Intraperitoneal administration of the anti-IL-23 antibody prevents the establishment of intestinal nematodes in mice.

Previous studies have established that an increased Th-9 response creates a hostile environment for nematode parasites. Given that IL-23, a cytokine required for maintenance of the IL-17-secreting phenotype, has inhibitory effects on IL-9 production, we hypothesized that reducing circulating IL-23 by treatment with anti-IL-23 antibodies would reduce the establishment and development of parasitic intestinal nematodes. In this study, we show that animals treated with anti-IL-23 monoclonal antibodies showed a drastic reduction in the number of mouse pinworms (Aspiculuris tetraptera) recovered from the intestine (p < 0.001) at 23 days post-infection compared to the untreated animals. The cytokine levels in Peyer's patches (PP) in treated and infected animals increase the expression of interleukins such as IL-25, IL-21, and IL-9, augmenting mucus production in the crypts, and boosting chemokines, such as OX40 and CCL20 in the mucosa. Our results suggest that the Th17/Th2 regulatory mechanism provoked by the administration of the anti-IL-23 antibody prevents the implantation of the intestinal nematode in mice. The diminished inflammatory IL-17 levels alter the Th9 environment perhaps as a consequence of IL-17 inhibiting IL-9 expression. These Th9 conditions may explain the successful treatment against Inflammatory Bowel Disease (IBD) both with antibodies against IL-23 or through parasitization with nematodes.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Induction of ulcerative colitis in mice influences the course of infection with the nematode Trichuris muris.

The aim of this study was to assess the effect of infection with the nematode whipworm Trichuris muris on the course of chemically induced acute ulcerative colitis in CBA/J mice, a strain proven to be highly resistant to infection with T. muris. Each mouse was infected with 50 embryonated eggs of T. muris by oral gavage. Acute colitis was triggered by administering 4% dextran sulphate sodium (DSS) in the drinking water for nine consecutive days at different times after infection. Concurrent infection and DSS administration exacerbate the severity of the colitis while favouring the permanence of parasites in the intestine. The induction of ulcerative colitis from days 54 to 62 post-infection (p.i.), when all worms had been expelled, ameliorated the course of the inflammatory disease. When ulcerative colitis was triggered earlier on, from days 27 to 35 p.i., the beneficial effects on inflammatory events were clearly shown with signs of mucosal epithelization and regeneration as early as day 1 after DSS administration. Previous infections by T. muris therefore accelerate recovery from subsequently induced inflammatory bowel disease and such an effect assists the nematode to persist in the intestinal niche.

Immunoproteomic analysis of Trichinella spiralis larval crude antigens recognized by sera from patients with trichinellosis after treatment with albendazole.

Antibody responses and antigen recognition were monitored during and after treatment with albendazole (ABZ) in nine patients selected from a trichinellosis outbreak that occurred in north-west Poland in 2007. Seven out of the nine patients yielded positive serum IgG response during treatment. One month after treatment, the IgG response decreased in most patients. Serum levels of ABZ and main metabolites greatly varied among patients without correlation with the IgG response. Two-dimensional electrophoresis and western blot with serum from each patient showed highly immunoreactive spots located between 3- 10 pI and 45-97 kDa in all patients. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/time-of-flight mass spectrometry (TOF MS) analysis identified actine, enolase, p49 protein, Caenorhabditis elegans-targeted antigen, and serine protease as the most reactive proteins. A minor spot located at 6 pI and 26 kDa identified as annexin I failed recognition in most patients showing decline in IgG response and clinical improvement after treatment. This protein could constitute a sensitive marker for the effectiveness of ABZ against trichinellosis.

Identification of proteins interacting with HSP70 mRNAs in Leishmania braziliensis.

HSP70 protein is involved in Leishmania differentiation, apoptosis, antimony-resistance and host-immune response. Therefore, this protein and the regulatory mechanisms of HSP70 gene expression are promising targets for therapeutic intervention against leishmaniasis. The regulation of mRNA expression in trypanosomatids operates mostly through the interaction of trans-acting proteins, and elements located in the untranslated regions of mRNAs. The aim of this work was to identify protein factors interacting specifically with the Leishmania braziliensis HSP70 mRNAs. Thus, the 5' UTR and the two types of 3' UTRs (UTR-I and UTR-II) from L. braziliensis HSP70 genes were used as baits in pull down assays using total protein extracts from parasites cultured at 26 or 35°C. The captured proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS) analysis. As a result, 52 different proteins were identified based on their binding to the L. braziliensis HSP70-mRNAs. As expected, several of the identified proteins were related to RNA metabolism (27%) and translation process (7%). In addition, five hypothetical conserved proteins having motifs related with RNA interaction were also identified (9.6%). Nevertheless, unexpected proteins, apparently unrelated to the mRNA expression, were also identified. The biological significance of these and others L. braziliensis detected proteins, including the HSP70 itself, is discussed. BIOLOGICAL SIGNIFICANCE: For the first time, a riboproteomic analysis of the proteins interacting with the untranslated regions of the heat shock protein 70 (HSP70) mRNA from Leishmania braziliensis was carried out. This work provides new insights related to protein factors putatively involved in the regulation of HSP70 gene expression in L. braziliensis, and thereby, contributes to a better understanding of the parasite biology, and ultimately to the development of novel therapeutic interventions for controlling the important diseases caused by this parasite.

