Do mice genetically selected for resistance to oral tolerance provide selective advantage for Schistosoma mansoni infection?

A highly evolved relationship exists between the parasitic flatworm Schistosoma mansoni and its vertebrate hosts that include the use of host immune signals by parasites. The S. mansoni infection was studied in two strains of mice genetically selected, over 18 generations of assortative mating, for extreme phenotypes of susceptibility (TS) and resistance (TR) to immunological tolerance. The objective was to observe whether the different host genetic backgrounds affected the outcome of experimental schistosomiasis. Fecal egg excretion, tissue egg count, worm recovery, and adult worm morphology and morphometry were monitored throughout the period of infection. TR mice presented total fecal egg excretion and thickness of tegument in adult male worms significantly higher than TS mice. Therefore, the comparative analysis of mice with extreme phenotypes of immunological response turns out to be useful in host-parasite relationship studies. Our results suggest that the TR mouse immunological profile provides a more favorable environment for the development of S. mansoni.     

Characterization of Schistosoma mansoni ATPDase2 gene, a novel apyrase family member

Schistosoma mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries. We have previously described the SmATPDase1 gene, encoding a protein from the external surface of the parasites. In this work, we describe the cloning and characterization of SmATPDase2, a novel CD39-like ATP diphosphohydrolase gene in S. mansoni. In silico analysis of the protein encoded by SmATPDase2 predicts a single N-terminal transmembrane domain similar to that described for secreted human apyrase isoforms. Immuno-colocalization experiments detected both SmATPDase proteins at the S. mansoni adult worm tegument basal and apical membranes, but only SmATPDase2 in the tegument syncytium. SmATPDase2 but not SmATPDase1 protein was detected by Western blot in culture medium supernatants following incubation of adult worms in vitro, indicating that SmATPDase2 was secreted by the parasite to the medium. Taken together these data suggest a non-redundant role for SmATPDase2 in the parasite-host interplay.     

Wound healing in the trematode Schistosoma

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

Characterization of proteins and immunogens released by adult Schistosoma mansoni

The proteins released in vitro by metabolically radiolabeled adult Schistosoma mansoni were identified by 2-dimensional gel electrophoresis. To determine the origin of these proteins, adult worms were fractionated into surface membrane, tegument, and remaining body components, and the electrophoretic patterns of the proteins in the 3 fractions were compared to those of the released proteins. The immunogens present in these fractions then were identified by immunoprecipitation with sera from humans infected with S. mansoni. This analysis indicated that essentially all of the proteins released from the worm were immunogenic, whereas most of the major membrane and tegumental proteins were not reactive with the immune sera. Thus, it appears that the adult worm is defended against immune attack by detection of the host's antibody response against released proteins rather than against proteins-exposed on the worm's surface.     

Antifibrotic effects of <Emphasis Type="Italic">Salvia miltiorrhiza </Emphasis>on dimethylnitrosamine-intoxicated rats

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-1 (TGF-1). The inhibitory effects of Sm (50~400 g/ml) on TGF-1-induced -smooth muscle actin (-SMA) secretion and the mRNA expressions of fibrosis-related genes, including -SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 g/ml) significantly inhibited TGF-1-stimulated -SMA secretion and the mRNA expressions of -SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 ± 0.2) or high (1.8 ± 0.1) dose of Sm, or silymarin (1.4 ± 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 ± 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of -SMA, TGF-1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection

Background

Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate.

Methods and Findings

We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins.

Conclusion

This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males

The excretory system of schistosomes has focused some attention during the last years since accumulating evidence suggests that it plays an important role in the host-parasite interaction. Signalling molecules such as phosphatases, but also proteases have been localised in the excretory system. To some extent, however, localisation studies are limited by the fact that sections of fixed specimens are used. In this study, we tested the fluorescent molecules FITC-dextran and Texas Red-BSA for their ability to enter the excretory system of living Schistosoma mansoni males. It is demonstrated that the dyes selectively stain the excretory tubules which are widely distributed along the worm body. This finding was used to investigate whether the staining of worms with Texas Red-BSA can help to localise transgene activity in worms which were transiently transformed by particle bombardment. A vector was used for transformation which contained the green fluorescent protein gene, under the control of the regulatory elements of the cysteine protease ER60 gene. After transformation and staining, confocal laser scanning microscopy revealed that ER60-induced green fluorescent protein activity colocalises with Texas Red-BSA in the excretory tubules. The results suggest a role for ER60 during the host-parasite interaction. Furthermore, the colocalisation approach introduced here opens further perspectives to characterise gene-expression profiles in this parasite.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.     

