Sex-dependent neutralizing humoral response to Schistosoma mansoni 28GST antigen in infected human populations.

The reduction of Schistosoma fecundity observed after experimental vaccination with the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28GST) antigen has been related to the inhibition of glutathione S-transferase (GST) enzymatic activity by specific antibody. The humoral immune response to the protective antigen Sm28GST and to the epitopes involved in the enzymatic site (amino acid ¿aa sequences 10-43 and 190-211) was evaluated in infected individuals before chemotherapy treatment. The capacity of the serum samples to inhibit GST enzymatic activity was assessed. Specific IgG3 response was predominant in the male population with a low intensity of infection and was associated with maximal GST inhibition. In contrast, the neutralizing activity of serum samples from women with a low intensity of infection was correlated with high specific IgA response specifically directed toward the 190-211 epitope. These results strongly support the hypothesis that GST-neutralizing IgG3 and IgA isotypes are sex dependent. The relationship of this specific acquired immune response with the level of intensity of infection is discussed.

Cellular immune responses of a Senegalese community recently exposed to Schistosoma mansoni: correlations of infection level with age and inflammatory cytokine production by soluble egg antigen-specific cells.

A recently reported epidemic of Schistosoma mansoni infection in Senegal provided an opportunity to study the dynamics of the development of immunity to human schistosomiasis. We report here on the cell-mediated immune response in a population of 99 females and 95 males, with particular emphasis on the relationship between intensity of infection and age. We found that the intensity of infection correlated negatively with age in females but not in males. In men and women, both Th1- and Th2-type cytokines were detected upon in vitro stimulation of PBMCs with soluble egg antigen (SEA) or soluble adult worm antigens (SWAP). In the female group, SEA-induced PBMC proliferation was associated with the production of IFN-gamma, IL-2 and IL-5, all of which correlated negatively with intensity of infection. Most cytokine production correlated positively with age. Spontaneous production of TNF-alpha, IL-6 and IL-10 was higher in the infected population than in an uninfected control group. Our results suggest that immunity to infection could be more pronounced in the female population and associated with a Th0/1 + 2 pattern of cytokine secretion mediated by soluble egg antigen (SEA).

Relationship of impairment of schistosome 28-kilodalton glutathione S-transferase (GST) activity to expression of immunity to Schistosoma mattheei in calves vaccinated with recombinant Schistosoma bovis 28-kilodalton GST.

Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) toward S. mattheei challenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinant S. bovis 28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinant S. bovis 28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinant S. bovis 28GST recognized the native S. mattheei 28GST and achieved comparable levels of inhibition of activity of recombinant S. bovis 28GST and S. matthei 28GST, indicating the presence of cross-reactive epitopes on these two molecules.

[Infectious etiology of diarrheal morbidity in a Senegalese region of high exposure to Schistosoma mansoni]. / Etiologie infectieuse de la morbidité diarrheique dans un foyer sénégalais fortement exposé à Schistosoma mansoni.

Endemic schistosomiasis due to Schistosoma mansoni has been observed in Richard-Toll (The Senegal River basin) in Senegal since 1990. Because of its high prevalence, schistosomiasis is assumed to be the cause of most cases of diarrhea observed in the region. The purpose of the present study carried out within the framework of the ESPOIR program for control of bilharziasis in the Senegal River region was to confirm the exact etiology of diarrhea in the region. A total of 109 subjects presenting diarrhea including 57 children under the age of 5 years were included in the study. In all cases, stool examination using appropriate techniques was performed to detect bacterial, viral, and parasitic agents. Schistosoma mansoni was identified in 47 cases (43.1%). Stool cultures were positive in 28 cases (25.6%) for Escherichia coli (n = 9), Shigella spp. (n = 18), and Salmonella spp. (n = 1). With regard to Shigella, a predominance of the Shigella dysenteriae type I stereotype (10/18) and a high incidence of co-infection involving Shigella spp. and Schistosoma was noted. Rotavirus infection was observed in 6 cases involving subjects under the age of 5 years. The relative incidence of the different infectious agents varied widely in function of age. This study in an endemic area of bilharziasis in Senegal demonstrates that Schistosoma mansoni should not be assumed to account for all cases of diarrhea occurring in the area.

IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized in part by circulating and tissue-bound IgG autoantibodies directed against the basement membrane zone. In addition, most of the patients with BP have increased serum IgE levels which seem to be correlated with the disease activity, whereas the presence of circulating anti-basement membrane zone IgE Abs has been reported in some patients. To elucidate whether IgE-dependent mechanisms play a role in the physiopathology of BP, we looked for the presence of IgE Abs specifically directed against the major BP Ag (BPAg1) in sera of BP patients at the onset and after remission of the disease. A radioimmunoassay and a 55-kDa recombinant protein (rBP55) obtained from a cDNA sequence, encoding the C-terminal region of the BPAg1 and containing the BPAg1 immunodominant epitopes, were used. Anti-rBP55 IgE Abs were found in 12 of the 19 sera tested. When the patients were divided into two groups according to the disease severity, anti-rBP55 IgE Abs were found only in patients with a severe form of the disease. Cytophilic IgE was detected on approximately 20% of peripheral blood eosinophils purified from BP patients. Immunohistochemistry studies suggested that some of the IgE-bearing cells in the lesional skin of BP patients are eosinophils. Immunostaining experiments revealed the existence of FcepsilonRI on both peripheral blood and tissue eosinophils. Taken together, these results suggest that IgE-dependent mechanisms could participate in the constitution of the lesions in BP.

[Presence of urinary Sm28GST antigen in Madagascar patients with Schistosomiasis mansoni]. / Présence d'antigène Sm28GST urinaire chez des patients malgaches atteints de bilharziose à Schistosoma mansoni.

Using an ELISA method, we quantitated the level of glutathion S-transferase 28 kDa (Sm28GST), specific of Schistosoma mansoni, in the urine of 13 malagasy patients. The presence of Sm28GST antigen has been demonstrated for the first time, in 8 patients (69%). No significant correlation could be found between antigen level and the parasitological burden in the feces. The use of an immunocapture ELISA technic on a larger number of samples will be necessary to assess if this test could be quantitative and reliable enough for the diagnosis or the follow up of patients infected with Schistosoma mansoni.

IgA antibodies to a protective antigen in human Schistosomiasis mansoni.

The specific IgA antibody responses to the protective recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) Ag and to derived synthetic peptides have been evaluated before and 6 mo after chemotherapy in S. mansoni-infected patients from Kenya. These studies revealed a parallelism between the age-dependent evolution of IgA antibody levels to Sm28GST and to one synthetic peptide (115-131) and the acquisition of resistance to reinfection. Functional analysis revealed that IgA antibodies to Sm28GST displayed a potent neutralizing effect on the enzymatic properties of the molecule, and also markedly impaired schistosome fecundity, by limiting both the egg laying of mature worms and the hatching capacity of schistosome eggs into viable miracidia. These results suggest that, in addition to IgE, IgA antibodies might participate in the protective immune response against schistosomiasis.

Synthetic vaccines and HIV-1 hypervariability: a "mixotope" approach.

The hypervariability of the gp120 envelope protein principal neutralizing domain, the V3 loop, represents a major problem in the design of vaccines against HIV-1. We have designed a mixed V3 loop peptide, termed "mixotope," obtained in a unique synthesis, and containing around 7.5 x 10(5) different sequences of 22 to 25 residues, organized around the conserved GPGR tetrapeptide. Free or coupled to a carrier protein, the "mixotope" induced in rabbits broadly specific antibodies, which recognized different individual V3 loop sequences, and the native gp120 protein. The "mixotope" approach may allow researchers to focus vaccine strategy against hypervariable functional epitopes of various pathogens.

A synthetic protein corresponding to the entire vpr gene product from the human immunodeficiency virus HIV-1 is recognized by antibodies from HIV-infected patients.

The 95 amino acid-protein encoded by the non-structural vpr gene of the human immunodeficiency virus type 1 (LAV-1BRU isolate) was chemically synthesized by solid phase methodology. The synthetic vpr protein was characterized by amino acid analysis, sequence analysis, RP-HPLC, and urea-SDS PAGE. Using a radioimmunoassay, antibodies to the synthetic protein were detected in sera of 25% of HIV 1-seropositive patients tested. Western blot analysis suggested that the antibodies preferentially recognize the dimeric form of vpr.

Antibody response of Schistosoma mansoni-infected human subjects to the recombinant P28 glutathione-S-transferase and to synthetic peptides.

The 28-kilodalton antigen of Schistosoma mansoni has been previously described as having importance as the basis for a potential vaccine. The P28 recombinant molecule and three peptides derived from its primary sequence, namely the 24-43, 115-131, and 140-153 peptides, have been tested to evaluate the humoral responses of Kenyan school children previously classified as susceptible or resistant to reinfection after chemotherapy. We report here that the P28 molecule and two of the peptides studied (peptides 115-131 and 140-153) can be used for detecting specific immunoglobulin G (IgG), IgE, and IgA antibodies. Moreover, the IgG4 response of the susceptible population was significantly greater than that of the resistant group, whereas no differences between the two populations were noticed in total IgG anti-P28 antibodies. This suggested that IgG4 could play a role in the lack of immunity of susceptible patients. A strong IgG3 response restricted to the 140-153 peptide was observed but did not discriminate between the resistant and susceptible populations. In contrast, a marked increase in the IgA response to the 140-153 peptide epitope(s) in sera of the resistant population was noticed. Taken together, these results suggest that the P28 antigen and two of the three peptides selected could give predictive information about the development of the disease or the efficiency of vaccination with P28 as the immunogen.

