Schistosoma japonicum reinfection after praziquantel treatment causes anemia associated with inflammation

There is a relationship between schistosomiasis and anemia, although the magnitude and exact mechanisms involved are unclear. In a cohort of 580 Schistosoma japonicum-infected 7- to 30-year-old patients from Leyte, The Philippines, we evaluated the impact of reinfection with S. japonicum after treatment with praziquantel on the mean hemoglobin level, iron-deficiency (IDA) and non-iron-deficiency anemia (NIDA), and inflammatory markers. All participants were treated at baseline and followed up every 3 months for a total of 18 months. At each follow-up, participants provided stools to quantify reinfection and venous blood samples for hemograms and measures of iron status and inflammation. After 18 months, reinfection with S. japonicum was associated with a lower mean hemoglobin level (-0.39 g/dl; 95% confidence interval [95% CI], -0.63 to -0.16) and 1.70 (95% CI, 1.10 to 2.61) times higher odds of all-cause anemia than those without reinfection. Reinfection was associated with IDA for high reinfection intensities only. Conversely, reinfection was associated with NIDA for all infection intensities. Reinfection was associated with serum interleukin-6 responses (P<0.01), and these responses were associated with NIDA (P=0.019) but not with IDA (P=0.29). Our results provide strong evidence for the causal relationship between S. japonicum infection and anemia. Rapidly reinfected individuals did not have the positive treatment effect on hemoglobin seen in nonreinfected individuals. The principle mechanism involved in S. japonicum-associated anemia is that of proinflammatory cytokine-mediated anemia, with iron deficiency playing a role in high-intensity infections. Based on the proposed mechanism, anemia is unlikely to be ameliorated by iron therapy alone.     

Characterization of IgG responses of rabbits to Sj14-3-3 protein after experimental infection with Schistosoma japonicum

An indirect enzyme-linked immunosorbent assay method was developed for detection of IgG against 14-3-3 protein in sera of rabbits. Rabbits infected with 500 cercariae of Schistosoma japonicum were grouped and the characterization of the IgG responses was observed. For the treated group, the IgG could be detected as early as 2–4?weeks post-infection and then their levels rose rapidly and reached a peak at around 6?weeks. After the infected rabbits were treated with praziquantel at 6?weeks post-infection, IgG levels in the sera significantly decreased. While in the untreated group, the IgG levels were constantly very low. For all infected rabbits, 60?% (six of ten) had positive reaction with 14-3-3 protein, and 40?% (four of ten) had high IgG levels. This finding would be more helpful to understand this 14-3-3 protein.     

Human T- and B-cell responses to Schistosoma mansoni recombinant glyceraldehyde 3-phosphate dehydrogenase correlate with resistance to reinfection with S. mansoni or Schistosoma haematobium after chemotherapy

Recently we reported that human T- and B-cell recognition of a 42-kDa protein (p42) in soluble extracts of adult Schistosoma mansoni worms correlates with resistance to reinfection with S. mansoni or S. haematobium. Amino acid microsequencing of p42 revealed that it consists predominantly of schistosome glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). We have expressed SG3PDH in Escherichia coli and purified the recombinant protein in a soluble and enzymatically active form. Recombinant SG3PDH (rSG3PDH) reacted with human monospecific antibodies to p42. Lymphoproliferation and production of interleukin-4 and gamma interferon (IFN-gamma) after in vitro stimulation with rSG3PDH and serum isotype responses to rSG3PDH were examined in individuals with extremes of resistance and susceptibility to reinfection after treatment of previous S. mansoni or S. haematobium infection. Lymphoproliferation and IFN-gamma production in response to rSG3PDH and the presence of serum immunoglobulin G1 (IgG1), IgG3, and IgA antibodies to rSG3PDH generally characterized individuals who are resistant to reinfection after chemotherapy. The data indicate that T- and B-cell immune reactivity to rSG3PDH correlates with resistance to reinfection, confirming previous studies identifying SG3PDH as a target of protective immunity in humans, and suggest that SG3PDH should be investigated as a possible vaccine for human schistosomiasis.     