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are locted within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.     

KCl-Extractable surface antigens of Schistosoma mansoni: immunological characterization and applicability in immunodiagnosis

A Schistosoma mansoni antigen preparation was obtained by extraction of adult worms with a 3 M KCl solution. An indirect immunofluorescence reaction on cryostat sections of adult worms showed that the extracted antigens mainly originated from the tegument. The complex antigenic composition of the tegument extract was shown by immunoelectrophoresis against serum from infected mice and immunized rabbits, which gave up to 9 and 17 precipitation lines, respectively. When we compared the use of adult worm antigens and the tegument antigen preparation in the DASS and ELISA tests for immunodiagnosis of human schistosomiasis, the average sensitivity of the tests with the two preparations was about equal, although considerable differences between individual sera occurred. Analysis of tegument antigens, fractionated by gel filtration, showed that the main serological activity of the tegument antigen preparation was due to high molecular weight antigens.     

Schistosoma mansoni: TGF-beta signaling pathways

Schistosome parasites have co-evolved an intricate relationship with their human and snail hosts as well as a novel interplay between the adult male and female parasites. We review the role of the TGF-beta signaling pathway in parasite development, host-parasite interactions and male-female interactions. The data to date support multiple roles for the TGF-beta signaling pathway throughout schistosome development, in particular, in the tegument which is at the interface with the host and between the male and female schistosome, development of vitelline cells in female worms whose genes and development are regulated by a stimulus from the male schistosome and embryogenesis of the egg. The human ligand TGF-beta1 has been demonstrated to regulate the expression of a schistosome target gene that encodes a gynecophoric canal protein in the schistosome worm itself. Studies on signaling in schistosomes opens a new era for investigation of host-parasite and male-female interactions.     

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man(7)GlcNAc(2) and probably Man(6)GlcNAc(2). These two products were also observed when Man(8)GlcNAc(2) isomer B instead of Man(9)GlcNAc(2) was used as substrate. Other oligosaccharides, such as Man(6)GlcNAc(2)-Asn, Man(5)GlcNAc(2)-Asn, and the alpha1,3- and alpha1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an alpha1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.     

Nitric Oxide Synthase Inhibition Promotes Carcinogen-Induced Preneoplastic Changes in the Colon of Rats

l-Arginine is metabolized either to polyamines through arginase and ornithine decarboxylase (ODC) activities or to citrulline and nitric oxide (NO, nitrogen monoxide) through the NO synthase (NOS) pathway. Polyamine levels and ODC activity are high in tumor cells. The aim of this study was to test whether N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, modulates colon carcinogenesis. Adult male Wistar rats were treated with azoxymethane (AOM, 15 mg/kg ip), a chemical carcinogen, once a week for 2 weeks. One week after the second injection the rats were randomly divided into two groups. One group (n = 8) received l-NAME (10 mg/kg body wt/day) in drinking water. The control group (n = 8) received tap water. After 5 weeks, the rats receiving l-NAME showed enhanced mean basal arterial blood pressure, decreased heart rate, and a significant decrease of the cGMP content in the colonic mucosa. In both groups, AOM induced the formation of colonic aberrant crypt foci (ACF). In l-NAME-treated rats, the number of ACF was higher than in controls by 47%. ODC activity was enhanced by 11-fold. S-Adenosyl-methionine-decarboxylase activity and putrescine concentration were significantly increased in the colonic mucosa of l-NAME-treated rats. The data suggest that l-NAME promotes carcinogen-induced preneoplastic changes in the colon by inhibiting NOS activity and by stimulating polyamine biosynthesis.