Claudia Mara Lara Melo Coutinho

Overexpression of ATP-activated P2X7 Receptors in the Intestinal Mucosa Is Implicated in the Pathogenesis of Crohnʼs Disease

Background:Extracellular nucleotides released in conditions of cell stress alert the immune system from tissue injury or inflammation. We hypothesized that the P2X7 receptor (P2X7-R) could regulate key elements in inflammatory bowel disease pathogenesis. Methods:Colonoscopy samples obtained from patients with Crohns disease (CD), ulcerative colitis, and controls were used to analyze P2X7-R expression by RT and real-time PCR, immunohistochemistry, and confocal microscopy. Inflammatory response was determined by the levels of cytokines by enzyme-linked immunosorbent assay in cultures of intestinal explants. Apoptosis was determined by the TUNEL assay. P2X7-R−/− C57BL/6 mice were treated with trinitrobenzene sulfonic acid or dextran sulfate sodium (DSS) for inducing colitis. Results:P2X7-R was expressed in higher levels in inflamed CD epithelium and lamina propria, where it colocalizes more with dendritic cells and macrophages. Basal levels of P2X7-R mRNA were higher in CD inflamed mucosa compared with noninflamed CD and controls and were upregulated after interferon-&ggr; in controls. Apoptotic rates were higher in CD epithelium and lamina propria compared with ulcerative colitis and controls. Levels of tumor necrosis factor-&agr;, interleukin (IL)-1&bgr;, and IL-17 were higher, whereas IL-10 was lower in CD compared with controls. Levels of tumor necrosis factor-&agr;-&agr; and interleukin-1&bgr; increased after adenosine-triphosphate and decreased after KN62 treatment in CD. P2X7-R−/− animals did not develop trinitrobenzene sulfonic acid or DSS colitis. Conclusions:The upregulation of P2X7-R in CD inflamed mucosa is consistent with the involvement of purinoceptors in inflammation and apoptosis. These observations may implicate purinergic signaling in the pathogenesis of intestinal inflammation, and the P2X7-R may represent a novel therapeutic target in CD.

Evidence for a perforin-mediated mechanism controlling cardiac inflammation in Trypanosoma cruzi infection

Summary.  CD8+ T lymphocytes are considered an important cell population involved in the control of parasitaemia and mortality after Trypanosoma cruzi infection. However, despite recent developments in this field, the mechanism whereby this control is exerted is still not completely understood. Here we have used perforin knockout (–/–) mice infected with Y strain T. cruzi in order to evaluate specifically the participation of the perforin‐based cytotoxic pathway in the destruction of cardiomyocytes, cellular inflammatory infiltration, and control of parasitaemia and mortality. We observed that although parasitaemia was equivalent in perforin (+/+) and (–/–) groups, survival rate and spontaneous physical performance were significantly lower in the perforin deficient mice. The cardiac inflammatory cell infiltration, mostly composed of CD8+ cells, was more evident in perforin (–/–) mice. Ultrastructural and immunofluorescence analysis, as well as plasma creatine kinase activity, revealed cardiomyocyte damage and necrosis, more evident in perforin (–/–) mice. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays performed in heart samples revealed similar and modest levels of apoptosis in both perforin (+/+) and (–/–) mice. These results indicate that perforin does not play a pivotal role in the control of parasitaemia and direct lysis of cardiomyocytes, but seems to be an important molecule involved in the control of cardiac inflammation and pathology induced by a highly virulent strain of T. cruzi.

Extracellular ATP induces cell death in human intestinal epithelial cells

BACKGROUND Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP. METHODS We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique. RESULTS ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells. GENERAL SIGNIFICANCE The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.

Scanning electron microscope study of biofilm formation at different flow rates over metal surfaces using sulphate‐reducing bacteria

Sulphate‐reducing bacteria (SRB) are implicated in biocorrosion in the oil industry. SRB biofilm formation was studied on metal coupons under a static system (SS) and a dynamic system (DS), using mixed populations of bacteria recovered from samplers installed in the water injection systems of offshore platforms on Brazils Atlantic coast. Adherent biofilms consisting of rod, vibrio, filamentous, spiral and coccoid‐shaped bacteria enmeshed in an extracellular polysaccharide matrix (EPM) formed over the coupon surfaces. Coupons subjected to SS, showed significant differences in the kinetics of biofilm formation, the nature of the initial adhesion process and the amount of EPM produced, compared with DS. After 4 h incubation, a reversible attraction of a large number of bacteria was observed under SS. However, by 24 h, true attachment of bacteria to the substrate had begun in both systems. Using DS, many more EPM elements were observed at given points in time. Biofilm maturation under DS showed a 6 days’ lea...

