5-HT3A serotonin receptor in the gastrointestinal tract: the link between immune system and enteric nervous system in the digestive form of Chagas disease.

Chagas disease is caused by Trypanosoma cruzi and remains one of the most neglected diseases in Latin America. One of its clinical forms is Chagas megacolon. Despite being known for more than half a century, detailed causes are still obscure. Recent evidence indicates a close relationship between the immune system and the enteric nervous system in the etiology of chagasic megacolon pathology. It is believed that low expression of the 5-HT3A serotonin receptor on lymphocytes could be linked to megacolon development. To test this hypothesis, this work investigated the distribution of CD4, CD8, and CD20 lymphocytes and their 5-HT3A receptor expression. The results demonstrated that Chagas patients without megacolon present a higher expression of the 5-HT3A receptor in all analyzed lymphocytes compared with Chagas patients with megacolon. These data suggest that the high expression of this receptor may lead to immunomodulation and prevent the development of Chagas megacolon.

Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization.

Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of ß-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

Comparison of parasitological, immunological and molecular methods for evaluation of fecal samples of immunosuppressed rats experimentally infected with Strongyloides venezuelensis.

Definitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

Entamoeba histolytica: gene expression analysis of cells invading tissues.

Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.

Entamoeba histolytica: cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels.

Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis.

Glial fibrillary acidic protein and S-100 colocalization in the enteroglial cells in dilated and nondilated portions of colon from chagasic patients.

After acute immunoreactive infestation with the Chagas' disease parasite, Trypanosoma cruzi, some patients develop chronic megacolon, whereas others remain asymptomatic. Chronic chagasic patients with gastrointestinal involvement exhibit inflammation and degeneration of enteric neurons. Our hypothesis is that enteric glial cells may be involved in the modulation of enteric inflammatory responses or even control the colon's dilatation. The aims of this study were to characterize the phenotype of enteric glial cells according to the expression of S-100 and glial fibrillary acidic protein and to look for correlation between these data and the neuronal loss in the colon of chagasic patients. We studied both dilated and nondilated portions of chagasic megacolon. We used a pan-enteric glial cell marker (anti-S-100), a subpopulation enteric glial cell marker (anti-glial fibrillary acidic protein), and a pan-neuronal marker (anti-Human protein C and protein D) with double-labeled sheets using a confocal microscope. Our results demonstrate that neuronal loss is similar in dilated and nondilated portions of chagasic megacolon. Moreover, the results indicate that neuronal destruction present in chagasic megacolon is preceded by glial component loss. The nondilated portion of chagasic megacolon exhibited increased expression of glial fibrillary acidic protein comparable with the dilated portion and also to the noninfected group. Our results suggest that glial fibrillary acidic protein enteric glial cells prevent dilatation of the organ and protect the enteric nervous system against the inflammatory process and neuronal destruction, preventing the destruction from expanding to unaffected areas of the colon.

Substance P and NK1 receptor expression in the enteric nervous system is related to the development of chagasic megacolon.

Chagasic megacolon is one of the most important forms of Chagas disease. This form is characterized by inflammation, neuronal destruction and organ dilatation. The aim of this study is to characterize the expression of substance P and its main receptor, NK1 receptor, in dilated and non-dilated samples of colon from chagasic patients with megacolon. Our results demonstrate that dilated portions of colon present high levels of substance P and low levels of NK1 receptor, whereas non-dilated portions and samples from non-infected individuals present low levels of substance P and high levels of NK1 receptor. We believe that this may indicate a neuro-immune relationship that occurs in Chagas disease.

Glycogen as a carbohydrate energy reserve in trophozoites of Giardia lamblia.

Although there is indirect evidence to suggest that glycogen is present in G. lamblia, to date it has not been purified and identified from this organism. In this study, a high molecular weight carbohydrate was purified and characterized and its physiological role as an energetic reserve was established. The monosaccharide constituents of the carbohydrate reserve were identified as glucose by two independent methods: thin layer chromatography and an enzymatic assay. The degree of branching of the molecule was evaluated by comparing its absorbance spectrum in the presence of lugol with spectra of standard solutions of glycogen and starch under the same conditions. The results strongly suggest that glycogen is present in G. lamblia and acts as an energy reserve in trophozoites of this organism.