Effect of four rounds of annual school-wide mass praziquantel treatment for schistosoma mansoni control on schistosome-specific immune responses.

This study evaluated potential changes in antischistosome immune responses in children from schools that received 4 rounds of annual mass drug administration (MDA) of praziquantel (PZQ). In a repeated cross-sectional study design, 210 schistosome egg-positive children were recruited at baseline from schools in western Kenya (baseline group). Another 251 children of the same age range were recruited from the same schools and diagnosed with schistosome infection by microscopy (post-MDA group). In-vitro schistosome-specific cytokines and plasma antibody levels were measured by ELISA and compared between the 2 groups of children. Schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) stimulated higher IL-5 production by egg-negative children in the post-MDA group compared to the baseline group. Similarly, anti-SEA IgE levels were higher in egg-negative children in the post-MDA group compared to the baseline group. Anti-SEA and anti-SWAP IgG4 levels were lower in egg-negative children in the post-MDA group compared to baseline. This resulted in higher anti-SEA IgE/IgG4 ratios for children in the post-MDA group compared to baseline. These post-MDA immunological changes are compatible with the current paradigm that treatment shifts immune responses to higher antischistosome IgE:IgG4 ratios in parallel with a potential increase in resistance to reinfection.

Immunology of human schistosomiasis.

There is a wealth of immunologic studies that have been carried out in experimental and human schistosomiasis that can be classified into three main areas: immunopathogenesis, resistance to reinfection and diagnostics. It is clear that the bulk of, if not all, morbidity due to human schistosomiasis results from immune-response-based inflammation against eggs lodged in the body, either as regulated chronic inflammation or resulting in fibrotic lesions. However, the exact nature of these responses, the antigens to which they are mounted and the mechanisms of the critical regulatory responses are still being sorted out. It is also becoming apparent that protective immunity against schistosomula as they develop into adult worms develops slowly and is hastened by the dying of adult worms, either naturally or when they are killed by praziquantel. However, as with anti-egg responses, the responsible immune mechanisms and inducing antigens are not clearly established, nor are any potential regulatory responses known. Finally, a wide variety of immune markers, both cellular and humoral, can be used to demonstrate exposure to schistosomes, and immunologic measurement of schistosome antigens can be used to detect, and thus diagnose, active infections. All three areas contribute to the public health response to human schistosome infections.

Adoptive suppression of granuloma formation.

Anti-egg granulomas formed in mice with chronic S. mansoni infection are smaller than those formed early (8 wk) after infection. Passive transfer of serum from mice with chronic infections to recipient mice with developing (6 wk) infections did not affect hepatic granuloma size at 8 wk of infection. In contrast, either spleen cells or lymph node cells from mice with chronic infections strongly suppressed the granulomatous process in recipient mice. Spleen cells, but not lymph node cells, of early-(7 wk) infected mice exhibited some ability to diminish granuloma formation in recipients. It appeared that the use of two sequential, weekly passive transfers of spleen or lymph node cells from chronic mice was even more effective in this suppressive capacity than a single transfer.

Cellular differentiation in the thymus. 3. Surface properties of rat thymus and lymph node cells separated on density gradients.

Young adult rat thymus and lymph node cell subpopulations were obtained by differential flotation on discontinuous BSA density gradients and assayed for properties characteristic of mature thymus-derived lymphocytes. One such subpopulation (C) of thymocytes was enriched in its ability to respond mitotically to a hemiallogeneic MLR stimulus, to localize in the parenchyma of lymph nodes and spleen, and to initiate a GVH reaction in a suitable host. These cells did not respond well to mitotic stimulation by PHA, they were lighter in density than the majority of mature lymph node thymus-derived lymphocytes, and they possessed a thymus-specific antigen (RTA) not present on peripheral lymphoid cells. We conclude that the acquisition of peripheral properties occurs sequentially, during an intrathymic differentiation cycle or shortly after the cells leave the thymus.

Neonatal idiotypic exposure alters subsequent cytokine, pathology, and survival patterns in experimental Schistosoma mansoni infections.

