HYPOXIC STRESS, HEPATOCYTES AND CACO-2 VIABILITY AND SUSCEPTIBILITY TO Shigella flexneri INVASION

SUMMARY Inflammation due to Shigella flexneri can cause damage to the colonic mucosa and cell death by necrosis and apoptosis. This bacteria can reach the bloodstream in this way, and the liver through portal veins. Hypoxia is a condition present in many human diseases, and it may induce bacterial translocation from intestinal lumen. We studied the ability of S. flexneri to invade rat hepatocytes and Caco-2 cells both in normoxic and hypoxic microenvironments, as well as morphological and physiological alterations in these cells after infection under hypoxia. We used the primary culture of rat hepatocytes as a model of study. We analyzed the following parameters in normoxic and hypoxic conditions: morphology, cell viability, bacterial recovery and lactate dehydrogenase (LDH) released. The results showed that there were fewer bacteria within the Caco-2 cells than in hepatocytes in normoxic and hypoxic conditions. We observed that the higher the multiplicity of infection (MOI) the greater the bacterial recovery in hepatocytes. The hypoxic condition decreased the bacterial recovery in hepatocytes. The cytotoxicity evaluated by LDH released by cells was significantly higher in cells submitted to hypoxia than normoxia. Caco-2 cells in normoxia released 63% more LDH than hepatocytes. LDH increased 164% when hepatocytes were submitted to hypoxia and just 21% when Caco-2 cells were in the same condition. The apoptosis evaluated by Tunel was significantly higher in cells submitted to hypoxia than normoxia. When comparing hypoxic cells, we obtained more apoptotic hepatocytes than apoptotic Caco-2 cells. Concluding our results contribute to a better knowledge of interactions between studied cells and Shigella flexneri. These data may be useful in the future to define strategies to combat this virulent pathogen.RESUMO A inflamação causada por Shigella flexneri pode causar danos à mucosa do cólon e morte celular por necrose e apoptose. Esta bactéria pode atingir a corrente sanguínea por esta via e o fígado através da veia porta. A hipóxia é uma condição presente em muitas doenças humanas, podendo induzir a translocação bacteriana a partir do lúmen intestinal. Nós estudamos a capacidade de S. flexneri invadir hepatócitos de rato e células Caco-2 nos microambientes de normóxia e hipóxia, bem como as alterações morfológicas e fisiológicas dessas células após a infecção sob hipóxia. Utilizamos a cultura primária de hepatócitos de ratos como modelo de estudo. Nós analisamos os seguintes parâmetros em condições de normóxia e hipóxia: morfologia, viabilidade celular, recuperação bacteriana e liberação de lactato desidrogenase (LDH). Os resultados mostraram menor quantidade de bactérias dentro das células Caco-2 do que em hepatócitos em condições de normóxia e hipóxia. Nós observamos que quanto maior foi a multiplicidade de infecção (MOI), maior também foi a recuperação bacteriana em hepatócitos. A condição hipóxica foi capaz de diminuir a recuperação de bactérias dos hepatócitos. A citotoxicidade avaliada pela liberação de LDH foi significativamente maior em células submetidas à hipóxia do que normóxia. As células Caco-2 em normóxia produziram 63% mais LDH do que os hepatócitos. O LDH aumentou 164% quando os hepatócitos foram submetidos à hipoxia e apenas 21% quando as células Caco-2 estavam na mesma condição. A apoptose avaliada por TUNEL foi significativamente maior em células submetidas à hipóxia que normóxia. Quando comparamos células hipóxicas houve mais apoptose entre hepatócitos do que nas células Caco-2. Concluindo, nossos resultados contribuem para um melhor conhecimento das interações entre as células estudadas e S. flexneri. Estes dados podem ser úteis no futuro, para definir estratégias de combate a este patógeno virulento

A Novel Organotellurium Compound (RT-01) as a New Antileishmanial Agent

Leishmaniasis is a neglected disease and endemic in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate the effect of RT-01, an organotellurane compound presenting biological activities, in 2 experimental systems against Leishmania amazonensis. The in vitro system consisted of promastigotes and amastigotes forms of the parasite, and the in vivo system consisted of L. amazonensis infected BALB/c mice, an extremely susceptible mouse strain. The compound proved to be toxic against promastigotes and amastigotes. The study also showed that treatment with RT-01 produces an effect similar to that treatment with the reference antimonial drug, Glucantime, in L. amazonensis infected mice. The best results were obtained following RT-01 intralesional administration (720 mu g/kg/day); mice showed significant delay in the development of cutaneous lesions and decreased numbers of parasites obtained from the lesions. Significant differences in tissue pathology consisted mainly of no expressive accumulation of inflammatory cells and well-preserved structures in the skin tissue of RT-01-treated mice compared with expressive infiltration of infected cells replacing the skin tissue in lesions of untreated mice. These findings highlight the fact that the apparent potency of organotellurane compounds, together with their relatively simple structure, may represent a new avenue for the development of novel drugs to combat parasitic diseases.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES