Search

Delivery of antigens by oral route is considered as the mainly strategy of immunization against enteric pathogens. However, it could result in the induction of oral tolerance, a state of unresponsiveness that could exacerbate the infection. Entamoeba histolytica is the enteric protozoan parasite responsible for the highest levels of morbidity and mortality worldwide. With the aim of induce a secretory immune response against amebic antigens, we immunized a group of C3H/HeJ mice by oral route with a 29 kDa cysteine-rich protein recognized by human salivary IgA fused to the cholera toxin subunit B (CREHP-CTxB). Mice were immunized once a week during a month with 100 ug of CREHP/CTxB in presence of bicarbonate. Two control groups of mice received CTxB and PBS, respectively. Serum and fecal samples were collected before immunization and the day of sacrifice, 7 days after the last dose. Analysis of the local and systemic humoral responses against the fused protein by ELISA showed the raising of both secretory IgA antibodies in feces and high levels of IgM antibodies in sera of immunized mice but not in the controls groups. However, the analysis of the cellular response by spleen cell proliferation and incorporation of 3H-thymidine, showed that T cell from the mice immunized with CREHP/CTxB were unable to respond to Con A and anti-CD3 antibodies. By contrast, T cells from control groups showed high levels of proliferation under those stimulus. The results suggest the generation of an oral tolerance of the suppressive type with an E. histolytica antigen fused to CTxB. [ABSTRACT FROM AUTHOR]

The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM(1), as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis., ((c) 2010 Elsevier Inc. All rights reserved.)

The purpose of this work was to clarify the taxonomy and phylogenetic relationship of the Phyllosoma complex and other important vectors in Mexico. The internal transcribed spacer 2 (ITS-2) of rDNA and the cytochrome B gene of mtDNA (mtCytB) were analyzed for the following species of triatomine: Triatoma bassolsae, T. longipennis, T. mazzottii, T. mexicana, T. pallidipennis, T. picturata, and T. phyllosoma belonging to the Phyllosoma complex, as well as T. dimidiata, T. rubida, T. infestans, and Rhodnius prolixus. The results obtained with the analysis of the ITS-2 sequences showed that the Phyllosoma complex species could not be phylogenetically separated, since T. bassolsae and T. pallidipennis, as well as T. phyllosoma and T. mazzottii were indistinguishable. In contrast, the mtCytB gene separates each one of these triatomine species. The results support the proximity of all seven species currently included in the Phyllosoma complex as well as the exclusion of T. dimidiata. For the first time T. lecticularia and T. rubida were analyzed and were also shown to be related to the Phyllosoma complex.

T cell mediated response is involved in a protective immune response against experimental cysticercosis conferred by immunization with Taenia solium paramyosin (TPmy) to BALB/c mice. In this study, we analysed the TPmy amino acid sequence for predicted CD4+ T cells epitopes. Five different regions of this protein showed that the residues anchor to bind the I-Ad molecule, synthetic peptides containing these epitopes were evaluated for their ability to induce lymphoproliferative responses of spleen cells from TPmy immunized mice. Among them, Tp176 (amino acids 176-192 sequence DDLQRQMADANSAKSRL) was the immunodominant T cell epitope of TPmy. Delineation of this epitope should facilitate analysis of the role of CD4+ T cell response in experimental cysticercosis.

Taenia solium paramyosin is an immunodominant antigen in human and porcine cysticercosis that has shown promise as a vaccine candidate against schistosomiasis and some filariasis. There are few studies to identify the immunologically relevant regions of paramyosin. In this work, we characterize the humoral and cellular response of neurocysticercotic patients against T. solium paramyosin. Western blots using different recombinant fragments of T. solium paramyosin, showed that the sera from neurocysticercotic patients were strongly reactive against the carboxyl end region, with poor recognition of the central and amino regions. In contrast, the cellular immune response of patients did not show preferential recognition of any region of paramyosin.

Some reports have suggested that human neurocysticercosis (NCC) induces immunosuppression. To test this hypothesis, we performed a study on active cases of NCC who had not received cestocidal or immunosuppressive treatments. We examined blood counts and specific T cell markers (CD3, CD4, and CD8) by flow cytometry and found no differences between patients with NCC and healthy individuals. Both groups responded to concanavalin A (Con A), and patients with NCC responded more to a parasite crude antigen than uninfected individuals. Peripheral blood mononuclear cells were examined for interleukin (IL)-2, interferon-gamma, IL-10, and IL-4 mRNA. Regardless of infection status, more than 60% of individuals synthesized IL-2 mRNA and, less frequently, the other cytokines. These data suggest that immunosuppression does not occur in NCC patients.