Exposure to maternal idiotypes (Ids) or antigens might predispose a child to develop an immunoregulated, asymptomatic clinical presentation of schistosomiasis. We have used an experimental murine system to address the role of Ids in this immunoregulation. Sera from mice with 8-wk Schistosoma mansoni infection, chronic (20-wk infection) moderate splenomegaly syndrome (MSS), or chronic hypersplenomegaly syndrome (HSS) were passed over an S. mansoni soluble egg antigen (SEA) immunoaffinity column to prepare Ids (8WkId, MSS Id, HSS Id). Newborn mice were injected with 8WkId, MSS Id, HSS Id, or normal mouse immunoglobulin (NoMoIgG) and infected with S. mansoni 8 wk later. Mice exposed to 8WkId or MSS Id as newborns had prolonged survival and decreased morbidity compared with mice that received HSS Id or NoMoIgG. When stimulated with SEA, 8WkId, or MSS Id, spleen cells from mice neonatally injected with 8WkId or MSS Id produced more interferon gamma than spleen cells from mice neonatally injected with HSS Id or NoMoIgG. Furthermore, neonatal exposure to 8WkId or MSS Id, but not NoMoIgG or HSS Id, led to significantly smaller granuloma size and lower hepatic fibrosis levels in infected mice. Together, these results indicate that perinatal exposure to appropriate anti-SEA Ids induces long-term effects on survival, pathology, and immune response patterns in mice subsequently infected with S. mansoni.

Complement-dependent platelet injury by staphylococcal protein A.

A new example of complement-mediated platelet injury has been described. Staphylococcal protein A (SPA) causes rabbit platelet injury as manifested by release of platelet 5-hydroxytryptamine (5HT). This reaction is complement-dependent and occurs over a very small range of SPA concentration, larger amounts being inhibitory. Complement fixation by SPA demonstrates the same narrow SPA concentration requirement whereas precipitation of IgG by SPA is roughly proportional to SPA concentration over a wide concentration range. The reaction can be separated into a sensitization step which requires SPA and plasma but not complement, and a release step which does require complement. Complement-mediated platelet damage induced by SPA is a new biologic property of this common component of the cell wall of pathogenic staphylococci which may contribute to the development of inflammatory and thromboembolic reactions complicating intravascular staphylococcal infection.

Relative imbalance between T regulatory cells and activated T cells in mice with differential morbidity in chronic Schistosoma mansoni infections.

Chronic (20-week) Schistosoma mansoni infections of CBA/J male mice present as two distinct forms of morbidity. Most mice develop moderate splenomegaly syndrome (MSS) resembling the intestinal form of chronic human schistosomiasis mansoni, while approximately 20% of mice develop hypersplenomegaly syndrome (HSS), more consistent with the severe hepatosplenic form of chronic human schistosomiasis mansoni. Here, we report the relative proportions of natural T regulatory cells (Treg) and activated CD4(+) T cells (Tact) for both splenic and granulomatous cell populations of MSS and HSS mice. Proportions of both Treg and Tact are greater in HSS than MSS mice. However, the ratios of Treg to Tact in both splenic and granulomatous cell populations from MSS mice are significantly higher than those of HSS mice. For both HSS and MSS mice, in vitro proliferation of their CD3(+) splenic cells induced by soluble egg antigens is inversely correlated with the ratio of Treg to Tact. Also, spleen or granuloma cells from MSS mice produced higher mean levels of IFN-gamma than those from HSS mice. Differential IFN-gamma productive capacities dictated by Treg : Tact ratios may contribute to the development of differential morbidities in this model of chronic experimental schistosomiasis mansoni.

Immune responses during human Schistosomiasis mansoni. Evidence for antiidiotypic T lymphocyte responsiveness.

We present a method for the examination of antiidiotypic cell-mediated reactivity during chronic human infections. Pooled and individual sera from patients with schistosomiasis mansoni were purified on immunoaffinity columns of schistosomal egg antigens (SEA). The eluates contained anti-SEA antibodies, but not SEA. These antibody preparations, and their F(ab)2 fragments, stimulated dose-dependent proliferation of peripheral blood mononuclear cells (PBMN) and T lymphocytes from some, but not all active or former schistosomiasis mansoni patients, and could do so autologously. Stimulation required presentation by plastic-adherent cells. The eluates did not stimulate PBMN from persons who had never had schistosomiasis. Affinity-purified anti-SEA antibodies from former patients (cured for greater than 10 yr) did not stimulate PBMN from patients with active infections. Reabsorption on SEA columns removed stimulatory activity from the eluates. We propose that multiclonal, SEA-related idiotypes expressed by some anti-SEA antibodies stimulate proliferation of T lymphocytes that express antiidiotypic specificities.

Idiotypic sensitization in utero of children born to mothers with schistosomiasis or Chagas' disease.