The kinetoplastid chemotherapy revisited: current drugs, recent advances and future perspectives.

Leishmaniasis, African sleeping sickness and Chagas disease, caused by the kinetoplastid parasites Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi, respectively, are among the most important parasitic diseases, affecting millions of people and considered to be within the most relevant group of neglected tropical diseases. The main alternative to control such parasitosis is chemotherapy. Nevertheless, the current chemotherapeutic treatments are far from being satisfactory. This review outlines the current understanding of different drugs against leishmaniasis, African sleeping sickness and Chagas disease, their mechanism of action and resistance. Recent approaches in the area of anti-leishmanial and trypanocidal therapies are also enumerated, new modulators from the mode of action, development of new formulations of old drugs, therapeutic switching and "in silico" drug design.

3D entropy and moments prediction of enzyme classes and experimental-theoretic study of peptide fingerprints in Leishmania parasites.

The number of protein 3D structures without function annotation in Protein Data Bank (PDB) has been steadily increased. This fact has led in turn to an increment of demand for theoretical models to give a quick characterization of these proteins. In this work, we present a new and fast Markov chain model (MCM) to predict the enzyme classification (EC) number. We used both linear discriminant analysis (LDA) and/or artificial neural networks (ANN) in order to compare linear vs. non-linear classifiers. The LDA model found is very simple (three variables) and at the same time is able to predict the first EC number with an overall accuracy of 79% for a data set of 4755 proteins (859 enzymes and 3896 non-enzymes) divided into both training and external validation series. In addition, the best non-linear ANN model is notably more complex but has an overall accuracy of 98.85%. It is important to emphasize that this method may help us to predict not only new enzyme proteins but also to select peptide candidates found on the peptide mass fingerprints (PMFs) of new proteins that may improve enzyme activity. In order to illustrate the use of the model in this regard, we first report the 2D electrophoresis (2DE) and MADLI-TOF mass spectra characterization of the PMF of a new possible malate dehydrogenase sequence from Leishmania infantum. Next, we used the models to predict the contribution to a specific enzyme action of 30 peptides found in the PMF of the new protein. We implemented the present model in a server at portal Bio-AIMS (http://miaja.tic.udc.es/Bio-AIMS/EnzClassPred.php). This free on-line tool is based on PHP/HTML/Python and MARCH-INSIDE routines. This combined strategy may be used to identify and predict peptides of prokaryote and eukaryote parasites and their hosts as well as other superior organisms, which may be of interest in drug development or target identification.

Generalized lattice graphs for 2D-visualization of biological information.

Several graph representations have been introduced for different data in theoretical biology. For instance, complex networks based on Graph theory are used to represent the structure and/or dynamics of different large biological systems such as protein-protein interaction networks. In addition, Randic, Liao, Nandy, Basak, and many others developed some special types of graph-based representations. This special type of graph includes geometrical constrains to node positioning in space and adopts final geometrical shapes that resemble lattice-like patterns. Lattice networks have been used to visually depict DNA and protein sequences but they are very flexible. However, despite the proved efficacy of new lattice-like graph/networks to represent diverse systems, most works focus on only one specific type of biological data. This work proposes a generalized type of lattice and illustrates how to use it in order to represent and compare biological data from different sources. We exemplify the following cases: protein sequence; mass spectra (MS) of protein peptide mass fingerprints (PMF); molecular dynamic trajectory (MDTs) from structural studies; mRNA microarray data; single nucleotide polymorphisms (SNPs); 1D or 2D-Electrophoresis study of protein polymorphisms and protein-research patent and/or copyright information. We used data available from public sources for some examples but for other, we used experimental results reported herein for the first time. This work may break new ground for the application of Graph theory in theoretical biology and other areas of biomedical sciences.

Trichinellosis survey in the wild boar from the Toledo mountains in south-western Spain (2007-2008): molecular characterization of Trichinella isolates by ISSR-PCR.