Heat shock and stress responses are documented for the first time in larval stages of the cestodes Taenia solium and Taenia crassiceps. Radioactive metabolic labelling after in vitro incubation of cysts at 43 degrees C, revealed the induction of heat shock proteins. In T. crassiceps, the major heat shock proteins were 80, 70 and 60 kDa. After prolonged incubation, a set of low molecular weight heat shock proteins (27, 31, 33 and 38 kDa), were also induced. In vitro incubation of cysts at 4 degrees C, induced the synthesis of stress proteins ranging from 31 to 80 kDa, indicating the parasite is also able to respond to cold shock. T. solium cysts exposure to temperature stress also resulted in an increased synthesis of 2 major heat shock proteins of 80 and 70 kDa. Western blots using the excretory-secretory products of T. solium showed that 2 heat shock proteins were recognized by antibodies in the sera of cysticercotic patients: one of 66 kDa and another migrating close to the run front. The T. solium 66 kDa protein was also recognized by specific antibodies directed to a 60 kDa bacterial heat shock protein, suggesting that it belongs to this family of proteins.

Acanthocephala (thorny-headed worms) is a phylum of endoparasites of vertebrates and arthropods, included among the most phylogenetically basal tripoblastic pseudocoelomates. The phylum is divided into three classes: Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala. These classes are distinguished by morphological characters such as location of lacunar canals, persistence of ligament sacs in females, number and type of cement glands in males, number and size of proboscis hooks, host taxonomy, and ecology. To understand better the phylogenetic relationships within Acanthocephala, and between Acanthocephala and Rotifera, we sequenced the nearly complete 18S rRNA genes of nine species from the three classes of Acanthocephala and four species of Rotifera from the classes Bdelloidea and Monogononta. Phylogenetic relationships were inferred by maximum-likelihood analyses of these new sequences and others previously determined. The analyses showed that Acanthocephala is the sister group to a clade including Eoacanthocephala and Palaeacanthocephala. Archiacanthocephala exhibited a slower rate of evolution at the nucleotide level, as evidenced by shorter branch lengths for the group. We found statistically significant support for the monophyly of Rotifera, represented in our analysis by species from the clade Eurotatoria, which includes the classes Bdelloidea and Monogononta. Eurotatoria also appears as the sister group to Acanthocephala.

To identify the Entamoeba histolytica antigens capable of inducing secretory IgA (sIgA) responses in humans, a cDNA library from the strain HM1:IMSS was immuno-screened with saliva from patients with intestinal amebiasis or amebic liver abscess. Clones isolated with sIgA antibodies from patients with intestinal amebiasis corresponded to the known serine-rich protein isoform, a 29 kDa cysteine-rich protein and 1-alpha elongation factor. Clones corresponding to enolase, cyclophilin, ribosomal protein L23a, and an Hsp70 family protein were isolated with sIgA from a patient with amebic liver abscess. A glutamic acid-rich peptide (EhGARP) positive with sIgA from a patient with amebic liver abscess was also isolated; for EhGARP, no homologs were found in the protein databases. The antigens isolated are potentially useful in the development of an oral vaccine or new diagnostic tools for amebiasis.

Cysticercosis is a parasitic disease caused by the larval stage or cysticercus of the cestode tapeworm Taenia solium. Cysticerci are able to survive in the host tissues for long periods in the presence of an immune response. Tissues of cestodes contain mineral concretions termed calcareous corpuscles. These corpuscles might serve for the focal deposition of exceeding amounts of calcium protecting the larvae against calcification. Studies on the morphology and composition of calcareous corpuscles in cestodes have resulted in a number of hypotheses on their origin and function. Calcareous corpuscles are believed to form either intracellularly or extracellularly and the cell type involved, the place of formation and the mechanism of mineral deposition seem to be also diverse. This review intends to provide an updated guide to the published literature on calcareous corpuscles in cestodes, giving emphasis on their role in larval physiology. Understanding biomineralization might lead to novel ways for the treatment of diseases caused by cestode larvae.

