Instituto Nacional Of Ciencia E Tecnologia Em Biologia E Bioimagem

Brazil

Instituto Nacional Of Ciencia E Tecnologia Em Biologia E Bioimagem

Brazil
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Silva-Filho J.L.,Federal University of Rio de Janeiro | Souza M.C.,Institute Tecnologia em Farmacos | Henriques M.G.,Institute Tecnologia em Farmacos | Morrot A.,Federal University of Rio de Janeiro | And 5 more authors.
Archives of Biochemistry and Biophysics | Year: 2015

Abstract Angiotensin II (Ang II) plays an important role in the regulation of the T-cell response during inflammation. However, the cellular mechanisms underlying the regulation of lymphocytes under physiologic conditions have not yet been studied. Here, we tested the influence of Ang II on T-cell migration using T cells from BALB/c mice. The results obtained in vivo showed that when Ang II production or the AT1 receptor were blocked, T-cell counts were enhanced in blood but decreased in the spleen. The significance of these effects was confirmed by observing that these cells migrate, through fibronectin to Ang II via the AT1 receptor. We also observed a gradient of Ang II from peripheral blood to the spleen, which explains its chemotactic effect on this organ. The following cellular mechanisms were identified to mediate the Ang II effect: upregulation of the chemokine receptor CCR9; upregulation of the adhesion molecule CD62L; increased production of the chemokines CCL19 and CCL25 in the spleen. These results indicate that the higher levels of Ang II in the spleen and AT1 receptor activation contribute to migration of naive T cells to the spleen, which expands our understanding on how the Ang II/AT1 receptor axis contributes to adaptive immunity. © 2015 Elsevier Inc. All rights reserved.


Silva-Filho J.L.,Federal University of Rio de Janeiro | Souza M.C.,Institute Tecnologia em Farmacos | Henriques M.D.G.,Institute Tecnologia em Farmacos | Morrot A.,Federal University of Rio de Janeiro | And 6 more authors.
Molecular Immunology | Year: 2011

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT1 and AT2, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4+ and CD8+ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10-8M Ang II did not change any of the parameters evaluated. However, 10-6M losartan, an AT1 receptor antagonist: (i) reduced the percentage of CD25+ and CD69+ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10-6M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10-8M). None of these responses was modified by 10-7M PD123319, an AT2 antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT1 receptor activation. Moreover, AT1 receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT2 receptor expression did not change after T cell receptor triggering. © 2011 Elsevier Ltd.


Ribeiro M.C.,Federal University of Rio de Janeiro | Costa-Alves M.S.,Federal University of Rio de Janeiro | Wengert M.,Federal University of Rio de Janeiro | Meyer-Fernandes J.R.,Federal University of Rio de Janeiro | And 6 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2012

Background: The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes. Methods: We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-32P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation. Results: This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl2 was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN3) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN3. The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg- 1 x min- 1 and K0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia. Conclusion: Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia. General Significance: This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia. © 2012 Elsevier B.V.


Silva L.D.S.,Federal University of Rio de Janeiro | Peruchetti D.D.B.,Federal University of Rio de Janeiro | Silva C.T.F.D.,Instituto Oswaldo Cruz | Ferreira-DaSilva A.T.,Instituto Oswaldo Cruz | And 5 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2016

Background: The molecular mechanisms involved in erythrocyte invasion by malaria parasite are well understood, but the contribution of host components is not. We recently reported that Ang-(1-7) impairs the erythrocytic cycle of . P. . falciparum through Mas receptor-mediated reduction of protein kinase A (PKA) activity. The effects of bradykinin (BK), a peptide of the kallikrein-kinin system (KKS), can be potentiated by Ang-(1-7), or angiotensin-converting enzyme (ACE) inhibitors, such as captopril. We investigated the coordinated action between renin-angiotensin system (RAS) and KKS peptides in the erythrocyte invasion by . P. . falciparum. . . Methods: We used human erythrocytes infected with . P. . falciparum to assess the influence of RAS and KKS peptides in the invasion of new erythrocytes. Results: The inhibitory effects of Ang-(1-7) were mimicked by captopril. 10-8 M BK decreased new ring forms and this effect was sensitive to 10-8 M HOE-140 and 10-7 M A779, B2 and Mas receptor antagonists, respectively. However, DALBK, a B1 receptor blocker, had no effect. The inhibitory effect of Ang-(1-7) was reversed by HOE-140 and A779 at the same concentrations. Co-immunoprecipitation assay revealed an association between B2 and Mas receptors. BK also inhibited PKA activity, which was sensitive to both HOE-140 and A779. Conclusions: The results suggest that B2 and Mas receptors are mediators of Ang-(1-7) and BK inhibitory effects, through a cross-signaling pathway, possibly by the formation of a heterodimer. General significance: Our results describe new elements in host signaling that could be involved in parasite invasion during the erythrocyte cycle of . P. . falciparum. . . © 2016.


Portella V.G.,Federal University of Rio de Janeiro | Silva-Filho J.L.,Federal University of Rio de Janeiro | Landgraf S.S.,Federal University of Rio de Janeiro | De Rico T.B.,Federal University of Rio de Janeiro | And 10 more authors.
Critical Care Medicine | Year: 2013

Objective: It is well known that sepsis causes damage in different organs, including kidneys. However, few studies have been conducted on the magnitude of the long-term effects of sepsis on the surviving population, in particular, in relation to kidney disease. In this study, we examined the impact of long-term effects of sepsis on a second kidney insult. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Wild-type mice were subjected to the cecal ligation and puncture sepsis model. Control animals underwent identical laparotomy but without ligation and cecum puncture. On days 0, 7, and 14 after surgery, the ratio between urinary protein and creatinine was measured. Fifteen days after surgery, surviving mice were subjected to a second kidney insult through intraperitoneal injections of bovine serum albumin for 7 days. On day 22 after surgery, urinary protein and creatinine, γ-glutamyl transpeptidase, lactate dehydrogenase, histologic parameters, macrophage infiltration, apoptotic cell, renal and plasmatic cytokines were determined. Measurements and Main Results: On days 7 and 14 after surgery, the urinary protein and creatinine observed in the septic animal group were higher than those observed in the control group. On day 22 after surgery, sepsis-surviving animals that were subjected to a second kidney insult showed more severe tubular injury compared with controls. This process seems to involve an immunosuppressive state because the concentrations of some renal cytokines, such as tumor necrosis factor-α, interleukin 6, interferon-γ and chemokine ligand 2, were decreased and leukocyte numbers were increased. Conclusions: These results suggest that sepsis induces long-term effects in kidney structure aggravating tubule damage in a second kidney insult. © 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins.

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