In Spain, trichinellosis represents a public health problem, with an average of five outbreaks per year, wild boar meat being the main source of infection. A trichinellosis survey (2007-2008 hunting campaign) was carried out on wild boars in the Toledo Mountains (south-western Spain, EU) in the context of a surveillance programme on wildlife diseases. A total of 2216 wild boars from different locations of the region were examined. The examination was carried out by veterinarians in the local abattoir (Matadero Municipal de Toledo). The positive samples were sent to the Department of Parasitology (Facultad de Farmacia, UCM) for experimental isolation and specific identification by inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR). Using this technique we identified 17 isolates as Trichinella spiralis with an electrophoretic profile indistinguishable from the T. spiralis reference strain (ISS48). We confirmed that ISSR-PCR is a robust technique for the molecular identification of Trichinella isolates. According to our results, the prevalence of T. spiralis in wild boars from the Toledo Mountains (>800 m above sea level) during the hunting season was approximately 0.77%. The prevalence of T. spiralis (100% of our observations) is a good example of the persistence of this species in sylvatic conditions (coming from the domestic cycle), if a good wild host is abundant. Our observations confirm the major prevalence of T. spiralis over T. britovi in this region, as well as the risk to human health represented by the consumption of uninspected wild boar meat.

Identification of Spanish Trichinella isolates by ISSR-PCR: intra-specific variability of Trichinella britovi.

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification.

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.

Contrasting effects of Trichinella spiralis and Trichuris muris antigens on the infection by Leishmania infantum in BALB/c mice.

The interaction of Trichinella spiralis and Trichuris muris derived antigens with the infection by Leishmania infantum was investigated in BALB/c mice. Infection with 10(6) promastigotes of L. infantum did not induce relevant serum antibody (IgG subclasses), nor cytokine (IFN-gamma, IL-4) responses despite that mice could partially control the infection. Immunization with T. spiralis activated a moderate IgG1 and secondarily an IgG2a anti-leishmanial response whereas immunization with T. muris elicited only a weak and late activation of IgG1 anti-leishmanial response. Immunization with T. muris caused an elevation of serum IFN-gamma levels which was drastically reinforced by the L. infantum infection, and that was accompanied by almost complete parasitological cure of infected mice. Immunization with T. spiralis induced an elevation of serum IL-4 levels but this response was greatly (about 60%) neutralized by the infection with L. infantum, and this was associated to exacerbation of the infection.

Setting new immunobiological parameters in the hamster model of visceral leishmaniasis for in vivo testing of antileishmanial compounds.

To establish suitable immunobiological parameters for in vivo testing of new antileishmanial compounds in the golden hamster model of visceral leishmaniasis, two groups of 8 animals were infected each with 10(5) or 10(7) stationary promastigotes by the intracardiac route and the clinical and immunoparasitological features were monitored up to day 155 after infection. All animals became infected at both doses, although significant differences were observed between parasite burdens in liver and spleen. The mean number of parasites in animals infected with 10(7) promastigotes increased by 9.5 times in liver and by 43.1 times in spleen compared with those infected with 10(5) promastigotes. In animals given the higher dose, the outcome of the disease occurred between days 75 and 90 after infection, whereas no signs of disease were apparent in those given the lower infecting dose. Positive antibody (IgG) responses were detected earlier (week 5-7 after infection) in animals infected with the higher dose than in those infected with the lower dose (week 8-10 after infection), but these responses did not correlate with individual parasitological loads in liver and spleen. An inverse correlation was observed between infecting doses and in vitro spleen lymphocyte proliferation against mitogens (ConA). The proportion of CD4(+) and CD19(+) spleen cell increased in animals given the higher infection, whereas it decreased in those given the lower infection compared to naive controls.

Microcapsules formulated in the enteric coating copolymer Eudragit L100 as delivery systems for oral vaccination against infections by gastrointestinal nematode parasites.

Microcapsules using the copolymer of methacrylic acid (Eudragit L100) were formulated for oral delivery of vaccines against the enteral/parenteral nematode parasite Trichinella spiralis. Antigenic preparations from first stage larvae (L1) of T. spiralis were microencapsulated in Eudragit L100. The microcapsules prepared by the spray drying method were resistant to acid pH, although the antigen was rapidly released under neutral and basic environmental conditions. The native protein conformation and biological activity was preserved in the microcapsules, as assessed by SDS-PAGE and ELISA. When administered to NIH mice, the antigen loaded microcapsules protected against infection by T. spiralis at both the intestinal and muscular levels, the worm burden diminishing by 45.58 and 53.33%, respectively. Furthermore, following administration of the microparticles an increase of the serum IgG1 response, a marker for the Th2 type response, was evident. These results indicate that microcapsules formulated with anionic biocompatible polymers such as Eudragit may be useful for oral vaccination against nematode infections.