Sulphate-reducing bacteria associated with biocorrosion: a review

Biocorrosion means any process of corrosion in wich microorganisms are somehow involved. As far as the petroleum industry is concerned, the anaerobic type is the more important, with Sulphate-Reducing Bacteria (SRB) accouting for half of the described processes. SRB are obligate anaerobs that use sulphur, sulphate or other oxidized sulphur compounds as oxidizing agents when decomposing organic material. A typical product of SRB metabolism, hydrogen sulphide -H2S-, is extremely toxic. In the present work we review the literature on mechanisms underlying biocorrosive process in wich SRB are involved and summarize some of the ultrastructural and eletrochemical work developed using SRB obtained from water injection flow in wells located on PETROBRAS offshore marine plataforms, sampled directly in the field over metallic probes, or cultured under laboratory conditions. Biofilms develop when SRB adhere to inert surfaces. A high diversity of morphological types is found inside these biofilms. Their extracellular matrix is highly hydrated and mainly anionic, as shown by its avid reaction with cationic compounds like ruthenium red. We have noted that variations in iron contet lead to interesting changes in the ultrastructure of the bacterial cell coat and also in the rate of corrosion induced in metallic test cupons. Since routine methods to prevent and treat SRB contamination and biodeterioration involve the use of biocides that are toxic and always have some environmental impact, an accurate diagnosis of biocorrosion is always required prior to a treatment decision. We developed a method that detects and semi-quantifies the presence of living or dead SRB by using free silver potentials as an indicator of corrosive action by SRB-associated sulphides. We found a correlation between sulphide levels (determined either by spectrophotometry, or using a silver electrode -E(Ag)- that measured changes in free potentials induced by the presence of exogeneously added sulphide) and SRB concentration (enumerated by a culturing method). E (Ag) was characterized under a variety of conditions andwas found to be relatively immune to possible interference resulting from aeration of media or from the psence of iron corrosion products. The method offers a simple, rapid, and effective means of diagnosing biocorrosive processes prior to their control.

P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells

P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RBlow. Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

Sulfate-reducing bacteria stimulate gut immune responses and contribute to inflammation in experimental colitis

ABSTRACT The intestinal microbiota is critical for mammalian immune system development and homeostasis. Sulfate‐reducing bacteria (SRB) are part of the normal gut microbiota, but their increased levels may contribute to colitis development, likely in association with hydrogen sulfide (H2S) production. Here, we investigated the effects of SRB in the gut immune response in germ‐free mice, and in experimental colitis. After 7 days of colonization with Desulfovibrio indonesiensis or with a human SRB consortium (from patients with colitis), germ‐free mice exhibited alterations in the colonic architecture, with increased cell infiltration in the lamina propria. SRB colonization upregulated the Th17 and Treg profiles of cytokine production/cell activation, in T cells from mesenteric lymph nodes. These alterations were more pronounced in mice colonized with the human SRB consortium, although D. indonesiensis colonization produced higher levels of H2S. Importantly, the colon of C57BL/6 mice with colitis induced by TNBS or oxazolone had increased SRB colonization, and the administration of D. indonesiensis to mice with TNBS‐induced colitis clearly exacerbated the alterations in colonic architecture observed in the established disease, and also increased mouse weight loss. We conclude that SRB contribute to immune response activation in the gut and play an important role in colitis development.

Periodate‐oxidized ATP modulates macrophage functions during infection with Leishmania amazonensis

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate‐oxidized ATP (oATP) on parasite control in Leishmania‐infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor‐independent manner, as macrophages from P2X7−/− mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP‐treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning.

Construção de espaços de escuta, diagnóstico e análise coletiva de problemas de saúde pública com a linguagem teatral: o caso das oficinas de jogos teatrais sobre a dengue

As caracteristicas historicas, sociais e ecologicas da dengue tem motivado acoes de educacao em saude visando medidas preventivas. Acoes educativas e comunitarias que partam do contexto da populacao envolvida tem sido apontadas como cruciais. No presente estudo, investigou-se a utilizacao da linguagem teatral como estrategia para caracterizar as concepcoes de educadores envolvidos na prevencao da dengue, por meio de Oficinas de Jogos Teatrais. A linguagem teatral foi escolhida por ser dialogica, estabelecer relacao com a inventividade da ciencia, estimular a colaboracao e provocar a acao espontânea dos participantes. A analise dos resultados evidenciou o relato dos educadores sobre a sensacao de isolamento profissional bem como a desconfianca quanto a credibilidade do poder executivo. Concluimos que e possivel utilizar experiencias teatrais para organizar espacos propicios a analise coletiva de situacoes ligadas a problemas de saude publica, estimulando acoes cooperativas por parte dos educadores.