Modulation of cell surface expression of stimulation-associated and costimulatory mole-cules on monocytes by Onchocerca volvulus microfilarial products. Downregulation of HLA-DR and B 7-1(CD80) molecules and upregulation of IL-2Ra chains were induced by microfilarial but not by ubiquitous nonparasite antigens. The unshaded area represents fluorescein-labeled cell controls. FI = fluorescence index ratio of fluorescence intensity of antigen-exposed cells and non-exposed cells.

Investigators’ Reports     Renal Handling of Cationic Proteins of Granulocytic Effector Cells F. W. Tischendorf, N. W. Brattig, M. Lintzel, W. Groenwoldt, A. Pieper Eosinophils function as effectors to damage larval stages of helminth parasites. The toxic potential includes the release of several major granule cationic proteins. In a comparative study comprising diseases with and without peripheral blood eosinophilia we found, that one of these granule proteins, eosinophil cationic protein (ECP), is highly specific for eosinophils. The protein is absent or occurs only in negligible amounts in neutrophils. Serological analyses in worm diseases demonstrated, that ECP serum levels reflect the ongoing disease and are related to the functional activity of the respective eosinophil effector system. ECP is a low molecular weight protein like the neutrophil constituent lysozyme. For lysozyme it is known, that its serum and urinary levels are dependent on renal function. To evaluate renal handling of ECP, we compared serum and urinary ECP levels with those of lysozyme in non-eosinophilic patients with glomerular and tubular renal dysfunction. Whereas serum lysozyme levels positively correlated with serum creatinine and urinary albumin and IgG, i.e. markers of glomerular filtration rate, and urinary lysozyme levels with the urinary tubular marker protein alpha-1-microglobulin, no statistical correlation could be revealed for serum and urinary ECP levels and the respective substances including lysozyme. The results indicate that ECP levels are not handled by the kidney and that ECP is a suitable clinical chemical marker of the functional activity of the eosinophil effector system. ECP levels determined in serum and urine samples of persons with eosinophilic helminth infection and malaria tropica, who often develop renal dysfunction, are currently analysed.    Proteolytic Enzymes in Onchocerca volvulus Stages A. Haffner, U. Rathjens, E. Taege, W. Groenwoldt, A. Wywiol*), F. W. Tischendorf, N. W. Brattig Parasite secreted proteases catalyze a broad spectrum of important biological reactions including invasion and migration of the parasite in host’s tissue or interference with host’s metabolism and defence mechanisms. We could demonstrate O. volvulus proteolytic enzymes degrading gelatine, casein and albumin. The cleavage of the matrix proteins fibronectin and elastin indicated a possible biological function in tissue invasion. In order to characterize a microfilarial protease we collected larger quantities of biological material in the endemic area of O. volvulus infection in Guinea (West Africa). We prepared females and males and procured three types of microfilariae: microfilariae from cut onchocercomata which were purified by use of Percoll gradient centrifugation; microfilariae hatching from intact cleaned onchocercomata, and thirdly, microfilariae leaving skin biopsies. Nodular and extranodular microfilariae as well as adult females and males were cultured at varying densities, numbers, in varying volumes of culture medium and time periods. Proteolytic activity was detected in a proportion of samples. Microfilariae and males were found to release different proteolytic enzymes preferentially after 48h and 72h cultivation. The proteolytic compound of microfilariae was characterized as serine protease-type with a pH optimum of 6.5-7.5 and a suspected molecular mass of about 45 kDa as shown by overlay gelatine gel electrophoresis. The quantity of the liberated microfilarial protease was, however, very low. Extract of adult females comprises multiple proteolytic activities, which could be partially inhibited by inhibitors for metalloproteases or serine proteases.

Analysis of Excretory-Secretory Proteins of Onchocerca volvulus Stages

A. Haffner, E. Taege, T. F. Kruppa*), F. W. Tischendorf, N. W. Brattig

Parasite excretory-secretory (E-S) products represent first line components of the parasite confronting the human host. They have been shown to comprise parasite defence molecules and appear to be stage and species-specific diagnostic antigens. The release of E-S proteins of O. volvulus was tested under varying culture conditions. Males and microfilariae were shown to liberate proteins during 24h to 48h. All culture supernatants were screened for the pattern of the E-S proteins in SDS gel electropheresis with silver staining. Male extract exhibited similar protein bands as adult female extract at 21, 28, 46, 55 and 70 kDa, but of less intensity. The extract of microfilariae showed strong protein bands below 23 kDa with a prominent band at 12 kDa, furthermore at 23 and 80 kDa, while the 46 kDa band  was less pronounced. Female E-S showed major protein bands at 12, 17, 31, 36, 52 and 66 kDa while male E-S exhibited similar patterns of bands but with lower intensity. Microfilaria E-S products predominantly showed the major band at 13 kDa, bands between 28-36 kDa and 60-66 kDa. By ELISA and western blotting the E-S products and extracts of the three stages were tested for the presence of the O. volvulus proteins OvGST1, OvSOD1, Ov33, OvL3-1 and S1 using monoclonal or polyclonal antibodies. OvGST1, OvSOD1 and S1 could be detected in the extracts as well as E-S products of all stages. OvL3-1 was detected in all extracts and female E-S but not in E-S products of males and microfilariae, while Ov33 was prominently found in female and male extracts but only weakly in microfilariae. Ongoing analysis of antibody reactivities of O. volvulus-exposed persons showed marked differences in the pattern of proteins recognized in western blots. While onchocerciasis sera recognized a regular pattern of bands in the microfilarial extract, they were reacting predominantly with proteins at 21, 28-33 and higher than 50 kDa in male extracts.
Supported in part by BMBF
*) Provisional Research Station of the Institute in Macenta, Guinea
 
