Microarray analysis identifies genes preferentially expressed in the lung schistosomulum of Schistosoma mansoni

The lung schistosomulum of Schistosoma mansoni is a validated target of protective immunity elicited in vaccinated mice. To identify genes expressed at this stage we constructed a microarray, representing 3088 contigs and singlets, with cDNA derived from in vitro cultured larvae and used it to screen RNA from seven life-cycle stages. Clustering of genes by expression profile across the life cycle revealed a number of membrane, membrane-associated and secreted proteins up-regulated at the lung stage, that may represent potential immune targets. Two promising secreted molecules have homology to antigens with vaccine and/or immunomodulatory potential in other helminths.     

Schistosoma mansoni: control of extramedullar eosinophil myelopoiesis in chronically infected mice by inflammatory macrophages

Extramedullar proliferation of eosinophil granulocytes can be induced in mice chronically infected with Schistosoma mansoni, by intraperitoneal implants of glass coverslips. Immature eosinophils are located in discrete foci on glass implants; they are not correlated with the eosinophil population of the peritoneal cavity, where only mature eosinophils can be observed. The same induction of eosinophil proliferation can be obtained in normal mice, by the transfer of macrophages elicited by glass implants in mice with chronic schistosomiasis. This induction could not be done with cells mobilized in normal mice, either after transplant into normal mice or into schistosome infected ones. Stimulated macrophages of mice with chronic schistosomiasis have a capacity to induce peripherical proliferation of eosinophil granulocytes. This capacity is independent of the quality of the intraperitoneal environment. It can be expressed after transferring macrophages elicited in schistosome infected mice into normal mice.     

Schistosoma mansoni: extramedullar eosinophil myelopoiesis induced by intraperitoneal glass implants in chronically infected mice

Intraperitoneal glass implants in mice with chronic Schistosoma mansoni infections induce intense local myeloid reactions involving essentially myeloid granulocytes. The same phenomenon is not observed in normal mice nor in mice with acute schistosomal infection. An association of myelopoiesis with differentiated macrophages mobilized on glass implants and with dense ameboid cells located inside myeloid foci was detected. A macrophage-dependent induction of this eosinophil reaction is postulated.     

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.     

Histopathological and ultrastructural studies of cutaneous reactions elicited in naive and chronically infected mice by invading schistosomula of Schistosoma mansoni

Naive and chronically infected CBA mice were challenged percutaneously with cercariae and biopsied at varying times thereafter to provide skin samples for light and electron microscopy. The epidermis and dermis doubled in thickness in both groups; this change occurred within 3 h in immune mice and by 48 h in controls. Immune skin showed a 5-fold increase in total thickness by 72 h. Primary reaction sites were characterised by neutrophil infiltrates but in immune mice, eosinophils replaced neutrophils by day 2. Granulocytic micro-abscesses formed in the epidermis in both naive and immune skin; they entrapped cast cercarial tails and schistosomula and were eventually sloughed from the skin surface. An early loss of challenge parasites may occur in this way. Not all penetrated schistosomula completed transformation by developing the double outer membrane and these may constitute additional casualties. Schistosomula in immune but not naive skin were invested by a surface coat; this is suggested to represent an antigen/antibody complex. Significant numbers of larvae in immune skins were associated with intact granulocytes or free eosinophil granules and dead, infiltrated parasites occurred in the dermis. Such individuals may account for the additional attrition recorded in immune mice. Mast cells became associated with granulocytes in both groups of animals; they degranulated by simple exocytosis in naive skin but compound exocytosis in immune skin.     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.     

Alterations of the intestinal innervation in mice infected with Schistosoma mansoni.

Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation.     

Schistosoma mansoni: reduced efficacy of chemotherapy in infected T-cell-deprived mice

The effect of host immunosuppression on the efficacy of schistosomicidal chemotherapy has been tested in T-cell-deprived CBA mice infected with Schistosoma mansoni. The drugs hycanthone, oxamniquine, and praziquantel were found to kill fewer adult S. mansoni worms in deprived mice than in comparably infected strain-, age-, and sex-matched, immunologically intact controls. Inconsistent results were obtained with niridazole, and amoscanate was as effective in deprived mice as in controls. The possibility that hycanthone, oxamniquine, praziquantel, and previously studied antimony act synergistically with immune effector mechanisms in killing adult schistosomes is discussed.     