Identification of paramyosin T cell epitopes associated with human resistance to Schistosoma mansoni reinfection

Paramyosin, a Schistosoma mansoni myoprotein associated with human resistance to infection and reinfection, is a candidate antigen to compose a subunit vaccine against schistosomiasis. In this study, 11 paramyosin peptides selected by TEPITOPE algorithm as promiscuous epitopes were produced synthetically and tested in proliferation and in vitro human leucocyte antigen (HLA)-DR binding assays. A differential proliferative response was observed in individuals resistant to reinfection compared to individuals susceptible to reinfection in response to Para (210-226) peptide stimulation. In addition, this peptide was able to bind to all HLA-DR molecules tested in HLA-DR binding assays, confirming its promiscuity. Para (6-22) and Para (355-371) were also shown to be promiscuous peptides, because they were able to bind to the six and eight most prevalent HLA-DR alleles used in HLA-DR binding assays, respectively, and were also recognized by T cells of the individuals studied. These results suggest that these paramyosin peptides are promising antigens to compose an anti-schistosomiasis vaccine.     

IFN-inducible p47 GTPases display differential responses to Schistosoma japonicum acute infection

Interferon gamma induced GTPase (IGTP) (also named Irgm3) and interferon gamma inducible protein 47 (IRG-47) (also named Irgd) are interferon (IFN)-inducible p47 GTPases that have been shown to regulate host resistance to intracellular pathogens. Little knowledge has been known about the role of p47 GTPases in host responses against extracellular pathogens. To investigate possible roles of IGTP and IRG-47 in the course of Schistosoma japonicum infection, IGTP and IRG-47 knockout and wild-type (WT) mice were challenged with cercariae of S. japonicum, and host responses were analyzed. At the acute stage of S. japonicum infection, mice that lacked IGTP displayed similar parasite burden and pathological damage to WT mice. Importantly, S. japonicum-infected IRG-47-deficient mice, in contrast to IGTP-deficient mice and WT mice, showed significantly reduced worms and lower egg-burden, but intense granulomatous reaction evoked by schistosome eggs in peripheral parts of liver lobes. In addition, upregulation of inflammation-related gene expression was observed in the spleen of IRG-47-deficient mice using oligonucleotide microarrays, in which multiple pathways of cytokine–cytokine receptor interaction, T-cell receptor signaling, complement, coagulation cascades and cell adhesion molecules were highlighted. Taken together, these data suggest that IGTP and IRG-47 might have distinct features that were differentially required for resistance to S. japonicum.     

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-gamma were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.     

Approaches to genotyping individual miracidia of Schistosoma japonicum

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.     

Increased T-helper-1-type immunity and decreased T-helper-2-type immunity in patients with preeclampsia.

PROBLEM: To examine whether preeclampsia involves type-1 T-helper (TH1) immune hyperactivity. METHOD OF STUDY: Expression of HLA-DR, a cell-surface marker of activation, was analyzed on CD3+, CD4+, and CD8+ T cells in 15 preeclamptic patients and 15 normal pregnant women using flow cytometry. Additionally, peripheral blood mononuclear cells from preeclamptic patients and normal pregnant women were cultured with or without phytohemagglutinin (PHA) stimulation, and interleukin (IL)-2, IL-4, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha concentrations were determined in the supernatant by immunoassays. RESULTS: HLA-DR antigen was expressed more strongly on CD3+ T cells in preeclamptic patients than in normal subjects. In preeclampsia, HLA-DR was expressed more strongly in CD8+ T cells than in CD4- T cells. More TNF-alpha, IL-2, and IFN-gamma were produced by unstimulated and stimulated cultured peripheral blood mononuclear cells from preeclampsia patients than by those from normal subjects. TNF-alpha/IL-4, IL-2/IL-4, and IFN-gamma/IL-4 ratios were higher in preeclamptic patients than in the normal group. Significant positive correlations were observed between mean blood pressure and concentrations of the Th-1 type cytokines IL-2, IFN-gamma, and TNF-alpha. CONCLUSION: Up-regulation of Th1 responses and down-regulation of Th2 responses occur in preeclampsia.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