 

Onchocerca volvulus Microfilarial Products Modulate the Expression of Stimulation-associated and Costimulatory Surface Molecules on Monocytes

N. W. Brattig, U. Rathjens, F. Geisinger

O. volvulus microfilariae survive a long period of time unfazed by immune and inflammatory responses of the human host. The majority of O. volvulus-infected persons shows signs of cellular anergy, exhibiting deficient T cell proliferative response and IL-2 production to onchocercal antigens. The underlying mechanisms of the cellular hyporesponsiveness are still poorly defined. Considerable evidence has grown that signals generated through the T-cell receptor and MHC-molecules are not sufficient for full activation of T cells; additional costimulatory signals are required for IL-2 production, proliferation and differentiation to effector function. Thus, the costimulatory pathway via CD28 on T cells and B7 (CD80/CD86) on monocytes/macrophages is critical in T cell activation as well as lymphokine production and the absence of CD28 engagement was shown to lead to incomplete activation, anergy, and unresponsiveness. We were interested to investigate the role of O. volvulus microfilariae and their products in immunomodulation and down-regulation observed in O. volvulus infection. In cocultures of O. volvulus microfilariae with mononuclear cells from infected persons we observed reduced proliferative responses to mitogen and IL-2. In the supernatants of microfilariae-exposed cells we detected IL-10 but not IL-4 and IL-5. Examining the expression of activation and costimulatory molecules on immune cells, we could demonstrate that the exposure of normal peripheral blood mononuclear cells to O. volvulus microfilarial products resulted in downregulation of the expression of CD80 and HLA-DR-molecules on monocytes. In contrast, the IL-2 receptor a (CD25), a T cell activation receptor, appeared upregulated on monocytes but not on T cells after exposure to the parasitic antigens. In preliminary experiments, O. volvulus antigens did not induce the expression of CTLA-4 (CD152) on T cells of onchocerciasis patients. The present results of ongoing experiments indicate that microfilarial constituents comprise immunomodulatory activity which may contribute to the state of unresponsiveness in the host facilitating microfilaria evasion.
Supported in part by BMBF
 
 

Induction of Basophil Degranulation by Onchocerca volvulus Microfilarial Allergens

N. W. Brattig, M. T. Rubio P. de Krömer, F. Geisinger, D. Meineke, F. W. Tischendorf

The skin-dwelling O. volvulus microfilariae are the causative agents for immediate hypersensitivity reactions leading to acute and chronic dermatitis in onchocerciasis. Thus, in the diagnostic Mazzotti reaction the anthelminthic compound diethylcarbamazine elicits tissue inflammatory responses which in their intensity depend on the microfilarial load of the treated subject. This hypersensitivity reaction is supposed to be induced by released products of degenerating microfilariae. Histamine release could be induced in peripheral blood basophils from onchocerciasis patients by adult total extract in a dose-related fashion up to concentrations of 100-10 ng/ml, indicating the strong allergenic potential of O. volvulus constituents. Vital microfilariae and released products of vital microfilariae, in contrast to the released products of adult females, were also shown to induce histamine release. Significantly higher degranulation rates, however, were elicited by dead microfilariae indicating that additional hidden allergens may be included. These findings are in accordance with the allergic Mazzotti reaction. The specificity of the histamine release was shown by the lack of histamine degranulation after exposure of the basophils to allergens derived from the house-dust mite Dermatophagoides pteronyssinus, and Aspergillus.
*) Provisional Research Station of the Institute in Macenta, Guinea
 

Monocyte- and Granulocyte-Mediated Killing of Onchocerca volvulus Microfilariae: Dependency on the Microfilarial Density of the Serum Ligand but not Cell Donor

Rathjens, U., A. Haffner, F. W. Tischendorf, F. Geisinger, A. Wywiol*), N. W. Brattig

