The Synaptic Leap

Parasitology Research (17 January 2012), pp. 1-6.

Echinostomiasis is a food-borne intestinal, snail-mediated parasitosis caused principally by ingestion of snails infected with digenean trematodes of the Echinostoma genus. The treatment and control of trematodiasis is usually done by administration of praziquantel (PZQ). In this study, we evaluated the effect on Echinostoma paraensei of different doses of praziquantel through analysis of morphological parameters using light microscopy, scanning electron microscopy, and confocal scanning laser microscopy along with parasitological data. We used 30 female mice aged 4 weeks. Each animal was given 40 metacercarie of E. paraensei by gavage. The animals were divided into five groups, each group containing six animals, where one group was utilized as untreated control. Two weeks after infection, the mice were given praziquantel by gavage at total dosages of 12.5, 25, 50 or 100 mg/kg by body weight. Two days after treatment, the mice were euthanized in a CO 2 chamber for recovery of helminths in the small intestine. The doses of 50 and 100 mg/kg of praziquantel eliminated all the worms. There were significant differences ( p < 0.05) between all the treated groups when compared to the control group. The body morphology showed contraction with vacuolization of the parenchyma, and the spine of the peristomic collar was not evident by light microscopy. The scanning electron microscopy revealed that the other doses caused retraction of spines of the peristomic collar and also the tegument spines at the body edge, as well as the development of vesicles and peeling; all these alterations were more evident at the dose of 25 mg/kg. In turn, the confocal scanning laser microscopy revealed vacuolization and disorganization of spines and vitelline glands. E. paraensei responds differently to experimental treatment with praziquantel according to the doses utilized causing morphological alteration and even worm elimination.
Juliana Ferraz, Joyce Souza, Michele Costa-Silva, Eduardo Torres, André Santana, Reinalda Lanfredi, Arnaldo Maldonado, Juberlan Garcia

Parasitology Research (17 January 2012), pp. 1-6.

Echinostomiasis is a food-borne intestinal, snail-mediated parasitosis caused principally by ingestion of snails infected with digenean trematodes of the Echinostoma genus. The treatment and control of trematodiasis is usually done by administration of praziquantel (PZQ). In this study, we evaluated the effect on Echinostoma paraensei of different doses of praziquantel through analysis of morphological parameters using light microscopy, scanning electron microscopy, and confocal scanning laser microscopy along with parasitological data. We used 30 female mice aged 4 weeks. Each animal was given 40 metacercarie of E. paraensei by gavage. The animals were divided into five groups, each group containing six animals, where one group was utilized as untreated control. Two weeks after infection, the mice were given praziquantel by gavage at total dosages of 12.5, 25, 50 or 100 mg/kg by body weight. Two days after treatment, the mice were euthanized in a CO 2 chamber for recovery of helminths in the small intestine. The doses of 50 and 100 mg/kg of praziquantel eliminated all the worms. There were significant differences ( p < 0.05) between all the treated groups when compared to the control group. The body morphology showed contraction with vacuolization of the parenchyma, and the spine of the peristomic collar was not evident by light microscopy. The scanning electron microscopy revealed that the other doses caused retraction of spines of the peristomic collar and also the tegument spines at the body edge, as well as the development of vesicles and peeling; all these alterations were more evident at the dose of 25 mg/kg. In turn, the confocal scanning laser microscopy revealed vacuolization and disorganization of spines and vitelline glands. E. paraensei responds differently to experimental treatment with praziquantel according to the doses utilized causing morphological alteration and even worm elimination.
Juliana Ferraz, Joyce Souza, Michele Costa-Silva, Eduardo Torres, André Santana, Reinalda Lanfredi, Arnaldo Maldonado, Juberlan Garcia

Acta Tropica (January 2012)