Cord blood mononuclear cells (CBMC) of neonates born of mothers with Chagas' disease or schistosomiasis exhibited strong proliferative responses against idiotypes expressed on antibodies with specificity for Trypanosoma cruzi or Schistosoma mansoni antigens, respectively. These immunoaffinity-purified preparations were stimulatory if they were prepared from pools of patients' sera or from the mother's own serum, taken early during her pregnancy. These CBMC did not respond to normal immunoglobulin, and CBMC of neonates born of uninfected mothers did not respond to antibodies against either T. cruzi or S. mansoni, or normal immunoglobulin preparations. We propose that in utero exposure of a fetus to some idiotypes expressed on placentally transferred antibodies induces anti-Id T lymphocyte sensitization, which we detect as a proliferative response by CBMC exposed to immunoaffinity-purified antibodies expressing the relevant idiotypes. This is the first experimental evidence that children born of mothers with chronic infections undergo natural in utero idiotypic manipulations and are born possessing cellular anti-Id reactivity.

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils.

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.

The effect of treatment of schistosomiasis on blood plasma HIV-1 RNA concentration in coinfected individuals.

OBJECTIVE: To determine whether drug treatment of Schistosomiasis mansoni infection leads to a reduction in plasma HIV-1 RNA concentration in coinfected individuals. METHODS: Stool and plasma samples were obtained prospectively from a cohort of HIV-infected persons (n = 30) in Kisumu, Kenya, before and after treatment of schistosomiasis with praziquantel (mean follow-up, 5.6 months; range 1-15 months). Schistosomal circulating cathodic antigen (CCA) concentrations in plasma were determined by ELISA and fecal egg counts were determined by microscopy. HIV-1 RNA concentrations were measured in pre- and post-treatment plasma samples obtained from the patients whose stool samples remained free of schistosomal eggs for the great majority of the follow-up period. RESULTS: Comparison of pretreatment and follow-up samples revealed that mean +/- SD fecal egg burden was reduced by 96.7% (481.5+/-803.5 versus 16.1+/-24.4 eggs/g feces) and mean plasma CCA concentration decreased by 90.1% (3.22+/-3.26 versus 0.32+/-0.38 microg/ml). In contrast, mean plasma HIV-1 load increased from 3.60+/-0.90 to 3.93+/-0.95 log10 RNA copies/ml (P< 0.001). Although no correlation was found between changes in HIV-1 load and changes in schistosomal burden, there was a significant correlation between changes in plasma HIV load and the time interval between pretreatment and follow-up samples (r = 0.41; P = 0.027). CONCLUSIONS: Treatment of schistosomiasis was not associated with a reduction in plasma HIV-1 load. This study does not, however, exclude the possibility of an adverse effect of helminthic infections on HIV-1 pathogenesis.

Primary in vitro sensitization to heterogeneous soluble antigens of Schistosoma mansoni adult worms.

Primary in vitro sensitization of normal mouse spleen cells was accomplished using a saline soluble heterogeneous antigenic preparation from the whole adult worms (SWAP) of Schistosoma mansoni. A SWAP-specific rosette-forming cell (RFC) assay, using antigen on sheep erythrocytes, was used to detect sensitization due to S. mansoni infection and primary in vitro SWAP exposure. The latter exposure was facilitated by SWAP adsorption to the culture vessel via poly-L-lysine. RFC expression was maximal on day 3 of culture. It was antigen-specific in regard to unexposed controls, SWAP and human gamma globulin, and as controlled by RFC detected by parallel treated (control) erythrocytes. The RFC assay was best read between 90 and 180 min after initiation of rosette formation. Assay erythrocytes gave reproducible results for at least 3 weeks after preparation.

Immunopathogenesis and immunoregulation in schistosomiasis. Distinct chronic pathologic syndromes in CBA/J mice.

Inbred CBA/J mice with chronic (20-week) Schistosoma mansoni infections demonstrate two distinct syndromes. Hypersplenomegaly syndrome (HSS), characterized by a massive spleen, liver fibrosis, ascites, and anemia, resembles hepatosplenic human schistosomiasis, complete with portal hypertension and shunting. Moderate splenomegaly (MSS) syndrome, with less severe pathology, parallels most chronic human infections. Phenotypic analyses of spleen cells for CD44, CD62L, CD45RB, Ia, and CD25 indicate that HSS mice have more activated and memory CD4+ T cells than do MSS mice. HSS animals also have more B cells that highly express B7-2. Anti-CD3 stimulated spleen cells from 8-week or chronically infected mice produce IL-4 and IL-10 in a manner that appears not to involve the CD28/B7-2 costimulation pathway. By contrast IFN-gamma production is augmented in the presence of anti-CD28 and decreased in the presence of anti-B7-2. Infected mice make very little IL-2 to anti-CD3, even with added anti-CD28. As cytokines affect resultant B-cell responses and HSS and MSS mice display distinctive isotypes, differential regulatory or anergy hypotheses may best explain MSS/HSS differences.

