Immunological cross reactivity between Schistosoma mansoni and cholera toxin.

Intranasal administration of schistosome antigens in combination with appropriate adjuvant may be an effective route for immunization against schistosomes, since the lungs represent an important site of elimination of schistosomulae. Our previous studies have shown that in mice intranasal administration of cholera toxin (CT) before infection with Schistosoma mansoni results in an enhancement of the worm burden in comparison to nontreated infected animals. In the present study, it was shown that mice treated intranasally with CT displayed high numbers of schistosome-reactive IgM-secreting cells in the spleen as well as high levels of schistosome-reactive serum IgM antibodies, whereas no significant immunological response against two other antigens, ovalbumin (OVA) or keyhole limpet haemocyanin (KLH) was noted. Sera from mice treated intranasally with CT recognized a 22 kDA antigen on SWAP blots. This band was not demonstrable after absorption of the sera with SWAP. These findings indicate a possible cross reactivity between cholera toxin and schistosome antigens. Further analysis by Western blot revealed that a 22 kDa antigen was detected on CT blots by sera from mice and humans infected with S. mansoni. This band was not demonstrable after absorption of the mouse or the human sera with CT. The 22 kDa cross reactive antigen was heat-stable. The antibodies against the 22 kDa antigen were only found within the IgM class but not within other Ig isotypes. Our findings also indicate that the 22 kDa antigen detected by anti-S. mansoni antibodies represents the A1 fragment of the cholera toxin.

Polypropylene fibre web, a new matrix for sampling blood for immunodiagnosis of schistosomiasis.

Blood sampling on filter paper is widely used for immunodiagnostic and epidemiological purposes. However, elution of conventional filter papers impregnated with sera containing Schistosoma mansoni circulating anodic antigen (CAA) recovered only a small fraction of the antigen, thereby reducing the sensitivity of the assay. Polypropylene-based non-woven fibre web is a new sampling material with a low density of fibres and with a small surface area of contact. When it was impregnated with serum containing CAA, approximately 90% of the antigen could be extracted. The yield of antibodies against S. mansoni from the new sampling material did not differ from that from conventional filter papers.

Intranasal administration of Schistosoma mansoni adult worm antigen in combination with cholera toxin induces a Th2 cell response.

Mice immunized with soluble adult worm antigen (SWAP) in combination with cholera toxin (CT) displayed significantly larger numbers of IgG1, IgM and IgA secreting cells in the spleen and in the lungs as compared to mice which had received SWAP only. The ratio of SWAP-specific IgG1 to IgG2a antibody-secreting spleen cells was also significantly higher in the SWAP-CT group. Analysis of cytokine responses revealed that SWAP-stimulated spleen and lung cells from the SWAP-CT group produced lower levels of IFN-gamma but higher levels of IL-4 and IL-5 as compared to cells from the SWAP group. These findings indicate that intranasal administration of SWAP-CT induces a Th2 cell response in the spleen and in the lungs. Our findings also suggest that CT was responsible for induction of this Th2 cell response, since intranasal administration of SWAP alone induced a Th1 type response in the spleen and in the lungs.

A comparative study on specific antibodies and circulating antigen (CAA) in serum and parasitological findings for diagnosis of schistosomiasis mansoni in an endemic area in Tanzania.