Nigeria is highly endemic for infection with Schistosoma haematobium, which most commonly manifests itself with blood in urine. To monitor the impact of annual mass drug administration (MDA) with Praziquantel for S. haematobium in Delta State, Nigeria, cross-sectional hematuria surveys of school children were conducted in 8 sentinel villages (SVs) at baseline (n = 240) and after two annual doses (n = 402). We assessed the comparability of three assessments of hematuria (child's reported history, nurse visual diagnosis (NVD) and dipstick) to determine the need for mass treatment. Dipstick was considered to be the gold standard. Prior to treatment, history and NVD each identified only the 3 most highly prevalent SVs, and overall this represented just 37.5% of the 8 SVs in need of treatment. Following treatment, after dipstick prevalence decreased by 88.5% (p < 0.001), and history and NVD identified only one of two villages still needing treatment. The study suggests that dipsticks should be the recommended method for launching and monitoring mass treatment for S. haematobium. Praziquantel treatment for Schistosoma haematobium decreased dipstick prevalence of hematuria by 88.5%. Urine dipsticks were superior to history and NVD for identifying communities needing treatment. ⺠We monitored impact of annual Praziquantel for Schistosoma haematobium in Delta State, Nigeria. ⺠Cross-sectional surveys of school children conducted at baseline and after 2 years. ⺠Compared three assessments of hematuria to determine the need for mass treatment. ⺠Following treatment, dipstick prevalence of hematuria decreased by 88.5% (p < 0.001). ⺠History and NVD failed to identify all communities in need of treatment.
Emmanuel Emukah, Julie Gutman, John Eguagie, Emmanuel Miri, Paul Yinkore, Ndudi Okocha, Victoria Jibunor, Nebe Obiageli, Nwoye Ikenna, Frank Richards

Trends in Parasitology (January 2012)

In a review of the studies on genital schistosomiasis, the cervix, the Fallopian tubes, and the vagina are the most common gynaecological sites to harbour Schistosoma haematobium. Lesions are caused by host responses to dead or viable schistosomiasis eggs and may render women with genital schistosomiasis susceptible to HIV. The typical genital changes, such as sandy patches and pathological blood vessels may make women susceptible to super-infection, cause contact bleeding, decreased fertility, abortions, discharge and bleeding. Further research is needed to find simple, low-tech diagnostic methods, treatment for chronic lesions, and to explore the preventive effects of mass drug administration on symptoms, sandy patches, HPV and the HIV epidemic.
Eyrun Kjetland, Peter Leutscher, Patricia Ndhlovu

Bioorganic & Medicinal Chemistry Letters (January 2012)

A praziquantel analog 10-hydroxy praziquantel and eight praziquantel/peroxide conjugates were synthesized. The biological activity of these compounds was evaluated against juvenile and adult stages of Schistosoma japonicum. Unlike praziquantel, 10-hydroxy praziquantel exhibits activity against both juvenile and adult Schistosoma japonicumin. All hybrid compounds displayed modest to significant worm killing activity. The present study has important significance for the development of hybrid antischistosomal drugs.
Wen-wen Duan, Si-jie Qiu, Yue Zhao, Huan Sun, Chunhua Qiao, Chao-ming Xia

PLoS Negl Trop Dis, Vol. 5, No. 1. (25 January 2011), e951.

Although accurate assessment of the prevalence of Schistosoma mansoni is important for the design and evaluation of control programs, the most widely used tools for diagnosis are limited by suboptimal sensitivity, slow turn-around-time, or inability to distinguish current from former infections. Recently, two tests that detect circulating cathodic antigen (CCA) in urine of patients with schistosomiasis became commercially available. As part of a larger study on schistosomiasis prevalence in young children, we evaluated the performance and diagnostic accuracy of these tests—the carbon test strip designed for use in the laboratory and the cassette format test intended for field use. In comparison to 6 Kato-Katz exams, the carbon and cassette CCA tests had sensitivities of 88.4% and 94.2% and specificities of 70.9% and 59.4%, respectively. However, because of the known limitations of the Kato-Katz assay, we also utilized latent class analysis (LCA) incorporating the CCA, Kato-Katz, and schistosome-specific antibody results to determine their sensitivities and specificities. The laboratory-based CCA test had a sensitivity of 91.7% and a specificity of 89.4% by LCA while the cassette test had a sensitivity of 96.3% and a specificity of 74.7%. The intensity of the reaction in both urine CCA tests reflected stool egg burden and their performance was not affected by the presence of soil transmitted helminth infections. Our results suggest that urine-based assays for CCA may be valuable in screening for S. mansoni infections. Control efforts for schistosomiasis have in part been hampered by the lack of a sensitive and accurate test that can be utilized to rapidly map the prevalence of the disease in different areas. Recently, new tests have become commercially available that may address this problem. This study was designed to compare the new tests, which detect a schistosome antigen in patients' urine, with more traditional tests that look for parasite eggs in stool or anti-parasite antibodies in serum. We found that the new tests performed very well to detect schistosomiasis in children in western Kenya, an area with a high prevalence of Schistosoma mansoni infections. There was no apparent effect of soil transmitted helminth infections on the performance of the tests and the intensity of the antigen detection assays correlated well with the levels of S. mansoni eggs in the stool and schistosome-specific antibody in serum. Additional evaluation is needed in areas with lower schistosomiasis prevalence and intensity levels but we believe that point of contact testing of urine for schistosome antigen could be an effective tool in schistosomiasis mapping and control efforts.
Hillary Shane, Jennifer Verani, Bernard Abudho, Susan Montgomery, Anna Blackstock, Pauline Mwinzi, Sara Butler, Diana Karanja, Evan Secor