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b-C9. The order of emergence of the homologous components of TCC (C6, C7, C8alpha, C8beta, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6-C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism.

Nucleotide sequences of 18S ribosomal RNA from 71 species of Platyhelminthes, the flatworms, were analyzed using maximum likelihood, and the resulting phylogenetic trees were compared with previous phylogenetic hypotheses. Analyses including 15 outgroup species belonging to eight other phyla show that Platyhelminthes are monophyletic with the exception of a sequence putatively from Acoela sp., Lecithoepitheliata, Polycladida, Tricladida, Trematoda (Aspidobothrii + Digenea), Monogenea, and Cestoda (Gyrocotylidea + Amphilinidea + Eucestoda) are monophyletic groups. Catenulids form the sister group to the rest of platyhelminths, whereas a complex clade formed by Acoela, Tricladida, "Dalyellioida", and perhaps "Typhloplanoida" is sister to Neodermata. "Typhloplanoida" does not appear to be monophyletic; Fecampiida does not appear to belong within "Dalyellioida," nor Kalyptorhynchia within "Typhloplanoida." Trematoda is the sister group to the rest of Neodermata, and Monogenea is sister group to Cestoda. Within Trematoda, Aspidobothrii is the sister group of Digenea and Heronimidae is the most basal family in Digenea. Our trees support the hypothesis that parasitism evolved at least twice in Platyhelminthes, once in the ancestor to Neodermata and again in the ancestor of Fecampiida, independently to the ancestor of putatively parasitic "Dalyellioida."

Arsenic and vanadium are important environmental and industrial pollutants. Due to their widespread occurrence and potential genotoxicity, we studied the aneuploidy-inducing effects of these elements in cultured human lymphocytes using a variety of techniques including fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 1 and 7, immunostaining of the lymphocyte spindle apparatus, and an in vitro assay measuring the polymerization and depolymerization of tubulin. Dose-related increases in hyperdiploidy were seen in lymphocyte cultures treated with sodium arsenite (NaAsO2) or vanadium pentoxide (V2O5) over concentrations ranging from 0.001 to 0.1 microM. NaAsO2-treated cells from different donors exhibited similar hyperdiploid frequencies, whereas substantial inter-individual variability was seen in the V2O5-treated cells. Examination of the spindle apparatus using an anti-beta-tubulin antibody indicated that these compounds might disrupt spindle formation by interacting with microtubules. Additional in vitro assays using purified tubulin indicated that both compounds inhibited microtubule assembly and induced tubulin depolymerization. These results indicate that in vitro exposure to both NaAsO2 and V2O5 can induce aneuploidy in human lymphocytes, and that this effect may occur through a disruption of microtubule function.

Amebiasis is one of the main causes worldwide of morbidity and mortality by parasites. Application of recombinant DNA technology to the study of Entamoeba histolytica is bringing new light into our understanding of this remarkable protozoan parasite and of the disease it causes. New achievements affect the way we approach many essential questions about E. histolytica, from the mechanism of its pathogenicity to the definition of E. histolytica as a separate species from the nonpathogenic E. dispar. To give a single example, transfection of trophozoites is now possible and a new generation of studies taking advantage of this capability of manipulation is expected in the short term. Our goal with this review is to provide an updated and simple guide to the growing information on the molecular biology of E. histolytica.

As our appreciation of the diversity within the flatworms has grown, so too has our curiosity about the ways in which these varied creatures are related to one another. In particular, the parasitic groups (trematodes, cestodes and monogeneans have been the focus of enquiry. Until recently, morphology, anatomy and life histories have provided the raw data for building hypotheses on relationships. Now, ultrastructural evidence, and most recently, molecular data from nucleic acid sequences, have been brought to bear on the topic. Here, David Blair, Andrés Campos, Michael Cummings and Juan Pedro Laclette discuss the ways in which molecular data, in particular, are helping us recognize the various lineages of flatworms.