A baseline study to evaluate the prevalence of Schistosoma mansoni infection as well as the diagnostic efficacy of serodiagnostic tests was performed in Kabaganga village, Kome island, Lake Victoria, Tanzania. A total of 1108 individuals were examined parasitologically and clinically. Egg excretion was demonstrated by one-sample Kato-Katz test. Specific IgG1 and IgG4 antibodies against S. mansoni adult worm (SAWA) and egg (SEA) antigens as well as circulating anodic antigen (CAA) were determined in serum samples from 250 of these subjects. As a control population 41 individuals from a non-endemic area were examined parasitologically, clinically and serologically. In the parasitologically examined Kabaganga population 45% were excreting eggs. The pattern of egg excretion was typical for an endemic area with a peak in the age group 10-14 years. Sixty-five percent of the serologically tested villagers were positive in the CAA test. A total of 80% were positive in either of the two tests, indicating an active infection. In 67-95% of these individuals the levels of isotype specific antibodies were increased. The prevalence of CAA positivity corresponded fairly well with that of Kato-Katz results in the age groups 10-29 years, but in the younger age groups a considerably greater number of individuals were positive in the CAA test than in the Kato-Katz test. The results obtained indicate that virtually all of the Kabaganga villagers, regardless of age, had an ongoing, active infection or had previously been infected with S. mansoni. This population, therefore, may be useful for evaluation of the diagnostic efficacy of various antibody tests. The highest degree of discrimination between the endemic and the non-endemic village populations was noted for anti-egg IgG4 antibodies. It is concluded that the combined determination of parasite eggs in faeces and CAA in serum provides high sensitivity as regards active infection. Increased levels of isotype-specific antibodies, particularly of the IgG4 subclass, is a sensitive indicator of past or present infection, and the prevalence of individuals with such increased levels may be a simple and reliable indicator of the frequency of schistosomiasis in a community.

Visceral leishmaniasis in Somalia. Significance of IgG subclasses and of IgE response.

We have determined the levels of IgG subclasses and IgE as well as specific antibodies of these isotypes in sera from 22 patients with clinical visceral leishmaniasis (VL) from Somalia. The results are compared with those obtained from 30 Somali and 23 Swedish controls. We found markedly increased concentrations of IgG1 in the VL sera, indicating that the pronounced increase in IgG in VL which is generally considered to be due to polyclonal B-cell activation is mainly restricted to this subclass. The IgG2 concentrations were significantly decreased. The IgG3 and IgG4 concentrations, on the other hand, did not differ between the two groups of Somali sera. The Somali control sera contained higher concentrations of IgG1 and IgG3, but significantly lower concentrations of IgG2 as compared to Swedish controls. The IgG4 values, on the other hand, were not different between the two groups of control sera. Anti-leishmania antibodies belonging to all IgG subclasses, were found in the patients' sera. There was no significant difference in total IgE between sera from VL patients and controls and specific IgE antibodies were only detected in a few patients. The Western blot assay (WB), revealed the presence of two bands corresponding to 74 kDa and 88 kDa in all patients' sera, indicating a possible diagnostic role for WB in this particular population.

Lack of interferon-gamma receptor does not influence the outcome of infection in murine schistosomiasis mansoni.

Studies of vaccine-induced immunity in experimental schistosomiasis in mice have suggested that interferon-gamma (IFN-gamma) is an important factor for the induction of protective immunity against schistosomiasis. The present study compares some parameters during primary schistosome infection in IFN-gamma receptor deficient mice and wild type mice. No significant difference in worm burden between the two groups was found. Almost the same number of eggs in the liver as well as typical granulomas with numerous macrophages and eosinophils were observed in both groups of mice. Furthermore, IFN-gamma receptor deficient mice infected with S. mansoni displayed a significant reduction in the number of IgG2a secreting cells in the spleen and a significant enhancement of IgA secreting cells in the spleen and in the lungs. These findings suggest that the lack of IFN-gamma activity may result in an enhanced dominance of Th2 cells which, however, does not influence the development of a primary schistosome infection.

Visceral leishmaniasis in Somalia: prevalence of markers of infection and disease manifestations in a village in an endemic area.