PLoS Negl Trop Dis, Vol. 5, No. 8. (9 August 2011), e1269.

Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+regulatory T cells (Tregs) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2 –associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+Tregs: CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+Tregs in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+Tregs down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+Tregs appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis. Schistosomiasis is an important parasitic helminth disease afflicting more than 200 million people worldwide. Infections are typically chronic and in the case of Schistosoma mansoni and S. japonicum the majority give rise to an intestinal form of disease caused by the deposition of parasite eggs in the colon and terminal ileum. The eggs cause Th2-associated inflammatory immune granulomas to form, which as the disease develops, are down-regulated by cells of the immune system. However, the mechanisms which underpin the down-regulation of granulomas in the large intestine are not known. In order to investigate the phenomenon of Th2-associated colonic inflammation, we utilized a murine model of infection with S. mansoni and compared immune responses at the acute and chronic phases of infection. We show that a type of regulatory T helper lymphocyte (CD4+CD25+FoxP3+Treg) contributes to regulation of colonic inflammation. These cells modulate anti-egg Th2 responses within the mesenteric lymph nodes and granulomatous pro-fibrotic Th2 responses within the colon. Our study highlights the importance of CD4+CD25+FoxP3+Tregs as a source of regulatory pressure on granuloma formation in the colon and by implication humans with chronic intestinal schistosomiasis.
Joseph Turner, Gavin Jenkins, Karen Hogg, Sarah Aynsley, Ross Paveley, Peter Cook, Mark Coles, Adrian Mountford

PLoS Negl Trop Dis, Vol. 5, No. 11. (15 November 2011), e1399.

The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4+ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4+ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection. Levels of Th17 and the other three CD4+ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice. Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology. Th17 immune cells secrete the IL-17 cytokine and contribute to host defenses against certain infections. Recent studies linked IL-17 with the severity of liver inflammation and suggested that Th17 cells contribute to the pathology in schistosomiasis, a serious disease caused by parasitic worms such as Schistosoma japonicum widespread in vertebrates including humans. However, the role of Th17 cells in protection against S. japonicum infection is still unclear. For the first time, we describe here the changes in Th17 cell levels during S. japonicum infection and suggest that the schistosome egg antigens are primarily responsible for stimulating the generation of host Th17 cells after S. japonicum infection. We further show that the level of Th17 cells in the host is determined by a combination of factors, namely exposure to complex parasitic antigens that either induce or suppress their generation. We also suggest that lowering IL-17 levels may favor the host's protective responses against S. japonicum infection. Our findings help to better understand the relationship between the host and parasite in terms of immune protection and pathology in schistosomiasis and may contribute to the future development of vaccination and therapeutic strategies.
Xiaoyun Wen, Lei He, Ying Chi, Sha Zhou, Jason Hoellwarth, Cui Zhang, Jifeng Zhu, Calvin Wu, Shawn Dhesi, Xuefeng Wang, Feng Liu, Chuan Su

PLoS Negl Trop Dis, Vol. 4, No. 5. (18 May 2010), e689.

