Relation between mast cells concentration and serotonin expression in chagasic megacolon development.

Chagas' disease is still reaching about 10 million people in the world. In South America, one of the most severe forms of this disease is the megacolon, characterized by severe constipation, dilated sigmoid colon and rectum and severe malnutrition. Previous data suggested that mast cells and serotonin (5-hydroxytryptamine [5-HT]) expression could be involved in intestinal homeostasis control, avoiding the chagasic megacolon development. The aim at this study was to characterize the presence of mast cells and expression of serotonin in chagasic patients with and without megacolon and evaluate the relation between mast cells, serotonin and megacolon development. Our results demonstrated that patients without megacolon feature a large amount of serotonin and few mast cells, while patients with megacolon feature low serotonin expression and a lot of mast cells. We believe that serotonin may be involved in the inflammatory process control, triggered by mast cells, and the presence of this substance in large quantities of the intestine could represent a mechanism of megacolon prevention.

Interstitial cells of Cajal: crucial for the development of megacolon in human Chagas' disease?

AIM: Megacolon, chronic dilation of a colonic segment,is accompanied by extensive myenteric neuron loss. However, this fails to explain unequivocally the formation of megacolon. We aimed to study further enteric structures that are directly or indirectly involved in colonic motility. METHOD: From surgically removed megacolon segments of seven Chagasic patients, three sets of cryosections from oral, megacolonic and anal zones were immunohistochemically quadruple-stained for smooth-muscle actin (SMA), synaptophysin (SYN, for nerve fibres), S100 (glia) and c-Kit (interstitial cells of Cajal, ICCs). Values of area measurements were related to the appropriate muscle layer areas and these proportions were compared with those of seven non-Chagasic control patients. RESULTS: Whereas nerve and glia profile proportions did not mirror unequivocally the changes of Chagasic colon calibre (nondilation/dilation/nondilation), the proportions of SMA (i.e. muscle tissue density) and c-Kit (i.e. ICC density) did so: they decreased from the oral to the megacolonic segment but increased to the anal zones (muscle tissue density: control 68.3%, oral 54.3%, mega 42.1%, anal 47.6%; ICC-density: control 1.8%, oral 1.1%, mega 0.4, anal 0.8%). CONCLUSION: Of the parameters evaluated, muscle tissue and ICC densities may be involved in the formation of Chagasic megacolon, although the mechanism of destruction cannot be deduced.

Neuronal plasticity of the enteric nervous system is correlated with chagasic megacolon development.

Chagas' disease is one of the few functional gastrointestinal disorders for which a causative agent has been identified. However, some pathological aspects of the chagasic megasyndromes are still incompletely understood. Chagasic megacolon is characterized by an inflammatory process, organ dilatation and neuronal reduction in both plexuses of the enteric nervous system (ENS). Although some studies on the ENS in Chagas' disease have been performed, the process of neuronal destruction and neuronal regeneration still remains unclear. Our hypothesis is that the regeneration process of the ENS may be involved with the mechanisms that prevent or retard organ dilatation and chagasic megacolon development. For that reason, we evaluated the neuronal regeneration with the marker GAP-43 in the colon's neuronal plexuses from chagasic patients with megacolon, and from non-infected individuals. Visual examination and quantitative analysis revealed an increased neuronal regeneration process in the dilated portion from chagasic patients when compared with the non-dilated portion and with non-infected individuals. We believe that this increased regeneration can be interpreted as an accentuated neuronal plasticity that may be a response of the ENS to avoid megacolon propagation to the entire organ and maintain the colon functional innervation.

Neurochemical coding of the enteric nervous system in chagasic patients with megacolon.

Neuronal destruction has been considered the hallmark of pathogenic mechanisms in chagasic megacolon. Characterization of neuropeptides in the enteric nervous system from chagasic patients with megacolon could elucidate some aspects of the development of this syndrome. In the present work we demonstrate the changes in expression of neuropeptides and neurochemical markers present in neuronal plexuses from the colons of chagasic patients with megacolon. Sections of frozen tissue samples were immunohistochemically labeled for anticalretinin, cChaT, substance P, VIP, NOS, and NPY. Immunoreactivity was observed using a confocal microscope. Our results demonstrate that in chagasic patients with megacolon, inhibitory motor neurons (VIP and NOS immunoreactive) are preferentially destroyed by Trypanosoma cruzi and/or the inflammatory process. These results suggest a selective destruction of enteric neurons in the colon of chagasic patients with megacolon, pointing to an important discovery in the mechanism of pathogenesis of Chagas' disease.

Morphometric study of eosinophils, mast cells, macrophages and fibrosis in the colon of chronic chagasic patients with and without megacolon.

