Cyclooxygenase-derived mediators regulate the immunological control of Strongyloides venezuelensis infection

The aim of this study was to define the immunoregulatory role of prostaglandins in a mouse model of Strongyloides venezuelensis infection. Strongyloides venezuelensis induced an increase of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid. Treatment with the dual cyclooxygenase (COX-1/-2) inhibitors indomethacin and ibuprofen, and the COX-2-selective inhibitor celecoxib partially blocked these cellular responses and was associated with enhanced numbers of infective larvae in the lung and adult worms in the duodenum. However, the drugs did not interfere with worm fertility. Cyclooxygenase inhibitors also inhibited the production of the T-helper type 2 (Th2) mediators IL-5, IgG1, and IgE, while indomethacin alone also inhibited IL-4, IL-10, and IgG2a. Cyclooxygenase inhibitors tended to enhance the Th1 mediators IL-12 and IFN-gamma. This shift away from Th2 immunity in cyclooxygenase inhibitor-treated mice correlated with reduced prostaglandin E(2) (PGE(2)) production in infected duodenal tissue. As PGE(2) is a well-characterized driver of Th2 immunity, we speculate that reduced production of this lipid might be involved in the shift toward a Th1 phenotype, favoring parasitism by S. venezuelensis. These findings provide new evidence that cyclooxygenase-derived lipids play a role in regulating host defenses against Strongyloides, and support the exploration of eicosanoid signaling for identifying novel preventive and therapeutic modalities against these infections.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

P>Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II(-/-) animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild-type (WT) and MHC I(-/-) mice. Histopathological analysis revealed that MHC II(-/-) mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I(-/-) animals. Additionally, levels of parasite-specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II(-/-) infected mice, while a non-significant increase in the level of IgG2a was found in comparison to WT or MHC I(-/-) infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.

Preliminary analysis of miRNA pathway in Schistosoma mansoni

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational at-rest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. in this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma Mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 Putative proteins in the parasite. In addition, the transcript levels of SinDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro Cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni. (C) 2008 Published by Elsevier Ireland Ltd.

Immunolocalization and pathological alterations following Strongyloides venezuelensis infection in the lungs and the intestine of MHC class I or II deficient mice

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II(-/-) and MHC I(-/-) mice were individually inoculated with 3000 larvae (U) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylineosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II(-/-) mice presented a significantly higher number of adult worms recovered from the small intestine on day 5 p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I(-/-) mice. In MHC II(-/-) mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I(-/-) on day 1 p.i. and in MHC II(-/-) mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I(-/-) and MHC II(-/-) on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection. (C) 2008 Elsevier B.V. All rights reserved.

Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins

The sumoylation pathway is a post-translational modification of nuclear proteins widespread among several organisms. SMT3C is the main protein involved in this process and it is covalently conjugated to a diverse assortment of nuclear protein targets. To date, 3 SUMO paralogues (SMT3C, A/B) have been characterized in mammals and plants. In this work we characterized two SUMO related genes, named SMT3B and SMT3C throughout Schistosoma mansoni life cycle. The SmSMTB/C encodes for proteins sharing significant amino acid homology with SMT3. Phylogenetical analyses revealed that both SmSMT3B/C are distinct proteins. Additionally, SmSMT3B and C are expressed in cercariae, adult worms, eggs and schistosomula however SinSMT3C gene showed an expression level 7 to 9 fold higher than SmSMT3B in eggs, schistosomula and adult worms. The comparison between the SmSMT3C genomic and cDNA sequences established that the encoding sequence is interrupted by 3 introns of 70, 37 and 36 bp. Western Blot has shown SMT3 conjugates are present in nuclear and total protein fractions of adults and cercariae. Therefore our results suggest a functional sumoylation pathway, and the presence of two paralogues also suggests the specificity of substrates for SMT3 in S. mansoni. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Immune responses in hookworm infections