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4raâ/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4raâ/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4raâ/lox liver granulomas, when compared to Il4raâ/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4raâ/lox and Il4raâ/â mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4raâ/lox and iLckcreIl4raâ/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4raâ/lox but not in iLckcreIl4raâ/lox granulomas. While aaMÏ were in close proximity to the parasite eggs in Il4raâ/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4raâ/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4raâ/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation. Schistosomiasis is a tropical disease caused by one of the species of the parasitic worm Schistosoma which infects over 200 million people worldwide. Signalling via the IL-4 receptor alpha (IL-4Rα), which is the common receptor chain for the ligands IL-4 and IL-13, is essential for inducing protective Type 2 immune response and granuloma formation in response to the parasite eggs. In experimental Schistosoma mansoni infection and egg-induced inflammation studies with cell type-specific IL-4Rα deficient mice, the role of IL-4Rα-activated alternative macrophages (aaMÏ) and IL-4Rα-responsive T cells was investigated with focus on the control of hepatic inflammation and granuloma formation. Interestingly, aaMÏ were not essential for the cellular composition or the Th1/Th2 cytokine profile in liver granulomas. In contrast, IL-4Rα-dependent T cell responses were important for predominant Th2 and IL-10 responses, as well as the presence of aaMÏ in the granulomas, avoiding major disruption in the granuloma cell composition. Moreover, a macrophage subpopulation was identified and those cells expressed the two aaMÏ markers, mannose receptor- and Ym1 in an IL-4Rα-independent but IL-10-dependent manner. These cells might be involved in the control of inflammation.
Benjamin Dewals, Reece Marillier, Jennifer Hoving, Mosiuoa Leeto, Anita Schwegmann, Frank Brombacher

PLoS Negl Trop Dis, Vol. 5, No. 6. (7 June 2011), e1178.

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis. Schistosomiasis is a significant cause of morbidity and mortality in the tropical world although its true burden has been historically underestimated. Millions of people currently endure severe pathology as a result of schistosome infections, although some individuals appear to be less susceptible to infection despite constant parasite exposure. A similar range of disease susceptibility is evident in different strains of inbred mice infected with schistosomes, thereby mirroring the clinical situation. Granuloma formation in the liver of both humans and mice is a characteristic manifestation of chronic schistosomiasis, and is largely controlled by gene signalling pathways. Certain genes expressed in particular cohorts of mice and humans may be associated with the development of severe pathology, or may confer a protective phenotype. This murine study highlights some key molecular aspects of chronic schistosomiasis which may be responsible for the development of both mild and severe pathology, and provides a bench mark for studying the mechanisms of schistosome-induced disease in humans.
Carly Perry, Melissa Burke, Deborah Stenzel, Donald McManus, Grant Ramm, Geoffrey Gobert

PLoS Negl Trop Dis, Vol. 4, No. 2. (9 February 2010), e598.

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of granulomas and fibrosis will be essential for understanding how schistosome antigens interact with the host environment. We report here the first whole genome microarray analysis of the murine liver during the progression of Schistosoma japonicum egg-induced granuloma formation and hepatic fibrosis. Our results reveal a distinct temporal relationship between the expression of chemokine subsets and the recruitment of cells to the infected liver. Genes up-regulated earlier in the response included T- and B-cell chemoattractants, reflecting the early recruitment of these cells illustrated by flow cytometry. The later phases of the response corresponded with peak recruitment of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11 (eotaxin 1), members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the Hepatic Stellate Cell/Fibrocyte chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively activated macrophages (e.g. Retnla) during this later phase provides further evidence of a role for these cells in schistosome-induced pathology. Additionally, we demonstrate that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggest the involvement of neutrophils in S. japonicum-induced hepatic fibrosis. These results further our understanding of the immunopathogenic and, especially, chemokine signalling pathways that regulate the development of S. japonicum-induced granulomas and fibrosis and may provide correlative insight into the pathogenesis of other chronic inflammatory diseases of the liver where fibrosis is a common feature. Schistosomiasis, a disease caused by parasitic worms, is a significant cause of illness and death in the developing world. Furthermore, recent reports suggest that the global burden of disease due to schistosomiasis has been significantly underestimated. Schistosomiasis of the liver arises due to inflammation and the deposition of scar tissue around parasite eggs trapped in this organ. In the current study we analysed the gene-expression profile of the mouse liver at several time points following infection with a virulent strain of Schistosoma japonicum to better understand the mechanisms that regulate this process. Progression of disease was associated with increased expression of different groups of genes with distinct biological functions. Specifically, we identified several genes encoding chemical signalling molecules that contribute to different phases of the response by recruiting key cell types to the site of inflammation. This study represents the most comprehensive report to date of the gene expression profile in the liver during schistosomiasis. These results provide further insight into the mechanisms that regulate the development of schistosome-induced inflammation and scarring and will aid in the development of novel treatments to alleviate the burden of disease caused by this parasite.
Melissa Burke, Donald McManus, Grant Ramm, Mary Duke, Yuesheng Li, Malcolm Jones, Geoffrey Gobert