Histological findings indicated the involvement of macrophages in host defence mechanisms against O. volvulus microfilariae (mf). Purified mf were co-cultured in vitro with monocyte-enriched peripheral mononuclear cells, neutrophilic or eosinophilic granulocytes from pa-
tients with onchocerciasis, putatively immune persons and non-endemic controls in the presence of sera from patients of varying mf densities and non-infected persons. Monocytes adhering to the surface of mf were identified by fluorescein-conjugated anti-CD14 anti-
bodies by immunofluorescence. Adherence of monocytes as well as granulocytes to the mf surface and killing of the mf were dependent on mediating serum ligands of O. volvulus-exposed persons from the endemic area (OvS) including complement components. The killing rates correlated inversely with the mf density of the serum donor. Cells obtained from healthy or O. volvulus-infected persons showed no differences in their capability to destroy mf. Killing occurred even more pronounced under autologous than homologous conditions where cells and mf derived from different donors. These results demonstrate the toxic potential of monocytes to destroy the mf stage of the filarial parasite which depended on the parasitological status of the serum donor as source of appropriate ligands. High parasite load was associated with low killing capacity not correlating with the titres of IgG antibodies to the tested O. volvulus antigens. Preliminary experiments showed that microfilarial culture supernatants can reduce cellular killing rates and that the treatment of mf with a protein biosynthesis inhibitor did enhance cellular killing in OvS. Mf, like the fourth stage larvae, are resistant to cellular interaction in the presence of normal human serum (NHS). Complement activation could not be achieved by treatment with the hydrogen peroxide or mellitin but by detergent, heat and high concentration of papain indicating the involvement of distinct surface molecules. Lectin binding studies were performed to detect carbohydrate entities which may be involved in the complement inhibition. Only the lectin of Limulus polyphemus was weakly bound to the mf surface suggesting the occurrence of low levels of sialic acid. Treatment of mf by sialidase, however, did not result in complement activation, and the binding of complement factor H could not be demonstrated on the surface of NHS-opsonized mf. In ongoing western blot experiments with O. volvulus extracts of mf and adults the binding of complement regulatory proteins to O. volvulus compounds will be examined.
Supported in part by BMBF
*) Provisional Research Station of the Institute in Macenta, Guinea
 
 

Neutrophil Chemotaxins in Onchocerca volvulus Female Worm Extracts

M. T. Rubio P. de Krömer, F. Geisinger, N. W. Brattig

Neutrophilic granulocytes represent the dominant inflammatory cells assembled around onchocercomata in O. volvulus infection. Recent studies showed that eosinophil accumulation around the female is dependent on the production of microfilariae by the females. Crude extract of O. volvulus females (OvE) was examined for chemotactic activity towards peripheral neutrophilic granulocytes from healthy individuals by use of an endogenous component (b-glucuronidase) chemotactic assay in Boyden chambers. Significant chemotactic responses of neutrophils were detected in OvE at 15-150 mg/ml in a dose-dependent manner (P<0.05). Checkerboard analysis demonstrated chemokinetic in addition to chemotactic activity. The chemotactic factors were stable to heating at 60° C for 30 min and 100° C for 5 min. Neutrophil migration was also elicited by excretory-secretory products of vital females. Fractionating of OvE by size exclusion FPLC technique using Superdex 2000 revealed two components with chemotactic activity, one with low molecular size of about 12 kDa and one with high molecular size >200 kDa. Further experiments will show whether the activity is related to the microfilarial components within the females and onchocercomata.
Supported in part by the BMBF
 

T Cell Responses in Co-infection with Onchocerca volvulus and the Human Immunodeficiency Virus Type 1 (HIV-1)

E. Sentongo*), D. W. Büttner*), T. Rubaale**), N. W. Brattig

Onchocerca volvulus and HIV-1 represent two important immunocompromising infective agents endemic in Africa. While predominant Th2 (type 2) responses characterise O. volvulus-infection, seropositive HIV-1 infection manifests as progressive loss of type 1 and increasing type 2 responses. To examine the effect of co-infection with O. volvulus and HIV on the immune response we studied T cell responses in 63 persons infected with O. volvulus and/or HIV-1 from Kabarole district in western Uganda. Their peripheral blood mono-
nuclear cells (PBMC) were analysed for phenotype by flow cytometry, proliferation as well as cytokine production to mitogenic PHA, parasite O. volvulus extract (OvE) and the ubiquitous antigens tuberculin (PPD), tetanus toxoid and Candida albicans wall extract. The proliferative response to PHA and PPD in O. volvulus/HIV co-infection (CD4+ 470/mm3) was less (P = 0.08, P < 0.01) and to C. albicans higher (P < 0.01) than in HIV-negative O. volvulus-infected persons (CD4+ 800/mm3). OvE-stimulated PBMC proliferation in HIV-negative O. volvulus-infected persons without microfilariae was not observed in the co-infected group. PPD-induced IFN-g, IL-4 and IL-5 production observed in HIV-negative O. volvulus-infected persons was reduced in co-infection (P < 0.01). The IL-4 and IL-5 response, elicited by OvE in O. volvulus-infected HIV-negative persons, was strongly reduced in co-infection (P = 0.02, P < 0.01). HIV-positive controls (CD4+ 200/mm3) from areas non-endemic for O. volvulus showed pronounced antigen-independent Th2 responses. IL-10 production was similar in all groups. Thus O. volvulus/HIV co-infected persons showed deficient antigen-specific Th1 responses and markedly impaired parasite-specific Th2 responses.
Supported in part by DAAD, Bonn
*) Parasitology Section
**) GTZ and Ministry of Health, Basic Health Services, Fort Portal, Uganda