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ABSTRACT: Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein β-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix.
Microbes and Infection 11/2011; 14(3):290-300. · 3.10 Impact Factor
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Elias Leiva-Salcedo,
Claudio Coddou, Felipe E Rodríguez,
Antonello Penna,
Ximena Lopez,
Tanya Neira,
Ricardo Fernández,
Mónica Imarai,
Miguel Rios,
Jorge Escobar,
Margarita Montoya,
J Pablo Huidobro-Toro,
Alejandro Escobar,
Claudio Acuña-Castillo
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ABSTRACT: The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.
Mediators of Inflammation 01/2011; 2011:152625. · 3.26 Impact Factor
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Jorge Escobar,
Lorena Varela-Nallar,
Claudio Coddou,
Pablo Nelson,
Kevin Maisey,
Daniel Valdés,
Alexis Aspee,
Victoria Espinosa,
Carlos Rozas,
Margarita Montoya,
Cristian Mandiola, Felipe E Rodríguez,
Claudio Acuña-Castillo,
Alejandro Escobar,
Ricardo Fernández,
Hernán Diaz,
Mario Sandoval,
Mónica Imarai,
Miguel Rios
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ABSTRACT: Arsenic has been associated with multiple harmful effects at the cellular level. Indirectly these defects could be related to impairment of the integrity of the immune system, in particular in lymphoid population. To characterize the effect of Arsenic on redox status on this population, copper smelter workers and arsenic unexposed donors were recruited for this study. We analyzed urine samples and lymphocyte enriched fractions from donors to determinate arsenic levels and lymphocyte proliferation. Moreover, we studied the presence of oxidative markers MDA, vitamin E and SOD activity in donor plasma. Here we demonstrated that in human beings exposed to high arsenic concentrations, lymphocyte MDA and arsenic urinary levels showed a positive correlation with SOD activity, and a negative correlation with vitamin E serum levels. Strikingly, lymphocytes from the arsenic exposed population respond to a polyclonal stimulator, phytohemaglutinin, with higher rates of thymidine incorporation than lymphocytes of a control population. As well, similar in vitro responses to arsenic were observed using a T cell line. Our results suggest that chronic human exposure to arsenic induces oxidative damage in lymphocytes and could be considered more relevant than evaluation of T cell surveillance.
Mediators of Inflammation 01/2010; 2010:403830. · 3.26 Impact Factor
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ABSTRACT: As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X(4) receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X(4) receptor. Application of 0.1-10 microM alfaxolone potentiated within 60-s the 1 microM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3-10 microM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the tau(off) of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17beta-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X(4) receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X(4) receptor more than 10-fold by 10 microM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.
Journal of Neurochemistry 06/2009; 110(2):734-44. · 4.06 Impact Factor