Antibodies to the nef protein and to nef peptides in HIV-1-infected seronegative individuals.

The silent period that follows infection by the human immunodeficiency virus (HIV-1) and precedes seroconversion remains a problem for the screening of blood supply, and knowledge about the mechanism involved in the maintenance of latency is only fragmentary. Using purified nef recombinant protein and six synthetic nef peptides, antibodies to the product of an HIV-1 regulatory gene, the negative regulatory factor (nef) involved in maintenance of proviral latency, were detected by Western blot and radioimmunoassay techniques in HIV-1-seronegative, viral antigen-negative, and virus culture-negative individuals at risk for HIV infection. This antibody response to nef was correlated in eight individuals with the detection of HIV-1 proviral DNA by oligonucleotide hybridization, following enzymatic amplification of HIV DNA in peripheral blood mononuclear cells. Such latent HIV infections have now been followed for up to 6 or 10 months in five individuals. In addition, retrospective and prospective analysis of HIV-1-seropositive individuals have shown (1) antibodies to nef preceding seroconversion, and (2) the persistence of antibodies to nef and of HIV-1 proviral DNA in a case of spontaneous complete HIV-1 seronegativation. Since DNA amplification cannot be currently considered for routine use, screening for anti-nef antibodies followed by confirmation by DNA amplification could represent a basis for new diagnostic strategies. Beyond their diagnostic implications, these findings, suggesting that regulatory genes of the HIV-1 provirus can be expressed prior to the initiation of virion synthesis, may also be applicable in the design of alternative vaccines against the acquired immunodeficiency syndrome.

Total and specific IgE in serum and cerebrospinal fluid of rats and guinea pigs infected with Angiostrongylus cantonensis.

The total and specific IgE response to Angiostrongylus cantonensis infection was evaluated according to host permissiveness. Total IgE levels measured by a double antibody radioimmunoassay (RIA) increased slowly in the permissive host (the rat), reaching a maximum between 4 and 8 weeks after infection. This maximum was earlier but significantly lower in the non-permissive host (the guinea pig). IgE antibodies specific for adult worms or L1 or L3 larvae of A. cantonensis were measured by a radioallergosorbent test (RAST). In the case of adult worms and L1 antigens, specific IgE antibody levels showed large variations in relation to the duration of infection in rats. In contrast to total IgE levels, the specific IgE response to L3 larvae was lower in rats than in guinea pigs in both the serum and cerebrospinal fluid (CSF). These results suggest variations in the total vs specific IgE response according to host permissiveness or non-permissiveness to A. cantonensis infection. These results are discussed in the context of the possible participation of IgE antibodies in immune defence.

Analysis of T and B cell epitopes of the Schistosoma mansoni P28 antigen in the rat model by using synthetic peptides.

The Schistosoma mansoni P28 molecule is an Ag inducing protective immunity in various experimental models. Three synthetic peptides, derived from the primary sequence of the recombinant P28 and comprising amino acids 24-43, 115-131, and 140-153, respectively, were synthesized according to their hydrophilicity, mobility, and accessibility profiles. The presence of B and T lymphocyte epitopes in these peptides has been examined in the rat model. The results showed that the 24-43 and the 115-131 peptides contained major epitopes for IgG but not for IgE. Moreover, the 24-43 peptide-specific IgG produced after injecting either the recombinant P28 Ag or the 24-43 peptide coupled to tetanus toxoid was essentially of the IgG2a subclass and to a lesser extent of the IgG1 subclass, whereas no IgG2c was detected. These 24-43 peptide-specific antibodies were cytotoxic in vitro for schistosomula in the presence of eosinophils as effector cells. The 24-43 and the 140-153 peptides contained major targets of T lymphocytes specific for the recombinant P28 Ag. T cell lines specific for the 24-43 peptide have been prepared. These cells proliferated in vitro when stimulated with various S. mansoni crude antigenic preparations or with the recombinant P28 Ag. Moreover, their passive transfer to rats immunized with the P28 Ag led to a significant increase in specific IgE without modifying the IgG response.

Protective role of IgE in immunocompromised rat toxoplasmosis.

In contrast to euthymic adult Fischer rats, immunocompromised Nu/Nu animals develop a lethal infection when inoculated with the RH strain of the protozoan Toxoplasma gondii. However, a significant period of survival is obtained when Nu/Nu rats are passively transferred with sera from 28-day infected Fischer +/+ (euthymic) animals. Specific IgE are involved since IgE-depleted sera are unable to afford such a protection. Only excreted/secreted Ag or living tachyzoites are able to induce a significant protective IgE response in intact animals. In addition, platelets or, to a lesser extent, eosinophil-rich populations from Toxoplasma infected or excreted-secreted Ag-immunized euthymic animals bear surface IgE and are cytotoxic for the parasite in vitro. Also, adoptive transfer of immune platelets confers a significant degree of protection to Toxoplasma-infected Nu/Nu animals. Our results clearly show the key role of Ag present in both living parasites and excreted-secreted Ag to induce, in this model, a protective IgE response. In addition, as in other parasitic infections, platelets and probably eosinophils are the effector cells involved in controlling parasitic dissemination during Toxoplasma infection in immunocompromised rats.

Properties of serine proteases of Schistosoma mansoni schistosomula involved in the regulation of IgE synthesis.

The regulation of IgE synthesis in vitro and in vivo by schistosomula-released products (SRP) has been shown to be dependent on the presence of serine proteases. The present paper concerns the characterization of the enzymes involved. The labelling of SRP with [3H]diisopropyl phosphofluoridate revealed two molecules, one major with an MW of 27,500 and one minor with an MW of 29,000. The same pattern was obtained by labelling of schistosomula or cercariae surfaces as well as of the total schistosomulum homogenate. The properties of these enzymes were studied by means of various specific substrates or inhibitors of serine proteases. The specificity was relatively narrow, but had some similarity with trypsin. When added to lymphoid rat cells in culture, SRP induced an increased expression of receptors for the Fc fragment of IgE (Fc epsilon RII). This suggests that the IgE-enhancing property of SRP was due to serine protease activity which may act by enhancing the lymphocyte Fc epsilon RII.

Role of serine proteases of Schistosoma mansoni in the regulation of IgE synthesis.

The regulation of the IgE response by schistosomula-released products (SRP) was studied either in vitro with rat and human cell cultures or in vivo by injection into rats of SRP with an unrelated allergen at primary or secondary immunization. The results obtained in vitro showed that non-dialysable factors present in SRP potentiate the IgE synthesis by rat and human cells. This enhancing effect was supported by molecules with serine protease activities. On the other hand, the inhibition or depletion of SRP in serine proteases induced a weak synthesis of IgM by rat cells in vitro. The injection of SRP into rats on day 0 with an unrelated allergen led to a potentiation of total IgE production, but an inhibition of specific IgE response. In contrast, a marked elevation of specific IgE response was obtained when SRP was injected upon secondary immunization. Serine proteases of SRP were partly responsible for this potentiative effect.

Measurement of picogram quantities of rat IgE from in vitro culture supernatants of B lymphocytes in experimental helminth infections.

A double-antibody liquid-phase radioimmunoassay was used to measure picogram quantities of rat IgE. The sensitivity of this method (approximately 10 pg/ml) allowed the determination of the production of IgE in in vitro culture supernatants of B lymphocytes of infected animals.

Development of a competitive radioimmunoassay for human plasma fibronectin.

A competitive radioimmunoassay (CRIA) for quantitating human plasma fibronectin levels has been developed and compared with a conventional immunoturbidimetry assay (IMTA). The assay ranges for CRIA and IMTA were 0.05-5 micrograms/ml and 100-1,000 micrograms/ml, respectively, and the lowest detectable amounts of fibronectin that differed significantly from zero were 17.6 ng and 50 micrograms. The correlation coefficient between CRIA and IMTA was r = 0.953 (p less than 0.001). We consider the CRIA as potentially useful in the identification and study of fibronectin in certain biological fluids where it may be present in low concentrations.

Specific IgE antibodies to Leishmania braziliensis in patients with mucocutaneous leishmaniasis.

A total of 116 sera of patients suffering from South American mucocutaneous leishmaniasis were analysed using the RAST (L. braziliensis-specific) and RIST techniques. Results of total and specific IgE levels were correlated with different clinical stages of the disease. The level of total and specific IgE appeared to be higher among patients with cutaneous ulcers than among those presenting mucocutaneous lesions, i.e. espundia. Furthermore, non-treated patients presented higher concentrations of total and specific IgE than did treated ones.