Prevalence and disease manifestations of visceral leishmaniasis (VL) were studied in a Somali village in an area which has long been known to be endemic for VL. Demographic data were collected from 102 households, comprising 438 inhabitants. Clinical examination was performed of 306 individuals, 72% of the 426 eligible persons. Of these, 276 (90%) agreed to give blood and 246 (80%) to be skin tested with leishmanin. Leishmanin reactions were positive; in 26% anti-Leishmania antibodies were detected in 11%, and splenomegaly was recorded in 14% (23% of those who were seropositive). Malaria was hypoendemic and therefore unlikely to be responsible for more than 10% of the cases with splenomegaly. Three of the seropositive villagers with splenomegaly complained of feeling ill. The remaining 91 sero- and/or leishmanin-positive individuals had no complaint regarding their health and had not experienced any long period of illness. There was a slight over-representation of males in the group of sero- and/or leishmanin-positive villagers, possibly due to a gender-associated difference in exposure to the parasite. Among the patients with clinical VL treated at Mogadishu hospitals during 1989 and 1990, the male/female ratio was 3.3:1, which may indicate a selection of male patients for hospital care. Most patients were < or = 15 years old, suggesting that the highest risk of becoming clinically ill was among children.

Visceral leishmaniasis in Somalia: prevalence of leishmanin-positive and seropositive inhabitants in an endemic area.

In an endemic area of Somalia both humoral and cell mediated immunity against Leishmania donovani was demonstrated in 246 inhabitants. In a study of 14 patients with active visceral leishmaniasis, we found that antibodies appear early in infection and that they are then demonstrable for a limited period only. Leishmanin positivity develops later and persists longer, but does not seem to be lifelong. The majority of the immunoreactive individuals were either sero- or leishmanin positive. This finding is in accord with the result obtained in recent experimental studies indicating a regulatory effect exerted on humoral and cell mediated immunity by different T lymphocyte subsets.

Visceral leishmaniasis in Somalia. Circulating antibodies as measured by DAT, immunofluorescence and ELISA.

Sera from patients with visceral leishmaniasis (VL) (n = 26), healthy residents of Mogadishu (n = 157), inhabitants of a village in an endemic area (n = 276) and healthy Swedes (n = 60) were examined using the direct agglutination test (DAT), immunofluorescence (IF) and ELISA for antibodies against Leishmania donovani. The study was carried out in order to provide baseline data for antibody responses in visceral leishmaniasis as existing in Somalia and to explore which one of these methods would be most suitable for diagnosis of clinical cases as well as for epidemiological population studies in Somalia. All patients had high levels of circulating antibodies, however, lower values were recorded in the early stages of the disease. High reactivity in ELISA was seen first after one year. All three tests distinguished well between sera from VL patients and healthy controls. Approximately 10% of the sera from villagers were reactive above the cut-off levels in the three tests. DAT is the simplest to perform and does not require much equipment. ELISA can be made simple and economic if performed in one serum dilution and read visually. IF requires more expensive and specialized equipment and is not suitable for large scale examination of sera. A complete evaluation of the three tests should also include the analysis of sera from various stages and manifestations of the disease.

Reference ranges for IgG, IgM and IgA in the serum of urban and rural Somalis.

In order to provide baseline data for an immuno-parasitological laboratory in Somalia, serum concentrations of IgG, IgM and IgA were determined in some key populations: healthy residents of Mogadishu (n = 157), inhabitants of the village of Daimo Samo (n = 276) and patients with malaria (n = 39) and visceral leishmaniasis (n = 26), both protozoan infections accompanied by hypergammaglobulinaemia and causing severe health problems in Somalia. Since the serum immunoglobulin concentrations in the Somali populations studied were not normally distributed, they were evaluated using medians and percentiles. Significantly higher values of IgG, IgM and IgA were demonstrated in healthy Mogadishu residents as compared to healthy Swedes. Daimo Samo villagers had significantly higher IgG and IgM values than healthy Mogadishu residents. Very high concentrations of IgG and IgM were demonstrated in sera from patients with visceral leishmaniasis. Somali patients with malaria also had marked hypergammaglobulinaemia, however, only in the IgG class. The high levels of IgG, IgM and IgA demonstrated in sera from Somalis, indicate the need for establishing local reference values and should be considered when introducing serological tests in tropical countries. Such methods are usually adopted to conditions in industrialized countries, where immunoglobulin contents of sera are lower.

Effect of cholera toxin on vaccine-induced immunity and infection in murine schistosomiasis mansoni.