Immune modulation of Th1, Th2, and T-reg transcriptional factors differing from cytokine levels in Schistosoma japonicum infection

Parasitology Research - In spite of long-term integrated control programs for Schistosoma japonicum infection in China, the infection is still persistent due to its zoonotic transmission and...     

Immunity after treatment of human schistosomiasis: association between cellular responses and resistance to reinfection.

Previous studies have demonstrated the development of an age-dependent resistance to reinfection after chemotherapeutic cure of the helminthic parasite Schistosoma mansoni. Here we report on a longitudinal investigation of cell-mediated responses in infected individuals before and after treatment which was designed to outline those parameters important in mediating a protective response. A well-defined study group of 89 individuals with an age range of 9 to 35 years was selected from an area of high S. mansoni transmission in the Machakos district of Kenya. Peripheral blood mononuclear cell proliferation and cytokine production (interleukin-2 [IL-2], gamma interferon IL-5, IL-4, and tumor necrosis factor) in response to different crude life cycle-stage antigens of S. mansoni were assessed longitudinally in vitro before, 3 months after, and 1 year after treatment. Detailed statistical analyses of the results from this study have indicated a clear negative association between the proliferative responses to adult- and schistosomulum-stage antigens and subsequent reinfection intensity in older individuals (14 to 35 years) which was not present in the younger individuals (9 to 13 years). This association was significant even after the effects of age, sex, and exposure had been accounted for in multiple regression analyses. Cytokines were detected predominantly in response to adult worm and egg antigen extracts. An inverse association between the two cytokines gamma interferon and IL-5 was detected in response to all antigens at the three time points investigated, indicating cross-regulation in the production of these two mediators. Differences in antigen-specific cytokine levels between the two age groups were detected, with significantly higher IL-5 levels detected in the older (more resistant) age group. An inverse correlation between this cytokine and reinfection was detected but could not be dissociated from the effects of age and exposure in multiple regression analysis.     

Antibody and cytokine responses to hydatid in experimentally infected Kazakh sheep with hydatidosis resistance haplotype

Different MHC haplotype of Kazakh sheep has different resistance and susceptibility of hydatidosis. Notably, the MvaIbc-SacIIab-Hin1Iab haplotype of MHC-DRB1 exon two was associated with resistance hydatidosis. In order to analyze the antibody and cytokine responses to hydatidosis in Kazakh sheep with hydatidosis resistance haplotype, eight Kazakh sheep with the haplotype of MvaIbc-SacIIab-Hin1Iab were chosen as the test group, and other eight, which were not associated with hydatidosis resistance or susceptibility, were taken as control. After experimentally infected with hydatid orally, the blood was collected on 0, 7, 14, 30, 45, 60, 75, 90, 105, and 120 days. Serum and mRNA level of the cytokines IL-2, IFN-γ, TNF-α, IL-4, and IL-10 were evaluated by ELISA and fluorescence quantitative real-time polymerase chain reaction, respectively. The total white blood cells and leukomonocytes were determined by automation cytoanalyze. The level of IgE, IgG, and IgM were evaluated by ELISA. The results showed that the total white blood cells and leukomonocytes in test group were significantly higher than in control on 7, 45, 90, and 105 days post-infection (p.i.). The serum level of IL-2 in test group was significantly higher than in control on 45 days p.i., while the difference of IL-2 mRNA expression between test and control group was not significant. The serum level of TNF-α in test group was significantly higher than in control at 90 and 105 days p.i., and the TNF-α mRNA in test group was also significantly higher than in control on 90 days p.i. The level of IgE, IgG, and IgM in test group was higher than in control, but none was significant. The results suggested that the test group, which was predominant of Th1, could induce the protective immunity, while the control, which was predominant of Th2, could induce the susceptibility to infection of hydatidosis.     