TSL-1 antigens of Trichinella: an overview of their potential role in parasite invasion, survival and serodiagnosis of trichinellosis.

The majority of studies on the immunobiology of Trichinella species have centred on the larval muscular phase (L1) with a view to identifying immunodominant antigens located on the surface of the cuticle and in the larval secretions; the nucleus of the parasite-host interaction. These antigens have been classified as eight groups (TSL-1-TSL-8), of which those belonging to the group TSL-1 have been most intensely studied. The principal constituents are glycoproteins, glycan carriers that contain a unusual sugar, the tyvelose (3,6-dideoxy-d-arabinohexose). Studies aimed at improving serodiagnostic techniques to detect trichinellosis indicate that these antigens are ideal candidates. They are capable of inducing a strong humoral response involving the generation of specific antibodies against beta-tyvelose, a sugar that seems to be exclusive to the Trichuroidea. Furthermore, these glycoproteins appear to fulfil an important function in the development and maintenance of the parasite in the muscular niche, and they appear to be fundamental for the invasion of the intestinal epithelium. It has also been demonstrated that specific monoclonal antibodies against tyvelose can mediate a degree of immunoprotection in the rat through the phenomenon known as rapid expulsion.

Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation.

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.

Two-dimensional electrophoresis and mass spectrometry for the identification of species-specific Trichinella antigens.

A proteomic approach was utilized for fine antigenic characterization of the closely related Trichinella genotypes Trichinella britovi T3 and Trichinella T8. Crude extract of muscle larvae L1 (LCE) from both isolates were analyzed by 2D-PAGE. Over 500 protein spots were reproducibly separated in both genotypes. These separated proteins were identified in Western blot with IgG1 and IgG3 from homologous and heterologous hyperimmune sera raised in BALB/c mice. A group of 20 and 15 spots migrating at 50--60 k Da and pH 5.5--6.5 in T. britovi and Trichinella T8 maps, respectively, reacted with the IgG1 from heterologous sera whereas a group of minor spots of similar migration patterns did not. Low cross-reactivity occurred for IgG3. MALDI-TOF and MALDI-TOF/TOF analysis of these antigens identified a predicted enolase, the protein P 49 and a predicted actin among the cross-reactive proteins and two hypothetical actins among the non cross-reactive proteins.

Anti-leishmanial activity of a new formulation of amphotericin B.

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post-infection (p.i.) to treat animals previously infected with 10(7) stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4(+) subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non-encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.

Ex vivo anthelmintic activity of albendazole-sulphoxide enantiomers.

The antiparasitic activity of racemic albendazole-sulphoxide (Ricobendazole = racRBZ) and its (+) and (-) enantiomers was tested in an ex vivo murine model for Trichinella spiralis infection. Larvae were isolated from the muscle of infected mice and exposed to concentrations between 0.01 and 1 microg/ml of the racemic mixture or to each of its enantiomers. The activity of each compound was then assayed by measuring the ability of the treated larvae to infect naive mice (larval viability). At a concentration of 0.5 microg/ml, all 3 compounds were highly effective in reducing the viability of the larvae, achieving reductions of 91.26% (racRBZ), 96.7% (+), and 89.2% (-), when compared with untreated controls. At lower concentrations (0.1 microg/ml), only treatment with (+)RBZ rendered a significant reduction in larval viability in comparison with controls (84.3%; P < 0.01), whereas at 0.01 microg/ml, none of the compounds altered larval viability (P > 0.05).

Biological variation in Trichinella species and genotypes.

At present, the genus Trichinella comprises seven species of which five have encapsulated muscle larvae (T. spiralis, T. nativa, T. britovi, T. nelsoni and T. murrelli) and two do not (T. pseudospiralis and T. papuae) plus three genotypes of non-specific status (T6, T8 and T9). The diagnostic characteristics of these species are based on biological, biochemical and genetic criteria. Of biological significance is variation observed among species and isolates in parameters such as infectivity and immunogenicity. Infectivity of Trichinella species or isolates is determined, among other considerations, by the immune status of the host in response to species- or isolate-specific antigens. Common and particular antigens determine the extent of protective responses against homologous or heterologous challenge. The kinetics of isotype, cytokine and inflammatory responses against T. spiralis infections are isolate-dependent. Trichinella spiralis and T. pseudospiralis induce different dose-dependent T-cell polarizations in the early host response, with T. spiralis initially preferentially promoting Th1-type responses before switching to Th2 and T. pseudospiralis driving Th2-type responses from the outset.