PLoS Pathog, Vol. 5, No. 4. (10 April 2009), e1000371.

Macrophage-specific expression of Arginase-1 is commonly believed to promote inflammation, fibrosis, and wound healing by enhancing L-proline, polyamine, and Th2 cytokine production. Here, however, we show that macrophage-specific Arg1 functions as an inhibitor of inflammation and fibrosis following infection with the Th2-inducing pathogen Schistosoma mansoni. Although susceptibility to infection was not affected by the conditional deletion of Arg1 in macrophages, Arg1â/flox;LysMcre mice died at an accelerated rate. The mortality was not due to acute Th1/NOS2-mediated hepatotoxicity or endotoxemia. Instead, granulomatous inflammation, liver fibrosis, and portal hypertension increased in infected Arg1â/flox;LysMcre mice. Similar findings were obtained with Arg1flox/flox;Tie2cre mice, which delete Arg1 in all macrophage populations. Production of Th2 cytokines increased in the infected Arg1â/flox;LysMcre mice, and unlike alternatively activated wild-type macrophages, Arg1â/flox;LysMcre macrophages failed to inhibit T cell proliferation in vitro, providing an underlying mechanism for the exacerbated Th2 pathology. The suppressive activity of Arg1-expressing macrophages was independent of IL-10 and TGF-β1. However, when exogenous L-arginine was provided, T cell proliferation was restored, suggesting that Arg1-expressing macrophages deplete arginine, which is required to sustain CD4+ T cell responses. These data identify Arg1 as the essential suppressive mediator of alternatively activated macrophages (AAM) and demonstrate that Arg1-expressing macrophages function as suppressors rather than inducers of Th2-dependent inflammation and fibrosis. While the function of NOS2 in Th1-type immunity has been investigated extensively, the role of Arg1 in the regulation of Th2-type responses is unclear. Previously, we showed that proline production in AAMs is regulated by Arg1 activity. Because proline is essential for collagen synthesis in myofibroblasts, numerous studies have suggested that Arg1-expressing AAMs regulate wound healing and fibrosis. The development of fibrosis in schistosomiasis is dependent on Th2 cytokines, and mice deficient in IL-4/IL-13 fail to upregulate Arg1. Nevertheless, although Arg1 expression is associated with Th2-dependent fibrosis, the contribution of macrophage-specific Arg1 to the pathogenesis of fibrosis in schistosomiasis was unknown. The studies conducted here with two different strains of mice deficient in macrophage-associated Arg1 demonstrate unequivocally that Arg1-expressing macrophages exhibit both anti-inflammatory and anti-fibrotic activity during Th2-driven inflammatory responses. In schistosomiasis, fibrosis, portal hypertension, and variceal bleeding are the primary pathological changes that characterize the severe hepatosplenic form of the disease in humans. It is widely believed that people who fail to adequately activate immune suppressive mechanisms when chronically infected with S. mansoni are the individuals who ultimately develop severe disease. Our data identify Arg1-expressing macrophages as critical mediators of immune downmodulation in chronic schistosomiasis.
John Pesce, Thirumalai Ramalingam, Margaret Mentink-Kane, Mark Wilson, Karim El Kasmi, Amber Smith, Robert Thompson, Allen Cheever, Peter Murray, Thomas Wynn

PLoS Negl Trop Dis, Vol. 1, No. 3. (26 December 2007), e32.