The mechanisms involved in the pathogenesis of chagasic megacolon are not completely characterized. Although autoimmunity may play a role in the pathogenesis of Chagas' disease, recent studies suggest a positive association of tissue parasitism, inflammation, and severity of lesions. The aim of this study was to evaluate the role of inflammatory cells and the occurrence of fibrosis in the colon of chagasic patients with and without megacolon. Samples from 26 patients were randomly selected and paraffin-embedded tissue blocks were sectioned and evaluated by histology and immunohistochemistry to analyse the occurrence and relation among eosinophils, mast cells, macrophages and fibrosis. Section analyses showed that the presence of eosinophils and mast cells in the analysed inflammatory cells has a direct correlation with fibrosis density in the chagasic megacolon. These data suggest that the megacolon's pathogenesis is based on a continuous process of cell damage. Our data propose that eosinophils, mast cells and macrophages may have a direct connection with the occurrence of fibrosis in the colon of chagasic patients. We believe that potential therapeutic agents against these cells could avoid the fibrosis process and contribute to prevent the development of chagasic megacolon.

Evaluation of antimicrobial activity in Paenibacillus spp. strains isolated from natural environment.

AIMS: Paenibacillus isolates were selected to test antimicrobial activity against bacteria, filamentous fungi and yeasts isolates, with the purpose of finding new bacterium species for microbiological control. METHODS AND RESULTS: Fifty-five strains belonging to 15 species of Paenibacillus were inoculated on trypticase soya agar, potato dextrose agar and sabouraud agar plates in order to evaluate their antimicrobial activity against 16 indicator bacteria, 14 filamentous fungi and six yeasts isolates, both reference and field strains. After these screening, culture supernatant of 17 isolates was prepared. Twenty-five Paenibacillus isolates presented antimicrobial activity, where seven species (Paenibacillus chibensis; P. koreensis; P. illinoiensis; P. validu; P. pabuli; P. brasilensis and P. peoriae) stood out inhibiting at least 13 of the 16 indicator bacteria. Only 14 of the 55 isolates exhibited antifungal activity. P. peoriae inhibited 13 among the 14 filamentous fungi and all yeasts indicator strains. Fourteen isolates produced culture supernatant with antimicrobial activity. CONCLUSIONS: Among the 55 isolates analysed, 25 exhibited a broad inhibition spectrum against bacteria and pathogenic fungi. P. validus, P. chibensis, P. koreensis and P. peoriae isolates proved to be the subject matter for studies on the production of antimicrobial agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study revealed other two species with antimicrobial activity: P. validus and P. chibensis, and it contributed to enhance Paenibacillus biocontrolling potential, proving that it exhibit a broad action spectrum.

Comparative study of the presence of Trypanosoma cruzi kDNA, inflammation and denervation in chagasic patients with and without megaesophagus.

Neuronal lesions have been considered the hallmark of chagasic megaesophagus, but the role of Trypanosoma cruzi and the participation of the inflammatory cells in this process are still debated. In the present study we counted neurons in the oesophagus from patients with and without megaesophagus and further examined these samples for the presence of parasite kDNA and cells with cytolytic potential (Natural Killer cells, cytotoxic lymphocytes and macrophages). The presence of parasite kDNA was demonstrated in 100% of cases with megaesophagus and in 60% of patients without megaesophagus. When analysed for the number of neurons, the patients without megaesophagus could be classified into 2 groups, as having normal or a decreased number of neurons. The former group did not show any inflammatory process, but interestingly, all patients without megaesophagus presenting decreased number of neurons also presented both parasite kDNA and inflammatory process in the organ. We further observed that the numbers of cytotoxic cells in the myenteric plexus region inversely correlate with the number of neurons. These data together strongly suggest that chronic lesions in chagasic megaesophagus might be a consequence of immune-mediated mechanisms, that last until the chronic phase of infection, and are dependent on the persistence of parasite in the host's tissue.

Smear layer influence on the apical seal of four different obturation techniques.

The purpose of this study was the in vitro evaluation of four techniques for the obturation of the root canal system in the presence or absence of a smear layer. Ninety-six human upper central incisors were instrumented using the pressureless crown-down technique and irrigated with 5.25% NaOCl. The teeth that had the smear layer removed were irrigated with this solution in combination with 17% EDTA. The teeth were obturated with lateral condensation with an accessory or standardized cone as the main cone, with vertical condensation of warm gutta-percha or with thermoplasticized injectable gutta-percha. Apical leakage was assessed by measuring the linear penetration of methylene blue dye with a stereomicroscope. The results showed no significant differences in the degree of leakage with and without the smear layer when the samples were considered as a whole. However, when the groups were assessed separately, teeth in the lateral condensation with an accessory main cone group and teeth in the thermoplasticized group leaked less with a smear layer present. In contrast teeth with lateral condensation and a standardized main cone leaked more with a smear layer present. In the vertical condensation groups there was no difference attributable to the smear layer.