Effect of somatostatin on gastrointestinal contractility in Schistosoma mansoni infected mice

Schistosoma mansoni infection induces severe gastrointestinal motility disturbances which are characterised by hyperactivity of intestinal muscle, abdominal pain, diarrhoea, vomiting and nausea. During schistosomiasis, the neuropeptide somatostatin is generated within inflammatory granulomas. However, somatostatin is also an important inhibitory modulator of gastrointestinal motility. In the present study, we have investigated the potential of somatostatin to reduce schistosomiasis-induced hyperactivity of gastrointestinal smooth muscle. Organ bath experiments were performed to study the contractility of isolated smooth muscle strips of intestine from control mice and from mice that were infected with S. mansoni for 2, 4, 8 and 16 weeks. Electrical field stimulation (0.5-8 Hz) of enteric nerves induced frequency-dependent neurogenic contractions of cholinergic origin in all regions of the small intestine. Somatostatin (0.1-1 microM) concentration-dependently inhibited the contractions to enteric nerve stimulation in the small intestine from uninfected control mice and from acutely S. mansoni infected mice (2 and 4 weeks of infection). After 8 weeks of infection with S. mansoni, this inhibitory effect of somatostatin was less pronounced and after 16 weeks of infection it was completely abolished. Histology demonstrated that chronic infection of mice with S. mansoni was associated with significant alterations in the musculature of the small intestine. These alterations may be associated with physiological changes in the responsiveness to somatostatin and suggest that the somatostatin neuroregulatory circuit of enteric neurotransmission in the small intestine is disturbed during chronic schistosomiasis mansoni.     

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

Schistosoma mansoni: higher free proline levels in the livers of infected mice

The concentration of L-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free L-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-delta-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free L-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000g x 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free L-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-delta-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free L-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.     

Demonstration of splenic auto-anti-idiotypic plaque-forming cells in mice infected with Schistosoma mansoni

Mice exposed to 35 cercariae of the human helminth Schistosoma mansoni develop chronic (greater than 16wk) infections characterized by immunoregulation of their cell-mediated granulomatous responses to schistosome eggs. Evidence was sought regarding the possible development of anti-idiotypic responses against the responses to soluble egg antigens (SEA). Sera were collected from CBA/J mice with chronic S. mansoni infections. Multiclonal idiotypic, anti-SEA antibody (id) was prepared from these pooled sera by affinity chromatography on an SEA immunoadsorbent column. Analysis of the id preparations by polyacrylamide gel electrophoresis demonstrated that this material contained only immunoglobulin heavy and light chains. A modified reverse plaque-forming cell (PFC) assay was developed to quantify anti-idiotypic (anti-id) PFC in spleen cell preparations from infected and age-matched control CBA/J mice. Expression of anti-id PFC began 2 to 3 wk after onset of egg production and continued throughout the course of infection. Positive selection of anti-id-reactive spleen cells by panning cell preparations from chronic mice on id-coated plates resulted in an enrichment of anti-id PFC in the id-adherent population. Conversely, the number of PFC reactive with SEA (id-producing PFC) was lowered by panning on id-coated plates. These data demonstrate the occurrence of anti-id responses during schistosomiasis mansoni. It is possible that such an immunoregulatory mechanism could play an important role in how an animal modulates the granulomatous response that leads to the formation of pathologic lesions and in the maintenance of this chronic infection.     

Further observations on ultrastructural changes in hepatocytes of mice infected with Schistosoma mansoni

The effect of hepatic granulomas initiated by eggs of Schistosoma mansoni on the ultrastructure of hepatocytes of murine hosts was studied. Specimens of infected livers were collected at half week intervals, starting at week 7 postinfection and terminating at week 9 postinfection. Only the hepatocytes adjacent to granulomas showed any alteration in structure. The most obvious change was the proliferation of smooth-surfaced endoplasmic reticulum, especially striking in samples collected 8 1/2 and 9 weeks postinfection. The hypothesis was presented that the material secreted by the egg of the organism might be responsible for what appeared to be a morphologic detoxification response by the hepatocytes. Other alterations evident in the hepatocytes were an increase in the lysosomal population, mitochondrial changes and a slight hypertrophy of the Golgi complexes. Previous related studies by other investigators were explored and the findings compared with the results of this study.     

Localization and identification of Schistosoma mansoni/KLH-crossreactive components in infected mice.

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.     