Hookworms infect perhaps one-fifth of the entire human population, yet little is known about their interaction with our immune system. The two major species are Necator americanus, which is adapted to tropical conditions, and Ancylostoma duodenale, which predominates in more temperate zones. While having many common features, they also differ in several key aspects of their biology. Host immune responses are triggered by larval invasion of the skin, larval migration through the circulation and lungs, and worm establishment in the intestine, where adult worms feed on blood and mucosa while injecting various molecules that facilitate feeding and modulate host protective responses. Despite repeated exposure, protective immunity does not seem to develop in humans, so that infections occur in all age groups (depending on exposure patterns) and tend to be prolonged. Responses to both larval and adult worms have a characteristic T-helper type 2 profile, with activated mast cells in the gut mucosa, elevated levels of circulating immunoglobulin E, and eosinoophilia in the peripheral blood and local tissues, features also characteristic of type I hypersensitivity reactions. The longevity of adult hookworms is determined probably more by parasite genetics than by host immunity. However, many of the proteins released by the parasites seem to have immunomodulatory activity, presumably for self-protection. Advances in molecular biotechnology enable the identification and characterization of increasing numbers of these parasite molecules and should enhance our detailed understanding of the protective and pathogenetic mechanisms in hookworm infections.

Cleavage of hemoglobin by hookworm cathespin D aspartic proteases and its potential contribution to host specificity

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Experimental human infection with the dog hookworm, Ancylostoma caninum

Objective: To investigate possible routes for human infection by the dog hookworm (Ancylostoma caninum). Design, setting and participant. Relatively small numbers of infective larvae were administered orally and percutaneously to an informed healthy volunteer (J K L) under medical supervision, at intervals between May 1998 and May 1999. Main outcome measures: Symptoms; weekly blood eosinophil counts; faecal microscopy. Results: A marked blood eosinophilia followed a single oral exposure to 100 infective larvae, while faecal examination remained negative. Eosinophil counts then declined gradually, although a rapid, spontaneous rise several months later, at the beginning of spring, possibly indicated reactivation of dormant larvae. Blood eosinophil numbers did not rise significantly after percutaneous infection with 200 larvae. A subsequent, smaller, oral inoculum of 20 larvae provoked an eosinophil response similar to that of the first experiment. Conclusions: Our findings suggest that, following ingestion, some infective larvae of A. caninum develop directly into adult worms in the human gut (as they do in dogs). While the percutaneous route might be the most common means of human exposure to canine hookworm larvae, leading generally to subclinical infection, oral infection may be more likely to provoke symptomatic eosinophilic enteritis.

Strongyloses ratti and S. stercoralis: effects of cambendazole, thiabendazole and mebendazole in vitro

The effects of in vitro incubation of three henzimidazole anthelmintics, thiabendazole, mebendazole and cambendazole on Strongyloides were compared. No drug affected hatching of S. ratti eggs or the viability of infective larvae or parasitic adult worms, but all three inhibited moulting of S. ratti larvae. In addition, cambendazole, but not thiabendazole or mebendazole, impaired the viability of S. ratti first- and second-stage larvae. The three drugs had no effect on isolated S. stercorais free-living adult worms, but they all prevented development of S. stercoralis rhabditiform larvae. Thiabendazole and mebendazole had no effect on the infectivity of either S. ratti or S. stercoralis infective larvae, but infection with these worms was abrogated by prior incubation with cambendazole. These results indicate that cambendazole acts in a different manner to the other two drugs. Since it is active against larvae migrating through the tissues, it is potentially of much greater value than thiabendazole or mebendazole in the therapy of strongyloidiasis.

Serological studies on shistosomiasis mansoni in the Northeast Brasil

Sera from the patients (N = 10) with schistosomiasis mansoni of the hospital of Federal University of Pernambuco, the Schistosoma mansoni egg-positive (N = 51) and -negative (N = 452) inhabitants in Cabo City area, out-patients (N = 37) of the IMIP hospital and Japanese immigrants (N = 127) in Petrolina City area of northeast Brazil as well as Japanese healthy subjects (N = 30) were examined by serological tests including an enzyme-linked immunosorbent assay with antigens prepared from eggs (ELISA-egg) and adult worms (ELISA-adult). The ELISA with egg or adult antigen correctly identified 100% of the uninfected individuals lived in non-endemic area of schistosomiasis. Moreover, when examined cross-reactivity of our ELISA with sera isolated from 78 subjects infected with various intestinal parasitic infections, only one of these sera reacted with the egg and adult antigens. On the examination of 51 sera from the egg-positive subjects, the ELISA-egg revealed the highest sensitivity (98.0%), whereas a large number of false negative reactions of ELISA-adult, Ouchterlony method using adult antigen, circumoval precipitation and immediate intradermal skin test were observed. A low sensitivity of these serologic tests except for ELISA-egg appears to be primarily due to their inability to detect antibody in the sera from egg-positive infantiles. There was no positive correlation between the absorbance values of these two types of ELISA among the sera isolated from ELISA-positive subjects. Rather, by the reactivity of these sera to egg or adult antigen, they could be divided into two subgroups; one reacted more positively with egg antigen and the other with adult antigen. Moreover, it was confirmed that the sera from young subjects (under 20 years old) appear to be highly reactive to the egg antigen than did aged ones. These data suggest that the ELISA with egg antigen, but not with the adult antigen, appears to be useful for the serological survey of schistosomiasis mansoni in the endemic area of northeast Brazil.