The internalization of host macromolecules to the vesicular fluid of T. crassiceps cysticerci was studied in vitro. Uptake of purified class G immunoglobulin was not significantly affected by the specificity of its antigen-recognition site and bovine serum albumin was internalized at a similar rate. Internalization was inhibited at low temperature, being optimal at 37 degrees C and saturation was accomplished only at a protein concentration in the culture medium over 12 mg/ml which is close to the physiological concentration of serum proteins in the host. Morphological studies using markers for adsorptive endocytosis allowed visualization of endocytic vesicles and tracking of their movement across the bladder wall tissue. Degradation of internalized proteins was observed at longer times of incubation, suggesting that proteins are later processed and that degraded host macromolecules can be nutrients for cysticerci. Quantification of this capability of internalization suggests that it might play a role in the in vivo removal of potentially damaging host macromolecules, such as antibodies or complement factors, from the host-parasite interface.

A glycoprotein-enriched fraction was obtained by Concanavalin A-Sepharose 4B affinity chromatography from a crude extract of T. solium cysticerci. The six most prominent glycoproteins with molecular sizes of 180, 103, 96, 68, 55 and 45 kDa were purified by electro-elution from polyacrylamide gel slices. Ultrastructural localization assays using hyperimmune rabbit sera to each glycoprotein, demonstrated their presence on the tegumentary surface of the bladder wall of T. solium cysticerci. Similar studies showed that the 180 kDa glycoprotein is also present on the surface of the T. solium and T. saginata adult worms, as well as in T. saginata, T. pisiformis and T. crassiceps cysticerci. The 55 kDa glycoprotein, which is one of the most abundant on the cyst surface, was found to correspond to the heavy chain of pig IgG by Western blotting.

Antigen B, a major antigen of the cestode parasite Taenia solium, has been purified and a portion of amino acid sequence obtained. Paramyosin of the trematode parasite Schistosoma mansoni, an immunogenic protein that has shown promise as a vaccine candidate, has several biochemical and immunological properties in common with antigen B. A full-length cDNA clone of S. mansoni paramyosin has been obtained and the predicted translation product contains a sequence that is highly homologous to the sequence obtained for antigen B. The predicted amino acid composition and isolectric point of paramyosin are nearly identical to those established for antigen B. Recombinant S. mansoni paramyosin, expressed in Escherichia coli as a fusion protein with beta-galactosidase, was recognized by antisera against T. solium antigen B. We conclude from these results that S. mansoni paramyosin and T. solium antigen B are homologous proteins. Since S. mansoni paramyosin is thought to be a muscle protein and T. solium antigen B a secreted glycoprotein with anti-complement activity, this conclusion raises some interesting questions regarding the role of this class of proteins in the host-parasite relationship.

Crystals of virus-like particles (VLP) are described as occurring in the nuclei of damaged tegumentary cytons from carcasses of Taenia solium metacestodes that had been stripped of their teguments. The VLP are grouped as parallel lines of round particles in an hexagonal packaging of spheroids forming small or large crystals. The individual particles have an external diameter of 36-37 nm and a wall of 5-6 nm thick, which surround a cavity of lower electron density. As identical crystals were also observed in normal tissues of T. solium and of T. crassiceps, it is suggested that both species of cysticerci are normal carriers of a similar species of virus. The possible biological implications of this condition are discussed.

Genomic and cDNA actin clones were isolated from Taenia solium gene libraries. The actin genes are interrupted by intervening sequences. Protein coding regions of both genes predict the same amino acid sequence. cDNA sequence data indicate that at least one gene is expressed at the larval stage. Results from Northern and Western blots showed that T. solium expresses an actin transcript of about 1,400 bases and a protein of 45,000 Da.

A 1-step procedure for the easy and rapid purification of milligram amounts of antigen B from a crude extract of Taenia solium cysticerci is described here. This procedure takes advantage of the property of the antigen B to bind to collagen and is based on antigen adsorption to polymeric collagen.

The metabolic adaptability of Taenia solium cysticerci was studied in vitro, by measuring their respiratory rate before, during, and after trypsin-induced evagination. Under aerobic conditions, the oxygen consumption increased about 40% during evagination of the cysticeri and returned to basal rates after the process was completed. The percentage of evagination induced by trypsin was not affected under anaerobic conditions or in the presence of respiratory poisons such as cyanide and carbon monoxide. These data indicate that cysticerci use either aerobic or anaerobic pathways according to oxygen availability in the environment. Results from experiments of irreversible respiratory poisoning using cyanide suggest the presence of an alternative respiratory chain. Proteolytic action of trypsin on a fibrous layer surrounding the invaginated larvae is suggested by histological evidence.