Differences in adult Schistosoma mansoni worm burden requirements for the establishment of resistance to reinfection in inbred mice. I. CBA/J and C57BL/6 mice.

Schistosoma mansoni infections of CBA/J and C57BL/6 mice provided the hosts with partial protection against reinfection by S. mansoni cercariae. The level of protection, assayed by perfusion 21-25 days after challenge, varied over the course of infection. After patency the degree of resistance observed in CBA/J mice was related to the adult worm burden harbored by the mice. The same was initially true in C57BL/6 mice. Those C57BL/6 mice which lived until a chronic state of infection were rarely well protected and harbored few female worms. Strain differences were also apparent in that the number of worms required by C57BL/6 mice to establish the same level of protection as CBA/J mice was double that needed by CBA/J mice. Measurements were made of the newly formed egg-induced hepatic granulomas throughout the infection period (up to 30 weeks after infection). The data revealed that although the pattern by which C57BL/6 mice developed maximum granuloma formation and subsequent modulation was identical to that observed in CBA/J mice, C57BL/6 granulomas were consistently half the volume of their counterpart lesions in CBA/J mice.

Effects of immunomanipulations on resistance induced by irradiated Schistosoma mansoni cercarial sensitization of C57BL/6 and CBA/J mice.

Mice sensitized with radiation-attenuated Schistosoma mansoni cercariae were treated by a variety of procedures known to interrupt immunoregulatory circuitries in attempts to alter the resistance induced by irradiated cercarial sensitization. Both highly resistant C57BL/6 and the often poorly resistant CBA/J strains were examined. Immunomanipulations included adult thymectomy, or administration of different drugs in relation to the time of challenge infection (day 0). Adult thymectomy was performed 3 weeks prior to sensitization or 3 weeks prior to challenge. Drug treatment included cyclophosphamide given at a dose of 20 mg/kg on alternate days, day -11 through day +11, cimetidine at 50 mg/kg/day, day -7 through day +21, or indomethacin at 2.5 mg/kg/day, day 0 through day +10. In most experiments, irradiated cercarial sensitized C57BL/6 mice developed significant resistance which was not altered by the immunomanipulative regimens used. If however, as fortuitously occurred in two adult thymectomy experiments, irradiated cercarial sensitization did not induce significant resistance, immunomanipulation by adult thymectomy allowed the development of a significant protected state. Similarly, adult thymectomy or cimetidine treatment in concert with immunization of CBA/J mice conferred moderate, statistically significant levels of stable resistance in this normally "less resistant" strain.

Effects of anti-schistosomal chemotherapy on immune responses, protection and immunity. II. Concomitant immunity and immunization with irradiated cercariae.

Resistance of mice to challenge infections of Schistosoma mansoni was evaluated before and after elimination of their primary, established S. mansoni infections with the chemotherapeutic drug praziquantel. Mice treated after either 10 or 20 weeks of primary infection were challenged 6 or 10 weeks after treatment. Mice infected for for 10 weeks prior to treatment expressed progressively less resistance 6 and 10 weeks after treatment. By 10 weeks after treatment significant levels of protection were no longer observed. Resistance waned more slowly if mice were treated 20 weeks after infection, and there was still significant expression of resistance to challenge 10 weeks after treatment. A separate set of experiments evaluated the use of highly irradiated cercariae as a vaccine in mice that had been previously infected with S. mansoni and cured with praziquantel. It was observed that effective immunizations were possible in previously infected mice. These studies demonstrate that established resistance waned after treatment and the rate of loss of protection was dependent upon the duration of infection prior to treatment. Furthermore, the irradiated cercarial vaccine studies indicate that in the murine model induction of immunological resistance was feasible following chemotherapeutic treatment of infected populations.

Effects of anti-schistosomal chemotherapy on immune responses, protection and immunity. III. An effective regimen of praziquantel does not alter immune capabilities of normal mice.

Praziquantel is a broad spectrum anti-helminthic drug active against a variety of parasitic trematodes and cestodes. This report studies the effects of a schistosomacidal regimen of praziquantel on the generation of the cell-mediated and humoral response capabilities of normal, uninfected mice. Three doses of 100 mg praziquantel/kg body weight at 4-hr intervals cured several strains of mice with Schistosoma mansoni infections of several intensities. When this curative regimen was applied to normal, uninfected mice it did not alter peripheral blood leukocyte counts, circulating eosinophil numbers or splenic weights. Neither did this regimen alter the development of responsiveness to sheep erythrocytes (SRBC) as measured by dermal delayed-type hypersensitivity (DTH) or the number of splenic plaque forming cells. Furthermore, DTH and serum antibody responses to a soluble antigen (bovine serum albumin) were also comparable in treated and untreated mice. It appears that an anti-schistosomacidal regimen of praziquantel does not alter normal murine cellular or humoral immune responses.

