RESUMO

A imunopatologia das doenças inflamatórias intestinais (DIIs) está associada a níveis aumentados de citocinas pró-inflamatórias, alterações na microbiota local e perda da integridade da barreira epitelial. De fato, a relação parasita-hospedeiro, definida aqui pela interação da microbiota com o microambiente da mucosa, tem sido relatada como fator central na imunopatogênese das DIIs. Neste contexto, o presente trabalho teve como objetivo avaliar o efeito da interação de Desulfovibrio indonesiensis, bactéria redutora de sulfato (BRS), com a linhagem de célula epitelial intestinal Caco-2, sobre a integridade das junções oclusivas e seu papel na modulação da produção de IL-8. Células Caco-2 foram incubadas (3h a 37ºC) com D. indonesiensis (proporção 10:1 bactéria–célula hospedeira), e a interação foi interrompida após 24h e 48h. Microscopia correlativa foi empregada para avaliar a integridade das junções celulares, sendo as culturas controle e as que interagiram com D. indonesiensis processadas para imunofluorescência indireta e microscopia eletrônica de varredura (MEV). Análises da expressão de ocludina, proteína de junção oclusiva, e permeabilidade paracelular foram realizadas por Western blot e resistência elétrica transepitelial (RET), respectivamente. Microscopia de contraste interferencial diferencial (DIC) e microscopia de fluorescência das culturas de células Caco-2 revelaram intensa coesão entre células da monocamada celular, com junções oclusivas visualizadas pela detecção de ocludina e ZO-1. A presença da BRS não acarretou mudanças nas junções celulares e na distribuição espacial de ocludina e ZO-1...

The immunopathology of inflammatory bowel diseases (IBD) is associated withincreased levels of pro-inflammatory cytokines, changes in local microbiota and loss of epithelial barrier integrity. In fact, the host-parasite relationship, here defined by the interaction of microbes with the mucosal microenvironment, has been reported as a central factor in IBD immunopathogenesis. Herein, we aimed to evaluate the interaction of Desulfovibrio indonesiensis, a sulfate reducing bacteria (SRB), with anintestinal epithelial cell strain, Caco-2, and its effect on the integrity of tight junctions and on IL- 8 production. Caco-2 cells were incubated (3h at 37°C) with D. indonesiensis (10:1 bacteria-host cell ratio), and the interaction stopped after 24h and 48h. Correlative microscopy was used to assess the integrity of cell junctions, and both control and D. indonesiensis interaction cultures were processed for indirectimmunofluorescence and scanning electron microscopy (SEM). Expression analysis of occludin, a tight junction protein, and paracellular permeability were performed by Western blot and transepithelial electrical resistance (TEER), respectively. Differential interference contrast (DIC) and fluorescence microscopy analyses ofCaco-2 monolayer cultures revealed intense cell cohesion, with tight junctions visualized by occludin and ZO-1 labeling. Cell junctions and spatial distribution of occludin and ZO-1 were not altered in the presence of BRS. Ultrastructural analysisshowed isolated bacteria and biofilm structures bound to Caco-2 cells without anyalteration in intercellular association. This inaltered junctional integrity was also seenby TEER assays, and Western blot results revealed no changes in occludinexpression...

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RESUMO

The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).

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