Localization of leucine aminopeptidase in the Schistosoma mansoni eggs and in liver tissue from infected mice.

Infection with Schistosoma mansoni causes hepatic granuloma formation and fibrosis in response to parasite eggs. The present work localized the leucine aminopeptidase (LAP) in S. mansoni eggs and in liver tissue sections from infected mice. Fresh eggs and livers obtained from infected hamsters were processed and stained with the L-leucine-7-amino-trifluoromethyl-coumarin specific substrate. The L-argnine-7-amino-trifluoro-methylcoumarin and Bestatin (leucine aminopeptidase inhibitor) were used to test the LAP substrate specificity and reactivity. The staining pattern for that enzyme in the egg and liver tissue reflects that the leucine aminopeptidase is a major egg constituent distributed in nearly all the egg except the spine. The control substrates confirmed the substrate broad specificity of LAP. In conclusion, the LAP enzyme is a major egg antigen and the target antigen for the antipathology vaccine development studies.

Effect of combined praziquantel and recombinant glutathione S-transferase on resistance to reinfection in murine Schistosomiasis mansoni.

This study was undertaken to evaluate the effect of recombinant Schistosoma mansoni-26 Glutathione S-transferase (rSm 26 GST) or soluble egg antigen (SEA) alone and in addition to praziquantel (PZQ) on the state of resistance to S. mansoni reinfection. The associated changes in the immune responses were evaluated. The experimental group of mice were injected intravenously before S. mansoni infection (80 cercariae/mouse) either with rSm26 GST (1 microgx4) or SEA (10 microgx4) in addition to PZQ (2x500 mg/kg) administered 6 weeks post-infection. Seven control groups were used, three of them were the infected (80 cercariae/mouse), the challenged (240 cercariae/mouse) and the infected challenged controls (80+240 cercariae/mouse). The rest of the four groups were the treated controls receiving: the GST-Lyzate, rSmGST, SEA and PZQ in the same doses and at the same timings. Challenge infection was conducted for all the groups 8 weeks post-infection. Animals were sacrificed 3 weeks post-challenge. After sacrifice animals were perfused and percentage resistance to reinfection was calculated. Immune responses were assessed by the measurement of hepatic granuloma diameter, intralesional T-cell phenotypes and serum immunoglobulin isotypes. The highest percentage of resistance to reinfection was observed in rGST-treated group while the lowest percentage of resistance was detected in PZQ-treated group. Whereas in mice receiving combined rGST or SEA and PZQ, percentage resistance to reinfection was significantly higher than that in PZQ treated mice. The remarkable reduction in granuloma diameter in rGST-treated group with or without PZQ was associated with decrease in the intralesional L(3)T(4)(+) and increase in Lyt(2)(+) T-cell phenotypes. However, no special relationship was observed between the percentage of resistance and the changes in granuloma diameter or intralesional T-cell phenotypes. The increase in percentage resistance to reinfection was found accompanied by increased anti SWAP IgE. Combined rGST and PZQ provided the complementary goals of improved state of resistance to reinfection 'which was compromized after cure with PZQ' and the maximal reduction in granuloma diameter.

Characterization of Schistosoma mansoni 44.7/56.8 kDa egg antigens recognized by human monoclonal antibodies which induce protection against experimental infection and proliferation of peripheral blood mononuclear cells from schistosomiasis patients.

We described here the characterization of Schistosoma mansoni egg antigens recognized by human monoclonal antibodies B10 (HmAb-B10) and D5 (HmAb-D5). SDS-PAGE and Western blot analysis revealed that these monoclonals recognized two antigens of M W 44.7/56.8 kDa, with pI of 7.0 and 7.8, respectively. The passive transfer of B10 and D5 induced a significant protection of 48% and 54% in Balbic mice. Results of in vitro cytotoxicity assay showed that both monoclonals were able to kill schistosomula in the presence of rabbit complement. These monoclonals mediated 48% and 74% of schistosomula cytotoxicity, respectively. Egg antigens were purified by affinity chromatography using monoclonal antibodies B10 and D5. Treatment of purified antigens with periodate, galactose oxidase and trifluoromethane sulphonic acid did not prevent binding by B10 and D5 in ELISA assay. However, the treatment with protease K and 2-mercaptoethanol affects the antibodies binding, showing that the HmAbs B10 and D5 recognize polypeptide epitopes. Vaccination of mice with these antigens in Freund's adjuvant induced 43% reduction in worms burden after challenge with S. mansoni cercariae. In vitro blastogenesis assays with peripheral blood mononuclear cells from patients infected with S. mansoni revealed that purified antigens were able to induce significant cell proliferation.

Efficacy of an antipathology vaccine in murine schistosomiasis administered with and without chemotherapy.

This study was undertaken to study the efficacy of praziquantel (PZQ) in potentially tolerized Schistosoma mansoni infected, egg-injected C57BL/6 mice, receiving multiple administrations of soluble egg antigen (SEA) intravenously (i.v.). Four animal groups were studied. Experimental group I received four injections of SEA (10 micrograms) intravenously on days -7, -5, -3 and -2 before infection and PZQ orally (500 mg/kg over two consecutive days 7 weeks post-infection. Three control groups received the following treatment: group II received the same tolerizing dose of SEA without PZQ, group III received PZQ in the same dose and at the same timing. Group IV received S. mansoni infection and egg injection 8 weeks post-infection and served as an infected, egg-injected control. Egg injection was conducted 8 weeks post-infection using viable S. mansoni eggs via the tail vein. Animals were killed 16 days post-egg injection, i.e. 10 weeks post-infection. After sacrifice, lungs and livers were removed for histopathological study and measurement of granuloma diameters. Spleens and serum were collected for the assay of lymphoproliferative response to SEA and antischistosomal immunoglobulins. The worm and egg burdens were also studied. Compared to infected, egg-injected untreated controls, repeated i.v. administrations of SEA down-regulated egg-injected (pulmonary) and egg-deposited (hepatic) granulomas and the lymphoproliferative response to SEA. Antischistosomal IgG level was increased. Susceptibility to S. mansoni infection was not found to be different from that in the infected, egg-injected controls. PZQ in the dose used caused complete eradication of worms, disappearance of immature egg stages, decrease in the number of mature eggs and an increase in the number of dead eggs. Hepatic granuloma diameter, lymphoproliferative response to SEA and IgG level were reduced. In mice receiving a combined regimen of multiple SEA administrations and PZQ with down-regulated granuloma and reduced lymphoproliferative response to SEA, the efficacy of PZQ was the same as in mice receiving PZQ alone. This was shown by comparable grades of worm and egg reduction. The histopathological examination of liver and lung sections in the different treated groups revealed moderate to small-sized hypocellular granulomas. Although no statistically significant difference was recorded between the mean granuloma diameters of the experimental group receiving both the tolerizing dose of SEA and PZQ compared to the group receiving the tolerizing dose of SEA without chemotherapy, this experimental group showed the least associated histopathological parenchymal changes. It appears from this work that combined SEA and PZQ provided many complementary goals; a reduction of egg-induced pathology, minimal parenchymal changes and the eradication of worms.

HPLC fractionated soluble egg antigen from Schistosoma mansoni elicits a heterogeneous human immune response.

Peripheral blood mononuclear cells (PBMC) from five chronic schistosomiasis patients, three former patients, a SEA sensitized individual, and normal controls were tested in lymphoblastogenesis assays for their ability to proliferate in response to soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) from Schistosoma mansoni. Cells from all patients and the SEA sensitized individual gave significantly higher responses than the normal controls when stimulated with SEA and SWAP. However, the chronic patients' SEA responses were much lower than those of the former patients and the SEA sensitized individual. When cells from the same donors were tested in the in vitro granuloma assay, all produced significant granulomatous responses except the normal controls. Once it was established that all individuals in the study gave significant lymphoproliferative responses and granulomatous reactions, SEA was subjected to HPLC fractionation to identify immunogenic protein components of SEA. HPLC separation yielded 25 major fractions. SEA responses from the sensitized individual and former patients exhibited broad, unregulated responsiveness including fractions with neutral, less charged proteins while the chronic patients demonstrated a more restricted range of responsiveness. SEA-HPLC fractions 14, 21, and 22 contain the most immunodominant proteins based on cellular proliferation data from reactive individuals tested.

Human schistosomiasis: modulation of in vitro granulomatous hypersensitivity and lymphocyte proliferative response by macrophages undergoing differentiation.

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.

Recognition of different Schistosoma mansoni antigens by specific human T cell clones.