Differences in adult Schistosoma mansoni worm burden requirements for the establishment of resistance to reinfection in inbred mice. II. C57BL/KsJ, SWR/J, SJL/J, BALB/cAnN, DBA/2N, A/J, B10.A(3R), and B10.A(5R) mice.

Eight strains of mice (C57BL/KsJ, SWR/J, SJL/J, BALB/cAnN, DBA/2N, A/J, B10.A(3R), and B10.A(5R) were studied in regard to the development of resistance to secondary challenge with Schistosoma mansoni following a primary infection. Also analyzed were the number of eggs recoverable from the livers of these strains after primary infection and the size of the hepatic, schistosome egg-focused granulomas newly formed at various times after infection. As previously observed by us and others, most often the degree of resistance conferred by an acute primary infection is related to the number of adult worms harbored due to the initial infection. The mean levels of protection observed in the different strains were seen to cover a spectrum from moderate to strong resistance, rather than demonstrating high or low resistance strains. There was no observable effect of the major histocompatibility complex on this resistance state. The number of eggs recoverable from the livers of the various strains was calculated on a total liver, per female worm basis, and was seen to be less in C57BL/KsJ, SWR/J and SJL/J mice. Granulomatous reactivity also varied considerably in a strain-dependent manner. These data are compared with previously reported strain differences in S. mansoni infections. Although some similarities are observed between laboratories certain strain-related discrepancies also exist and are discussed.

The eosinophil in the inflammatory response to cercarial challenge of sensitized and chronically infected CBA/J mice.

Sensitized or chronically infected CBA/J mice were challenged percutaneously with cercariae of Schistosoma mansoni. Early inflammatory response was characterized by edema and neutrophil infiltration directed toward penetration tracks; chief pathology associated with schistosomula was localized disruption of epithelial cell relationships, except in areas of desmosomes. By 24 hours eosinophils were the prominent cells in the inflammatory reaction, with large numbers replacing neutrophils in penetration tracks. Extensive local eosinophilia occurred in areas of widespread epidermal destruction and collagen damage. Electron microscopy revealed degranulation of eosinophils in microabscesses and in areas of collagen damage and metabolically active fibroblasts. Eosinophils were rarely observed in contact with schistosomula. No marked difference could be observed between sensitized (but unprotected) mice and chronically infected mice in their response to cercarial challenge; a similar eosinophil response took place whether the mice were partially protected or totally unprotected. The data indicate that although eosinophils may be involved in this protection their presence alone is not sufficient to afford acquired resistance to murine schistosomiasis mansoni.

Reduced reproductive efficiency in mice with schistosomiasis mansoni and in uninfected pregnant mice injected with antibodies against Schistosoma mansoni soluble egg antigens.

Female CBA/J mice were infected with approximately 15 cercariae of Schistosoma mansoni and mated with normal syngeneic males 7 weeks later. Uninfected mice were bred in parallel, and both groups were subsequently bred several more times. Pregnancies were documented by formation of vaginal plugs, and daily records were kept concerning the status of the pregnancies, delivery, and offspring. One hundred thirty-two pregnancies in uninfected mice resulted in 101 (77%) viable litters. In contrast, 133 pregnancies in mice infected with S. mansoni lead to 45 (34%) viable litters. The decreased number of viable litters born or raised by infected mothers resulted from maternal death during pregnancy (5%), spontaneous abortion (20%), and infanticide (42%). Observations of multiparous infected mice indicated that this reduced rate of production of viable, surviving offspring was not different in subsequent pregnancies nor influenced by the duration of the infection. At 2 weeks of age, offspring of infected mice weighed significantly less than offspring of equal-sized litters from uninfected mice, but this weight differential was not sustained beyond 2 weeks of age. Subsequent studies compared the effects on uninfected pregnant mice of injections of antibodies against S. mansoni soluble egg antigens (immunoaffinity-purified from sera of mice with 16 week S. mansoni infections) vs. normal mouse immunoglobulin. After repeated injections of these antibodies during multiple pregnancies, lowered reproductive efficiencies (37%) were observed as compared to parallel studies of pregnant mice injected with normal mouse immunoglobulin (67%).