune persons suggests considerable immune activation of this group of O. volvulus-exposed persons. *) Provisional Research Station of the Institute in Macenta, Guinea

Onchocerca volvulus infection is characterized by tissue and peripheral blood eosinophilia. Eosinophilic granulocytes are supposed to function as a unique defence system. They are exclusively mobilized and attracted by microfilariae and microfilarial products. We analysed five groups of individuals for their eosinophilic potential with special emphasis on eosinophilic cationic granule proteins and their levels in serum and urine. Forty-two O. volvulus-infected (Ov-positive) individuals (31 patients with the generalized form, 11 with the sowda form) were investigated and 40 O. volvulus-negative persons (Ov-negatives) resident in an endemic area in Guinea (West-Africa). The groups included generalized onchocerciasis pa-tients exhibiting microfilarial densities <80 and >80 microfilariae (mf)/mg skin, patients with and without coexistent helminths other than O. volvulus, and individuals infected with Schistosoma haematobium. Increased levels of circulating eosinophils and serum eosinophil cationic proteins (ECP and EPX/CLA) were a feature of all groups characterized by peripheral blood eosinophilia, with the highest values of the cationic proteins in sowda type onchocerciasis, exceeding that of generalized patients with microfilarial densities >80/mg skin. The increase of eosinophil markers was also stated for O. volvulus-negative persons without other helminths indicating the occurrence of not detected helminths. Serum ECP and EPX/CLA were positively correlated, the levels of both proteins increasing with elevation of peripheral eosinophil counts. When Ov-negatives and generalized patients with mf densities <80/mg skin were divided into sections without and with intestinal helminths, the divided sections could only be distinguished by significantly higher IgE levels in the sections with secondary helminths. High IgE levels were particularly found in infection with hook worm. Urinary ECP and EPX/CLA as newly established parameters showed the following: EPX/CLA is excreted in all groups studied. The highest urinary levels were observed in onchocerciasis patients, especially in the sowda form, exceeding the levels documented for the Caucasian reference population by far. Urinary ECP was found elevated only in patients infected with S. haematobium. From these studies it appears, that extremely elevated IgE serum levels are characteristic for sowda-type of onchocerciasis and for the subgroups with coexistent intestinal helminth infection. EPX/CLA is a primary urine protein whereas urinary ECP is excreted secondary to urogenital manifestation of parasitic disease, for example S. haematobium infection. Both proteins are markers for blood and tissue eosinophilia. ECP-uria in infection with S. haematobium appears not to be related to renal dysfunction. Further studies are designed to elucidate the diagnostic value of these proteins in helminth and other disorders.

*) Molecular Medicine
 
 

Immunological studies on filarial infections are hampered by possible interference of the tissue-dwelling filariae and intestinal helminths with the immune system. We analyzed the antibody response to O. volvulus extracted antigens in 95 patients with generalized onchocercasis, 9 patients with a sowda form of onchocerciasis, 14 putatively immune persons (PI) who were exposed to O. volvulus but elicited no signs of onchocerciasis, and 26 individuals without O. volvulus-infection (Ov-negatives) resident in an endemic area in Guinea (West-Africa). Sowda patients showed the highest titers of anti-O. volvulus IgG1, IgG3 and IgE and the highest numbers of peripheral eosinophils. PI had significantly lower serum isotype titers than the group of generalized onchoceriasis but higher IgG1, IgG4 and IgE levels than the Ov-negatives who showed low IgG3 levels, markedly high total IgE as well as eosinophil counts. IgG1, IgG4 and IgE titers correlated with the microfilarial density of the skin.

Half of the total study group (49%) was co-infected with intestinal helminths, predominantly Ancylostoma duodenale (29%) and/or Ascaris lumbricoides (26%). Comparing the four study groups with respect to the presence or absence of intestinal coinfection we stated for the co-infected section twofold higher levels of eosinophils vs the non-coinfected section. A strong augmentation of total IgE levels was only found in the Ov-negatives with intestinal helminths but not in the other study groups. The overall pattern of the isotypic antibody re-sponses appea-red similar for the four study groups independent of intestinal helminth co-infection. However, the IgG4 response in the sowda group was higher in coinfected subjects as compared to non-coinfected persons but lower in the co-infected generalized subgroup suggesting interference of secondary helminth infection with the immune system in both polar groups. The low IgG4 levels were observed in patients co-infected with hook worm but not with A. lumbricoides. IgG4 levels, eosinophil counts and IgE levels in persons with a density >80 microfilariae/mg skin were similar in the coinfected and non-coinfected section suggesting that the O. volvulus-related responses are not influenced positively by secondary helminths in persons with high microfilarial densities. High IgG3 levels were preferentially found in O. volvulus-infected persons when coinfected with T. trichiura, Strongyloides stercoralis and Schistosoma mansoni. These observations indicate an alteration of O. volvulus-specific antibody responses by coinfection with intestinal helminths.

*) Molecular Medicine
 
 

The cellular composition of the nodule in Onchocerca volvulus infection indicates specific immunologic defense mechanisms. Factors which stimulate the extravasation, migration, accumulation, and activation of inflammatory cells, are particularly important in inflammatory lesions induced by parasites. In immunohistological analyses intact ("live") female O. volvulus - which contained in their uteri oocytes but no embryos or microfilariae - were examined to exclude chemotactic activity elicited by microfilarial components. Intact and degranulating neutrophils labelled specifically by defensin, elastase, myeloperoxidase or cathepsin G were seen in close proximity or adherent to O. volvulus females. Neutrophils were also found adjacent to male worms. The anterior ends of the females and the male worms usually lay in a small cyst filled with a semiliquid suspension of neutrophils. Within the cyst it may be easier for the adult worms to take up host cell constituents for their nutrition and to move freely for mating, which would be difficult in the fibrous tissue of the nodule. Neutrophils were also adjacent to the cuticle of productive females while eosinophils were only found in larger numbers in the tissue near to females producing microfilariae. Chemotactic responses of neutrophils were demonstrated using O. volvulus extracts and excretory-secretory products of vital females. Fractionating of extract from female worms by FPLC technique revealed two components with chemotactic activity, one with a molecular mass less than 12 kDa and another with a high molecular mass >200 kDa. It is assumed that the production and release of a neutrophil chemotactic factor attracts neutrophils and these cause a modulation of the granulomatous reaction in the nodule at the site of the male and female worm. It cannot be excluded that the neutrophil chemotactic factor(s) originated in intracellular bacteria found in the hypodermis of O. volvulus and are not produced by the filariae.

*) Department of Helminthology and Entomology
**) Department of Biochemical Parasitology