There is a long and rich history of research and control in the field of schistosomiasis that has resulted in major scientific and public health accomplishments. Examples of such findings and accomplishments include immunologic regulation in chronic infections [1], the association of helminth infections with Th1-regulating Th2-type immune responses [2], the critical role of interleukin-13 in fibrogenesis [3], and the development and validation of the “dose pole” for determining praziquantel dosages in the field [4],[5]. Perhaps in part because of this broad and successful history, those who work on schistosomiasis come from a wide variety of backgrounds and interests. While such variety is enriching to the field, it sometimes results in diverse opinions about which of the many research opportunities should be pursued. Such diversity, we believe, has at times led to a divisiveness that has harmed overall progress in the field. Partly in response to such events, we have worked with as many of those interested in schistosomiasis as we could identify to develop what we feel is a comprehensive and cohesive agenda for schistosomiasis research (Image 1).
Daniel Colley, Evan Secor

Immunity, Vol. 20, No. 5. (May 2004), pp. 623-635.

Macrophage/neutrophil-specific IL-4 receptor alpha-deficient mice (LysM(Cre)IL-4Ralpha(-/flox)) were generated to understand the role of IL-4/IL-13 responsive myeloid cells during Type 2 immune responses. LysM(Cre)IL-4Ralpha(-/flox) mice developed protective immunity against Nippostrongylus brasiliensis accompanied by T(H)2 development and goblet cell hyperplasia. In contrast, LysM(Cre)IL-4Ralpha(-/flox) mice were extremely susceptible to Schistosoma mansoni infection with 100% mortality during acute infection. Mortality was not dependent on neutrophils and occurred in the presence of T(H)2/Type 2 responses, granuloma formation, and egg-induced fibrosis. Death was associated with increased T(H)1 cytokines, hepatic and intestinal histopathology, increased NOS-2 activity, impaired egg expulsion, and sepsis. IL-10 was not able to compensate for the absence of IL-4/IL-13-activated alternative macrophages. Together, this shows that alternative macrophages are essential during schistosomiasis for protection against organ injury through downregulation of egg-induced inflammation.
De'Broski Herbert, Christoph Hölscher, Markus Mohrs, Berenice Arendse, Anita Schwegmann, Magda Radwanska, Mosiuoa Leeto, Richard Kirsch, Pauline Hall, Horst Mossmann, Björn Claussen, Irmgard Förster, Frank Brombacher

PLoS Negl Trop Dis, Vol. 4, No. 10. (19 October 2010), e850.

The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite. Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens. RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi.
Saša Štefanić, Jan Dvořák, Martin Horn, Simon Braschi, Daniel Sojka, Debbie Ruelas, Brian Suzuki, Kee-Chong Lim, Stephanie Hopkins, James McKerrow, Conor Caffrey

PLoS Negl Trop Dis, Vol. 5, No. 10. (25 October 2011), e1368.

Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs. The flatworm disease, schistosomiasis, is a major public health problem in sub-Saharan Africa, South America and East Asia. A hallmark of infection with Schistosoma mansoni is the immune response to parasite eggs trapped in the liver and other organs. This response involves an infiltration of cells that surround the parasite egg forming a “granuloma.” In mice deprived of T-cells, this granulomatous response is lacking, and toxic products released by eggs quickly cause liver damage and death. Thus the granulomata protect the host from toxic egg products. Only one hepatotoxic molecule, omega-1, has been described to date. We set out to identify other S. mansoni egg hepatotoxins using liver cells grown in culture. We first showed that live eggs, their secretions, and pure omega-1 are toxic. Using a physical separation technique to prepare fractions from whole egg secretions, we identified the presence of IPSE/alpha-1, a protein that is known to strongly influence the immune system. We showed that IPSE/alpha-1 is also hepatotoxic, and that toxicity of both omega-1 and IPSE/alpha-1 can be prevented by first mixing the proteins with specific neutralizing antibodies. Both proteins constitute the majority of hepatotoxicity released by eggs.
Maha-Hamadien Abdulla, Kee-Chong Lim, James McKerrow, Conor Caffrey

PLoS Negl Trop Dis, Vol. 2, No. 7. (30 July 2008), e267.