Human T lymphocyte clones (TLC) sensitized to Schistosoma mansoni soluble egg antigens (SEA) were developed from chronic schistosomiasis mansoni patients in order to investigate the regulatory mechanisms involved with in vitro granulomatous hypersensitivity to this parasite. All clones studied displayed CD4+ phenotype and required antigen-presenting cells in antigen-driven proliferation and granuloma formation. Each T lymphocyte clone has been shown to proliferate and generate in vitro granulomas in response to SEA, adult worm antigens (SWAP), or cercaria antigens (CAP). In contrast, no proliferation was observed in any of these T cell clones when unrelated S. mansoni antigens were used. Some SEA-generated TLC were not able to proliferate in the presence of SEA; however, S. mansoni SEA-reactive ones were able to recognize epitopes in Schistosoma japonicum SEA, indicating cross-reactivity between these two species. Using IFN-gamma ELISA, it was shown that TLC cells stimulated with SEA can secrete appreciable amount of this lymphokine. Further in vitro studies with these SEA-TLC will help us to understand in more depth the role of regulatory T cells in the human granulomatous hypersensitivity to S. mansoni eggs.

Granulomatous hypersensitivity to Schistosoma mansoni EGG antigens in human schistosomiasis. IV. A role for prostaglandin-induced inhibition of in vitro granuloma formation.

The prostaglandins (PG) are known to regulate immune cell function(s) and participate in the progression of both acute and chronic inflammatory reactions. Using an in vitro model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the role of immune complexes (IC) in the induction and release of PG and their inhibitory effects on granuloma development. The hypersensitivity-type granuloma reaction to soluble egg antigen (SEA) was examined using a model of in vitro granuloma formation. Our results show that granuloma formation was dramatically suppressed by the addition to the granuloma cultures of IC, PGE1, PGE2, while PGF2 alpha had no significant effect. The inhibition of the PG function was achieved by the introduction of anti-PG antibodies that blocked suppression of granuloma formation. It appears in this model system that IC may inhibit the activity of granuloma formation by stimulating the monocyte-macrophage lineage to release inhibitory mediators. Our results suggest that the prostaglandins E series may be important in the generation and maintenance of suppression of the granulomatous inflammatory response to S. mansoni egg antigens.

Human giant cell formation induced in vitro by Schistosoma mansoni antigens.

Although multinucleated giant cells have been described for many years in association with different chronic inflammatory responses, their participation in immunoregulatory mechanisms within the schistosome egg granulomas remains to be clarified. In this study we determined if soluble egg antigen (SEA) or adult worm antigen preparations (SWAP) from S. mansoni induce giant cell formation in vitro and their relationship with the intensity of granulomatous reactivity. Antigenic stimulation of peripheral blood mononuclear cells (PBMC) from patients (N = 9) with active schistosomiasis infection increased giant cell formation per field after the 12th day in culture when treated with S. mansoni SEA conjugated to polyacrylamide beads (PB-SEA) (17 +/- 1.2) and SWAP (PB-SWAP) (18.5 +/- 1.5). The increase in the number of giant cells was statistically significant when compared to the control polyacrylamide beads (PB) (9 +/- 1.1) and purified protein derivative conjugated to beads (PB-PPD) (11.6 +/- 1.7). We also observed a correlation between an increase in the number of giant cells and a decrease in in vitro granuloma index (GI) to PB-SEA (GI decreased from 4.3 +/- 0.2 on the 6th day to 3.2 +/- 0.2 on the 12th day) and PB-SWAP (GI decreased from 4.8 +/- 0.3 on the 6th day to 3.5 +/- 0.05 on the 12th day). These data suggest that giant cell formation may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.