Evaluation of the resistance to Schistosoma mansoni infection in Nectomys squamipes (Rodentia: Cricetidae), a natural host of infection in Brazil

The development of resistance in three stages throughout an active infection (pre-ovular, acute and initial chronic stages) was studied, comparing the total number of adult worms recovered from the reinfected group and the control groups. It was shown that Nectomys squamipes was unable to develop resistance in the tested conditions and, on the other hand, reinfection in the pre-ovular period of the parasite led the rodent to present the phenomenonacilitation, with reduction of natural resistance and an increase in the parasite load. These results suggest the existence of other forms of immunity diverse from the concomitant immunity in the host-parasite relationship, according to the employed model.

Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment

Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In general, the levels and/or positivity rate of circulating antigens and specific IgG antibodies were significantly higher in patients with hepatointestinal (HI) and hepatosplenic (HS) forms than in mild intestinal (I) forms. An association between Po antigens and clinical features of the disease was observed, as the level of these antigens was low (137 ng/ml) as well as the positivity rate (7.9%) in patients with I forms; values that were intermediate (593 ng/ml and 33.3%) in those with HI forms, and high (1.563 ng/ml and 50.0%) in more severe HS forms. The Pt antigens were detected in the studied clinical forms not differing statistically but, the positivity rate was significantly higher in HS forms comparatively to I forms. The antisera studied revealed distinct circulating antigen profiles, and the prognostic value of Po and Pt antigens was suggested.

Schistosoma mansoni Sambon, 1907: comparative morphologica studies of some Brazilian strains

The morphology of Schistosoma mansoni adult male worms from three strains which have been maintained in albino mice for several generations, was compared to a strain that has been isolated from the natural host Nectomys squamipes (Rodentia: Muridae) captured in Sumidouro (Rio de Janeiro State) and have been maintained in the same sylvatic rodent under laboratory conditions. Total length of specimens, distance between suckers, the number of testes and extention of testes grouping were the taxonomic characters analysed. The worms recovered from N. squamipes showed expressive differences (p< 0.01) compared to the other strains regarding the considered morphological characters. The strains that were maintained in mice presented statistical differences (p< 0.01) in several characters. Some adult worms besides the normal position of the testes also showed an atypical arrangement of these glands. It can be concluded that the morphology of adult worms may be used to distinguish S. mansoni strains and that morphological changes in adult worms are not induced by successive inoculations of a strain in mice.

Effects of non-specific immunopotentiators in experimental Schistosoma mansoni infection: II. Corynebacterium parvum

The effects of Corynebacterium parvum on host protection, tissue reaction and "in vivo" chemotaxis in Schistosoma mansoni infected mice were studied. The C. parvum was given intraperitoneally using a dose of 0.7 mg, twice a week (for 4 weeks), thirty days before (prophylactic treatment) or after infection (curative treatment). The host protection was evaluated through the recovery of adult worms by liver perfusion and was lower in the prophylactic group as compared to the control group (p = 0.018), resulting in 44% protection. The "in vivo" leukocyte response in both prophylactic and curative groups was higher as compared to the infected/non treated group (p = 0.009 and p = 0.003, respectively). Tissue reactions were described in the experimental and control groups, but there were not remarkable differences among them. The possible biological implications and relevance of the findings for the defensive response of the host and control of schistosomiasis are discussed.

Oviposition by Schistosoma mansoni during in vitro cultivation

Observation of Schistosoma mansoni oviposition during in vitro culture of adult worms for a maximum period of 10 days showed three well distinct phases in the kinetics of oviposition: an initial phase with low egg production, a period of maximum oviposition and finally a progressive reduction in the number of eggs during the late phases of culture. The kinetics of oviposition and the number of eggs laid by the parasites are influenced by the number of worm pairs per amount of RPMI 1640 medium, time of parasite development in the vertebrate host and type of serum utilized in the culture medium.