Evaluation of Escherichia coli STb toxin in an in vitro organ culture model (IVOC) / Avaliação de toxina STb de Escherichia coli em modelo de cultura de órgãos in vitro (IVOC)

ETEC pathogenesis comprises adhesion to the small intestine, colonization and enterotoxin production. STb is one of the heat-stable toxins produced by ETEC. Previous reports, using culture supernatants of STb-positive ETEC strains, indicated loss of villous absorptive cells causing mild atrophy and microscopic alterations in jejunum mucosa. These culture supernatants contained many other compounds beside STb toxin and in addition the quantity of toxin in these supernatants was not determined. Thus, this study was undertaken to evaluate the effect of pure STb toxin on piglet jejunum explants in an in vitro organ culture (IVOC) model. Tissues of piglets of 11 weeks were used and put into culture. Morphometric analysis of tissues revealed that villous epithelial area was reduced in tissues treated with pure STb after 3, 4 and 5 hours (p 0.0001) compared to untreated tissues. STb-treated tissues presented atrophic villi due to loss of villi tip. Our data shows that, in piglet jejunum explants, pure STb toxin can lead to cell modification and consequently to destruction, seen as villi atrophy. These changes result in a reduced absorptive area and could be partly responsible for the diarrhea observed in the animal following STb intoxication.

EX VIVO MODEL OF RABBIT INTESTINAL EPITHELIUM APPLIED TO THE STUDY OF COLONIZATION BY ENTEROAGGREGATIVE ESCHERICHIA COLI

ABSTRACT BACKGROUND The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of “stacked bricks” on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

U0126 Attenuates Cerebral Vasoconstriction and Improves Long-Term Neurologic Outcome after Stroke in Female Rats

Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.