Human giant cell formation induced in vitro by Schistosoma mansoni antigens

Although multinucleated giant cells have been described for many years in association with different chronic inflammatory responses, their participation in immunoregulatory mechanisms within the schistosome egg granulomas remains to be clarified. In this study we determined if soluble egg antigen (SEA) or adult worm antigen preparations (SWAP) from S. mansoni induce giant cell formation in vitro and their relationship with the intensity of granulomatous reactivity. Antigenic stimulation of peripheral blood mononuclear cells (PBMC) from patients (N = 9) with active schistosomiasis infection increased giant cell formation per field after the 12th day in culture when treated with S. mansoni SEA conjugated to polyacrylamide beads (PB-SEA) (17 +/- 1.2) and SWAP (PB-SWAP) (18.5 +/- 1.5). The increase in the number of giant cells was statistically significant when compared to the control polyacrylamide beads (PB) (9 +/- 1.1) and purified protein derivative conjugated to beads (PB-PPD) (11.6 +/- 1.7). We also observed a correlation between an increase in the number of giant cells and a decrease in in vitro granuloma index (GI) to PB-SEA (GI decreased from 4.3 +/- 0.2 on the 6th day to 3.2 +/- 0.2 on the 12th day) and PB-SWAP (GI decreased from 4.8 +/- 0.3 on the 6th day to 3.5 +/- 0.05 on the 12th day). These data suggest that giant cell formation may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs

The effect of zinc deficiency on granuloma formation, liver fibrosis, and antibody responses in experimental schistosomiasis.

The influence of various dietary zinc levels on the fibrotic aspects of granuloma formation and on the humoral response to schistosome egg antigens was investigated in C57Bl/6 mice by feeding groups of animals zinc-deficient diets. At six weeks of age, control and zinc-deficient mice were exposed individually to 50-60 cercariae of the Brazilian LE strain of Schistosoma mansoni. The animals were maintained on their respective diets for eight weeks postinfection, then all animals were killed and analyzed for body weight, spleen weight, collagen content of the liver, in vivo granulomatous histopathology, and antibody responses to soluble egg antigens. Zinc-deficient mice experienced stunted growth and reduced weight gain. Granulomatous hypersensitivity to schistosome eggs in the liver was measured in liver histopathologic sections using morphometric analysis and was found to be depressed in infected mice fed the moderately and the severely zinc-deficient diets. The low level of zinc in the diet also affected the humoral immune response of the host to schistosome egg antigens.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. II. In vitro granuloma modulation induced by polyclonal idiotypic antibodies.

We have previously reported on Id/anti-Id-receptor interactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive Id develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We report here on experiments that extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA Id and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA Id-reactive T cells were capable of regulating autologous in vitro granuloma formation. Both CD4 and CD8 T cells could be activated to regulate granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the presence of intact F(ab')2 Ig molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA-sensitized individuals. These data support the hypothesis that anti-SEA cross-reactive Id are important in regulating granulomatous hypersensitivity in chronic intestinal schistosomiasis patients and these cross-reactive Id appear to play a major role in cell-cell interactions that result in the regulation of anti-SEA cellular immune responses.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. III. In vitro granuloma modulation induced by immune complexes.

Granulomatous hypersensitivity to parasite eggs of Schistosoma mansoni is an important factor in the development of morbidity in chronic schistosomiasis. It has been demonstrated previously that the chronic, well-tolerated, intestinal form of schistosomiasis is associated with the establishment and maintenance of a variety of immunoregulatory mechanisms. We have used an in vitro model of granuloma formation for the purpose of studying the regulation of granulomatous hypersensitivity to S. mansoni egg antigens, mediated by immune complexes (IC). Our results show that the peripheral blood mononuclear cells (PBMCs) from patients with active schistosome infections, when treated with sera from chronic schistosomiasis patients, were able to induce an inhibitory activity on in vitro granuloma formation. Significant modulation of the in vitro granuloma reaction remained after treatment of PBMCs with isolated IC or manufactured IC with soluble egg antigen (SEA) and purified IgG from pooled chronic schistosomiasis sera. In contrast to granuloma modulation stimulated with whole molecule IgG-SEA IC, the incubation of PBMCs with F(ab')2 IgG-SEA IC did not induce any suppression of the granulomatous hypersensitivity to SEA. It appears in this model system that IC may inhibit the activity of granuloma formation by stimulating macrophages to release suppressive mediators. We have demonstrated this possibility by inhibition of prostaglandin activity using indomethacin. The addition of indomethacin to the granuloma culture significantly reduced in vitro granulomatous hypersensitivity to S. mansoni eggs in patients with chronic intestinal schistosomiasis and do so by inducing macrophages to secrete prostaglandins.

Production and characterization of human monoclonal antibodies against Schistosoma mansoni.