A bench scientist studying schistosomiasis must make a large commitment to maintain the parasite's life cycle, which necessarily involves a mammalian (definitive) host and the appropriate species of snail (intermediate host). This is often a difficult and expensive commitment to make, especially in the face of ever-tightening funds for tropical disease research. In addition to funding concerns, investigators usually face additional problems in the allocation of sufficient lab space to this effort (especially for snail rearing) and the limited availability of personnel experienced with life cycle upkeep. These problems can be especially daunting for the new investigator entering the field. Over 40 years ago, the National Institutes of Health–National Institute of Allergy and Infectious Diseases (NIH-NIAID) had the foresight to establish a resource from which investigators could obtain various schistosome life stages without having to expend the effort and funds necessary to maintain the entire life cycle on their own. This centralized resource translated into cost savings to both NIH-NIAID and to principal investigators by freeing up personnel costs on grants and allowing investigators to divert more funds to targeted research goals. Many investigators, especially those new to the field of tropical medicine, are only vaguely, if at all, aware of the scope of materials and support provided by this resource. This review is intended to help remedy that situation. Following a short history of the contract, we will give a brief description of the schistosome species provided, provide an estimate of the impact the resource has had on the research community, and describe some new additions and potential benefits the resource center might have for the ever-changing research interests of investigators.
Fred Lewis, Yung-san Liang, Nithya Raghavan, Matty Knight

PLoS Negl Trop Dis, Vol. 5, No. 10. (11 October 2011), e1348.

Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen. More people are infected with Schistosoma haematobium than other major human schistosomes yet it has been less studied because of difficulty in maintaining the life cycle in the laboratory. S. haematobium might be considered the ‘neglected schistosome’ since minimal information on the genome and proteome of S. haematobium is available, in marked contrast to the other major schistosomes. In this report we describe tools and protocols to investigate the genome and genetics of this neglected schistosome. We cultured developmental stages of S. haematobium, and investigated the utility of introducing gene probes into the parasites to silence two model genes. One of these, firefly luciferase, was a reporter gene whereas the second was a schistosome gene encoding a surface protein, termed Sh-tsp-2. We observed that both genes could be silenced – a phenomenon known as experimental RNA interference (RNAi). These findings indicated that the genome of S. haematobium will be amenable to genetic manipulation investigations designed to determine the function and importance of genes of this schistosome and to investigate for novel anti-parasite treatments.
Gabriel Rinaldi, Tunika Okatcha, Anastas Popratiloff, Mary Ayuk, Sutas Suttiprapa, Victoria Mann, Yung-san Liang, Fred Lewis, Alex Loukas, Paul Brindley

PLoS Negl Trop Dis, Vol. 5, No. 6. (7 June 2011), e1170.

Close to 800 million people in the world are at risk of schistosomiasis, 85 per cent of whom live in Africa. Recent studies have indicated that female genital schistosomiasis might increase the risk of human immunodeficiency virus (HIV) infection. The aim of this study is to quantify and analyse the characteristics of the vasculature surrounding Schistosoma haematobium ova in the female genital mucosa. Cervicovaginal biopsies with S. haematobium ova (n = 20) and control biopsies (n = 69) were stained with immunohistochemical blood vessel markers CD31 and von Willebrand Factor (vWF), which stain endothelial cells in capillary buds and established blood vessels respectively. Haematoxylin and eosin (HE) were applied for histopathological assessment. The tissue surrounding S. haematobium ova had a higher density of established blood vessels stained by vWF compared to healthy controls (p = 0.017). Immunostain to CD31 identified significantly more granulation tissue surrounding viable compared to calcified ova (p = 0.032), and a tendency to neovascularisation in the tissue surrounding viable ova compared to healthy cervical mucosa (p = 0.052). In this study female genital mucosa with S. haematobium ova was significantly more vascularised compared to healthy cervical tissue. Viable parasite ova were associated with granulation tissue rich in sprouting blood vessels. Although the findings of blood vessel proliferation in this study may be a step to better understand the implications of S. haematobium infection, further studies are needed to explore the biological, clinical and epidemiological features of female genital schistosomiasis and its possible influence on HIV susceptibility. Schistosomiasis is a fresh water parasite infection that affects millions of people, especially in Africa. Recent knowledge about the genital manifestations of schistosomiasis; especially its possible association with human immunodeficiency virus (HIV) infection, has led to increased focus on this neglected tropical disease. Millions of women remain undiagnosed for genital schistosomiasis, and may suffer from abnormal mucosal blood vessels, contact bleeding and lesions named sandy patches. This study analyses a unique selection of female genital biopsies containing parasite eggs. Protein detection and standard histopathological assessment are combined to quantify and study the characteristics of the mucosal blood vessels surrounding the eggs. Our results show that the genital mucosa with parasite eggs is more vascularised compared to healthy tissue, and that viable eggs tend to be surrounded by proliferating blood vessels. These findings have not yet been correlated directly to clinical manifestations. Further studies are needed in order to provide clinical advice on the risks and consequences of mucosal lesions particular to female genital schistosomiasis.
Peter Jourdan, Borghild Roald, Gabriele Poggensee, Svein Gundersen, Eyrun Kjetland