核酸疫苗Sj14-3-3联合CpG和mIL-12免疫小鼠抗日本血吸虫攻击感染的研究

目的 在证明核酸疫苗pcDNA3．1（＋）-Sj14-3-3具有部分抗血吸虫感染的基础上，联合使用CpG和pcDNA3．1（＋）-mIL-12，观察此二种佐剂在攻击感染小鼠的免疫效果及其抗血吸虫感染的保护机制。方法 分别用pcDNA3．1（＋）-Sj14-3-3＋pcDNA3．1（＋）-mIL-12、pcDNA3．1（＋）-Sj14-3-3＋CpG、pcDNA3．1（＋）-mIL-12和CpG免疫小鼠。攻击感染后6W计数成虫负荷和肝虫卵数；检测免疫后0W、6W和12W小鼠血清总IgG、IgG1和IgG2a水平；检测小鼠脾细胞培养上清中IFN-γ和IL-4；流式细胞术检测免疫鼠脾细胞中CD4^＋和CD8^＋T细胞的比率。结果 pcDNA3．1（＋）-Sj14-3-3＋pcDNA3．1（＋）-mIL-12和pcDNA3．1（＋）-Sj14-3-3＋CpG免疫小鼠的减虫率分别为41．2％和28．7％；减卵率分别为52．6％和41．2％。单独使用pcDNA3．1（＋）-mIL-12和CpG也有一定的免疫保护作用。保护性免疫主要通过诱导宿主产生CTL、TH1型和体液免疫应答。结论 pcDNA3．1（＋）-mIL-12和CpG具有较强的增强核酸疫苗pcDNA3．1（＋）-Sj14-3-3抗血吸虫攻击感染作用。     

Thymocytotoxic autoantibodies found in mice infected with Schistosoma japonicum.

Thymocytotoxic autoantibodies were demonstrated in sera of C3H/HeJms, C57BL/6J, and ddY mice infected with 50 cercariae of Schistosoma japonicum, using C57BL/6J thymocytes as target cells in the trypan blue dye exclusion test. Kinetic study revealed that thymocytotoxic activity began to increase at week 6 of infection, reached a maximum at 8 weeks, and thereafter decreased gradually. Thymocytotoxic antibodies had an optimal reactivity at 4 degrees C and were sensitive to 2-mercaptoethanol treatment, suggesting that they were immunoglobulin M in nature. The cytotoxicity was completely abolished by absorption with C57BL/6J thymocytes but not with S. japonicum parasites or eggs. The antigen reacting with thymocytotoxic antibodies was found in the thymus, brain, spleen, and, to a lesser extent, kidney and liver. In parallel with the appearance of thymocytotoxic antibodies, the increase of background plaque-forming cells to trinitrophenyl, polyvinyl pyrrolidone, and sheep erythrocytes in the spleen of S. japonicum-infected mice suggested that te induction of thymocytotoxic antibodies may be the consequence of polyclonal B-lymphocyte stimulation by the infection.     

Gene expression profile of LPS-stimulated dendritic cells induced by a recombinant Sj16 (rSj16) derived from Schistosoma japonicum

Sj16, a 16-kDa protein secreted from Schistosoma japonicum, has been demonstrated an anti-inflammatory effect in vitro and in vivo, but its mechanism is still not clear. In this study, microarray analysis was performed to investigate the effects of recombinant Sj16 (rSj16) on the gene expression of the lipopolysaccharide (LPS)-stimulated dendritic cells (DCs). Immature DCs were treated with LPS, LPS?+?recombinant Sj16 (rSj16), or rSj16 alone for 24 h, and the gene expression profiles were examined using complementary DNA (cDNA) microarrays. With the cutoff value of 2-fold change in the expression, 509 genes were affected, 226 genes upregulated, and 283 genes downregulated after adding rSj16. Analysis by functional annotation clustering tool showed that rSj16-affected genes mainly associated with inflammatory response, defense response, regulation of immune system process, apoptosis, and cell migration. The results revealed that rSj16 reduced the LPS-induced pro-inflammatory genes such as cytokines (e.g., IL6, IL18, IFN-γ, IL12a, IL1b), chemokines, and receptors (e.g., CXCL1, CXCL9, CCL5, CCR5, CCR1, CCR2, CXCR3) and increased the anti-inflammatory gene IL-10. Further data mining of these genes by pathway analysis showed that genes regulated by rSj16 were significantly involved in cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, antigen processing and presentation, and Jak-STAT signaling pathway. In addition, quantitative real-time PCR (qRT-PCR) and Western blot analysis showed that rSj16 downregulated the expression of inhibitor of nuclear factor kappa-β kinase subunit beta (IKKβ) and nuclear factor-kappa β p65 (NF-κβ) messenger RNA (mRNA) and inhibited the phosphorylation of IKKβ and the NF-κB p65 protein, which implied that rSj16 exerting immunomodulatory effects by suppressing NF-κB signaling pathway. These results provide useful information in further understanding of the immunoregulation mechanisms of Sj16, and it is indicated that Sj16 could be as a potential molecule for the immunosuppressant drug development.     

Human immunoglobulin E responses to a recombinant 22.6-kilodalton antigen from Schistosoma mansoni adult worms are associated with low intensities of reinfection after treatment.

Schistosoma mansoni-infected individuals who have low intensities of reinfection following treatment produce immunoglobulin E (IgE) antibodies against a range of S. mansoni adult-worm antigens. One of the targets of the IgE response is an adult-worm sodium dodecyl sulfate-polyacrylamide gel electrophoresis band of 22 kDa (Sm22), which contains an antigen(s) located within the tegument and gut lining of adult worms and relatively late schistosomula life cycle stages only. A significant negative correlation between the level of anti-Sm22 IgE and the intensity of reinfection following treatment suggests that IgE responses against this antigen(s) are characteristic of individuals who are resistant to reinfection. To identify the antigen(s) in the Sm22 band that are associated with these IgE responses, we have cloned and characterized a recombinant 22-kDa protein (rSm22) that cross-reacts immunologically with Sm22. There was a high correlation between native and recombinant Sm22 isotype responses, indicating that the correct antigen had been cloned and that responses against rSm22 made up the majority of the responses against Sm22. By analyzing human isotype responses to rSm22 with human sera from a longitudinal treatment and reinfection study and correlating the anti-rSm22 isotype responses, retrospectively, with the intensity of reinfection following treatment for each individual, we observed a negative correlation between the IgE response to rSm22 and the intensity of reinfection. This relationship remained significant after allowing for age and other isotype responses to rSm22, in particular IgG4.     

Oesophagostomum spp. in pigs: resistance to reinfection

For a study on the occurrence of resistance to reinfection with porcine nodular worm species, pigs were infected twice weekly with 1,000 infective larvae (L₃) of Oesophagostomumquadrispinulatum for 8 weeks. All pigs, including noninfected controls, were then treated with fenbendazole. At 10 days after treatment, all pigs received a single challenge inoculation of 5,000 L₃ of either O. dentatum or O. quadrispinulatum, respectively. Pigs were slaughtered at 6 weeks after the challenge infection for determination of their worm burdens. The pigs trickle- and challenge-infected with O. quadrispinulatum had significantly lower egg excretion levels (P < 0.01) and worm burdens (P < 0.05) than challenge control pigs, thus indicating some degree of host immunity against the homologous challenge infection. No resistance to reinfection was evident for the heterologous challenge infection. This study elucidates further aspects of the interaction between nodular worm species in the pig. Received: 17 April 1998 / Accepted: 8 June 1998     

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.