We have produced a panel of human monoclonal antibodies (MoAb) from patients infected with Schistosoma mansoni in order to analyse more carefully the human immune response to this helminth infection. This study describes the production, characterization and analysis of these MoAbs. Briefly, peripheral blood mononuclear cells from chronically infected patients were (1) isolated and stimulated with parasite antigens in vitro, (2) positively selected for B-cells on anti-Ig columns, and (3) then transformed with Epstein-Barr virus (EBV). Once EBV cell lines were established, they were selected for anti-S. mansoni antibodies using an ELISA, cloned, retested and then fused with the mouse-human heteromyeloma SHM-D33. In this study, we describe five MoAbs which have different antigenic specificities for life-cycle stages based on ELISA to soluble crude antigen preparations, membrane immunofluorescence on whole intact organisms, and immunofluorescent staining of cryostat frozen sections. The importance of these reagents with regard to the human immune response to S. mansoni is currently being evaluated.

Anti-idiotypic T cells in human schistosomiasis.

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of granulomatous hypersensitivity in patients infected with S. mansoni. Our previous studies have shown that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding antigen-conjugated polyacrylamide beads; and 2) this reactivity is a CD4 T cell dependent, macrophage dependent, B cell independent response which is antigenically specific for parasite egg antigens. We report here on idiotype - anti-idiotype receptor interactions involved in the regulation of granulomatous hypersensitivity in patients and former patients infected with S. mansoni. Five human monoclonal antibodies specific for parasite egg antigens were used to activate CD4 and CD8 T cell subsets. These activated anti-idiotypic T cells were then assayed for regulatory effector functions in an autologous in vitro granuloma model.

The ultrastructure of the Schistosoma mansoni egg.

Helminth eggs have resisted analysis by electron microscopy because fixatives, dehydrating agents, and embedding media penetrate these eggs poorly. Slam-freezing at liquid nitrogen temperature followed by freeze-substitution and Spurr's medium embedment provides preservation of the internal structure of the Schistosoma mansoni egg shell, developing miracidium, and perimiracidial structures. The egg shell consists of the three previously described layers (outer microspinous, middle intermediately dense, and inner dense layers) with cribriform pores. A newly described layer (Reynolds' layer) develops subjacent the egg shell and is comprised of densely-packed branching filaments. A single layer of squamous cells (von Lichtenberg's envelope) closely adheres to Reynolds' layer. Between von Lichtenberg's envelope and the embryo is a space (Lehman's lacuna); this space is initially filled with electron-lucent fluid, but subsequently masses of granulofloccular material (Cheever bodies) develop; Cheever bodies are partially membrane bound. Epidermal plates differentiate from superficial cells of the embryonal cell mass, while epidermal ridges differentiate from cells just below the surface of the embryonal cell mass. The cytoplasmic layer (von Lichtenberg's envelope) interposed between the host extracellular fluid and the developing miracidia effect a barrier against a simple passive diffusion; this infers that complex macromolecules, such as schistosomal egg antigen, undergo active, and perhaps selective, transport in or out of the egg.

The immune response to Schistosoma mansoni infections in inbred rats. VI. Regulation by T cell subpopulations.

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. CDF rats were depleted of RT 7.1+ (anti-Pan-T), W3/25+ (anti-T helper/inducer), or OX8+ (anti-T suppressor) cells by the in vivo administration of monoclonal antibodies (mAb). The development of parasites and immunity to challenge by S. mansoni were compared with results in undepleted normal and congenitally athymic rats. Discrete subpopulations of T lymphocytes were adoptively transferred to ascertain effects upon parasite development and the protective immune response. In vitro studies, involving utilizing cocultivation of cell subpopulations +/- cyclosporin A, were utilized to dissect mechanisms. Depletion of T lymphocytes by anti-RT7.1 mAb and anti-W3/25 mAb resulted in augmented initial worm development, suboptimal resistance, and decreased antibody and delayed-type hypersensitive reactivity directed against schistosome antigens. Depletion with OX8 mAb produced opposite effects. The adoptive transfer of T cell subpopulations produced concordant results with T cell regulation expressed B cell-dependent effector mechanisms. The coadoptive transfer of cells resulted in the suppression of resistance afforded by the W3/25+ cells by OX8+ cells, which could be augmented in vitro by cyclosporin A. Thus, protective immunity to S. mansoni in rats is regulated by discrete subpopulations of T lymphocytes. The findings suggest the possibility of selective immune regulation of resistance based on the manipulation of specific T cell subpopulation.