PLoS Negl Trop Dis, Vol. 5, No. 12. (6 December 2011), e1396.

Urogenital schistosomiasis, caused by infection with Schistosoma haematobium, is widespread and causes substantial morbidity on the African continent. The infection has been suggested as an unrecognized risk factor for incident HIV infection. Current guidelines recommend preventive chemotherapy, using praziquantel as a public health tool, to avert morbidity due to schistosomiasis. In individuals of reproductive age, urogenital schistosomiasis remains highly prevalent and, likely, underdiagnosed. This comprehensive literature review was undertaken to examine the evidence for a cause-effect relationship between urogenital schistosomiasis and HIV/AIDS. The review aims to support discussions of urogenital schistosomiasis as a neglected yet urgent public health challenge. We conducted a systematic search of the literature including online databases, clinical guidelines, and current medical textbooks. We describe plausible local and systemic mechanisms by which Schistosoma haematobium infection could increase the risk of HIV acquisition in both women and men. We also detail the effects of S. haematobium infection on the progression and transmissibility of HIV in co-infected individuals. We briefly summarize available evidence on the immunomodulatory effects of chronic schistosomiasis and the implications this might have for populations at high risk of both schistosomiasis and HIV. Studies support the hypothesis that urogenital schistosomiasis in women and men constitutes a significant risk factor for HIV acquisition due both to local genital tract and global immunological effects. In those who become HIV-infected, schistosomal co-infection may accelerate HIV disease progression and facilitate viral transmission to sexual partners. Establishing effective prevention strategies using praziquantel, including better definition of treatment age, duration, and frequency of treatment for urogenital schistosomiasis, is an important public health priority. Our findings call attention to this pressing yet neglected public health issue and the potential added benefit of scaling up coverage of schistosomal treatment for populations in whom HIV infection is prevalent. Urogenital schistosomiasis is a parasitic infection caused by a worm, Schistosoma haematobium, which lives in the bloodstream of infected individuals. It affects at least 112 million people, mostly in sub-Saharan Africa, and has been suggested to be a risk factor for becoming infected with HIV. We reviewed publications in order to examine whether it seems likely that this parasitic infection could be a risk factor for HIV. Evidence from many types of studies supports the hypothesis that urogenital schistosomiasis does increase a person's risk of becoming infected with HIV. Studies also suggest that individuals who have both urogenital schistosomiasis and HIV have a more aggressive HIV infection and can more easily transmit HIV to their sexual partners. Praziquantel is an oral, nontoxic, inexpensive medication that is safe in pregnancy and is recommended for treatment of schistosomiasis. In areas where both infections co-exist, regular administration of praziquantel both to young girls and to sexually-active women may be an important approach to reducing HIV transmission. Our findings support the importance of making praziquantel more available to people who live in areas of the world where both urogenital schistosomiasis and HIV infection are widespread.
Pamela Mbabazi, Olivia Andan, Daniel Fitzgerald, Lester Chitsulo, Dirk Engels, Jennifer Downs