Garlic attenuates histological and histochemical alterations in livers of Schistosoma mansoni infected mice

Interest in screening for new anti-schistosomal agents is growing because of increased concerns about resistance to and safety of praziquantel. We investigated the anti-schistosomal action of prophylactic and therapeutic doses of garlic on the histological and histochemical alterations caused by Schistosoma mansoni infection. Livers of infected mice were characterized by granulomas, periportal inflammation and fibrosis, hepatocyte vacuolation, fatty degeneration and necrosis, and hypertrophy and pigmentation of Kupffer cells. Significant depletion of carbohydrates and increased lipid vacuoles also were observed. All garlic regimens caused suppression of granuloma formation and amelioration of histological and histochemical changes; the continuous treatment protocol produced the best results. Garlic appears to be a safe and economical anti-schistosomal adjuvant for attenuating the pathogenicity of schistosomiasis.     

Epitopes rationally selected through computational analyses induce T‐cell proliferation in mice and are recognized by serum from individuals infected with Schistosoma mansoni

Schistosomiasis is the second leading cause of death due to parasitic diseases in the world. Seeking an alternative for the control of disease, the World Health Organization funded the genome sequencing of the major species related to schistosomiasis to identify potential vaccines and therapeutic targets. Therefore, the aim of this work was to select T and B‐cell epitopes from Schistosoma mansoni through computational analyses and evaluate the immunological potential of epitopes in vitro. Extracellular regions of membrane proteins from the Schistosoma mansoni were used to predict promiscuous epitopes with affinity to different human Major Histocompatibility Class II (MHCII) molecules by bioinformatics analysis. The three‐dimensional structure of selected epitopes was constructed and used in molecular docking to verify the interaction with murine MHCII H2‐IA^b. In this process, four epitopes were selected and synthesized to assess their ability to stimulate proliferation of CD4⁺ T lymphocytes in mice splenocyte cultures. The results showed that Sm041370 and Sm168240 epitopes induced significant cell proliferation. Additionally, the four epitopes were used as antigens in the Indirect Enzyme‐Linked Immunosorbent Assay (ELISA) to assess the recognition by serum from individuals infected with Schistosoma mansoni. Sm140560, Sm168240, and Sm041370 epitopes were recognized by infected individuals IgG antibodies. Therefore, Sm041370 and Sm168240 epitopes that stood out in in silico and in vitro analyses could be promising antigens in schistosomiasis vaccine development or diagnostic kits. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:804–814, 2017     

IgM antibodies recognizing carbohydrate epitopes shared between schistosomula and miracidia of Schistosoma mansoni that block in vitro killing

A series of monoclonal antibodies (mAb) was raised in mice against Schistosoma mansoni, which recognized a carbohydrate determinant on a major Mr greater than 200,000 schistosomulum surface antigen. These mAb cross-reacted with the surface of cercariae and miracidia and with schistosomula of S. haematobium and S. bovis. Other mAb were generated that only recognized a Mr 20,000 schistosomulum surface antigen; they did not cross-react with eggs or miracidia and were species specific. The anti-Mr 20,000 mAb of the IgG1 isotype exhibited high levels of complement-dependent cytotoxicity to schistosomula in vitro. IgM mAb that recognized carbohydrate epitopes of the Mr greater than 200,000 surface antigen blocked the lethal activity of the anti-Mr 20,000 mAb. The IgM anti-Mr greater than 200,000 mAb also reduced complement-dependent cytotoxicity of serum from mice vaccinated with irradiated cercariae.     

Antischistosomal and liver protective effects of Curcuma longa extract in Schistosoma mansoni infected mice

With a view to clarify the induction of the "Crabtree consequence" in liver cells of S. mansoni infected mice, the curative effect of oil extract of C. longa was tested and compared to praziquantel (PZQ) the effective drug against all schistosome species occurring in man. Protein, glucose, glucose-6-phopsphatase, AMP-deaminase, adensoine deaminase, urea concentration, pyravate kinase (PK), phosphoenol pyruvate carboxykinase (PEPCK) and PK/PEPCK ratio were estimated. In addition, worm burden and ova count in mice infected with S. mansoni were elucidated. The result showed that C. longa normalized the concentration of protein, glucose, AMP-deaminase and adenosine deaminase, which were changed by infection. Moreover, it lowered pyruvate kinase level, while PZQ-treatment induced more elevation of this enzyme. PZQ was more effective in lowering worm burden while C. longa extract was more potent in reducing egg count.