RESUMO
Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.
Assuntos
Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Brasil , Células Cultivadas , Meios de Cultura/química , Citocinas/análise , Fatores de Transcrição Forkhead/análise , Voluntários Saudáveis , Humanos , Subunidade alfa de Receptor de Interleucina-2/análise , Leucócitos Mononucleares/química , Receptor de Morte Celular Programada 1/análise , Subpopulações de Linfócitos T/química , Adulto JovemRESUMO
The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.
Assuntos
Trifosfato de Adenosina/metabolismo , Canais Iônicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/administração & dosagem , Animais , Células Cultivadas , Citometria de Fluxo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Receptores Purinérgicos P2X7 , Transdução de SinaisRESUMO
SUMMARY: In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.