Intradermal vaccination of mice with soluble adult worm antigen (SWAP) in combination with Mycobacterium bovis BCG (Swedish strain) induced significant protection against subsequent infection with Schistosoma mansoni cercariae. When cholera toxin (CT) was used as an adjuvant in combination with SWAP or fraction A, no significant protection was observed. However, intradermal vaccination in combination with CT triggered a strong anti-SWAP antibody response and induced a strong delayed-type hypersensitivity response to schistosome antigens (SWAP or fraction A), one significantly higher than that in the SWAP-BCG group. In addition, vaccinating mice intranasally with SWAP or cercarial antigen together with CT as adjuvant failed to induce any significant protection. Surprisingly, mice given CT alone intranasally revealed a significantly enhanced worm burden. These findings suggest that mucosal application of CT may modulate the host-parasite relationship in favor of parasite survival.

Comparative antigen analysis of different life stages of Schistosoma mansoni by crossed immunoelectrophoresis.

Two-dimensional (crossed) immunoelectrophoresis was used for analysis of soluble antigen extracts obtained from the three developmental stages, cercariae, adult worms and eggs, of Schistosoma mansoni by using homologous hyperimmune sera produced in sheep. The antigenic relationships between the three stages as well as the possible relationship to the intermediate snail host were studied. Seven antigen components were shown to be shared between all three life stages of S. mansoni. Furthermore, one antigen was common to adult worm and snail, and one other antigen was shared between cercaria and snail. By using an intermediate gel containing lectin in the antigen-antibody system or by enzyme staining of the immune precipitates it was possible to identify schistosome antigens possessing lectin reactivity or enzyme activity. Characterization of enzyme activities revealed three individual precipitating antigens in adult worm of S. mansoni possessing esterase, leucyl-glycyl-glycine peptidase and phenylalanyl-leucine peptidase activities, respectively. One further precipitinogen with malate dehydrogenase activity was identified for all three life stages.

Kinetics and isotype distribution of parasite-specific antibody responses in the spleen of mice during primary infection with Schistosoma mansoni. Studies at the single-cell level.

The solid-phase enzyme-linked immunospot assay has been adapted for enumerating cells secreting antibodies to crude antigenic extracts from the main developmental stages of Schistosoma mansoni. The frequencies of splenocytes secreting antibodies reactive with soluble antigenic extracts from cercariae, adult worms, and eggs, were examined in C57BL/6 mice during the first 12 weeks of a primary infection with S. mansoni. Despite the existence of cross-reactive moieties present in these antigenic preparations, the characteristics of the responses monitored differed with regard to kinetics, magnitude, and/or isotype distribution. The responses peaked between 8 and 10 weeks of infection, were characterized by a predominance of cells secreting IgM antibodies against all three antigens, and followed the patterns IgM greater than IgA greater than IgG for cercariae, IgM greater than IgG greater than IgA for adult worms and IgM greater than IgG greater than IgA for egg extracts. On the other hand, these patterns were not always reflected in serum, especially for cercariae-reactive circulating IgG and IgA responses. When examined for numbers of total IgM-, IgG- and IgA-producing cells, the spleen of S. mansoni-infected mice was shown to display considerable numbers of immunoglobulin-producing cells in all three isotypes studied. The most spectacular increases were noted for IgG-secreting cells (more than 10-fold) and for IgM-secreting cells (up to 6-fold) 4 weeks after initial cercarial exposure. Taken together, these observations indicate that primary infection with S. mansoni results in early immunoregulatory alterations which may contribute to the maintenance of specific as well as nonspecific B cell hyperactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells.

A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described.

Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells.

A reverse modification of ELISPOT assay using nitrocellulose membranes and epitope-specific monoclonal antibodies is described for the detection of single lymphokine-secreting cells. As a model, the production of gamma-interferon by mitogen stimulated human peripheral blood lymphocytes has been examined. The assay can also be modified to permit microscopic examination of spot-forming cells.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses.

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs.

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.