RESUMO
To persist, a virus must co-exist with the host that it infects, thus allowing the virus to survive and to subvert the programmed cell death of the host. In this study, we investigated whether the intrinsic pathway of the apoptotic process is suppressed in a previously reported macrophage cell line persistently infected with respiratory syncytial virus (RSV). To this end, after using staurosporine to induce apoptosis, we determined cell viability and the degree of annexin staining and DNA fragmentation between infected and mock-infected cells. RSV persistence leads to a subversion of apoptosis; whereas in mock-infected macrophages, apoptosis was evident. The cellular apoptotic pathway involve was searched by determining the activities of caspases and the expression of anti-apoptotic proteins. Although caspases-3 and -9 were expressed, their activities were altered; the activity of caspase-3 was reduced and that of caspase-9 could not be detected. Expression of anti-apoptotic proteins Bcl-2, Bcl-X, and XIAP was enhanced, with Bcl-X and XIAP being regulated post-transcriptionally; the induction of the anti-apoptotic factors and the reduced caspases activities might account for the subversion of apoptosis. The data implies that in our viral persistence model an anti-apoptotic program is induced relating alterations of caspases-3 and -9 activity and expression of anti-apoptotic proteins, suggesting that the intrinsic pathway is suppressed. These findings are of importance for understanding the intracellular genes involved in subversion of apoptosis by RSV persistence in macrophages.
Assuntos
Apoptose , Evasão da Resposta Imune , Macrófagos/imunologia , Macrófagos/virologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/patogenicidade , Animais , Anexinas/análise , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Camundongos , Estaurosporina/toxicidade , Regulação para CimaRESUMO
BACKGROUND: In the last few years, the study of microparticles (MPs)--submicron vesicles released from cells upon activation or apoptosis--has gained growing interest in the field of inflammation and in infectious diseases. Their role in the human malaria parasite Plasmodium vivax remains unexplored. Because acute vivax malaria has been related to pro-inflammatory responses, the main hypothesis investigated in this study was that Plasmodium vivax infection is associated with elevated levels of circulating MPs, which may play a role during acute disease in non-immune patients. METHODS: Plasma MPs were analysed among thirty-seven uncomplicated P. vivax infections from an area of unstable malaria transmission in the Brazilian Amazon. The MP phenotype was analysed by flow cytometry using the classical MP marker, annexin, and fluorochrome-labeled monoclonal antibodies against specific cell surface markers. The frequencies of plasma MPs in P. vivax patients (n=37) were further compared to malaria-unexposed controls (n=15) and ovarian carcinoma patients (n=12), a known MPs-inducing disease non-related to malaria. RESULTS: The frequencies of plasma circulating MPs were markedly increased in P. vivax patients, as compared to healthy age-matched malaria-unexposed controls. Although platelets, erythrocytes and leukocytes were the main cellular sources of MPs during vivax malaria, platelet derived-MPs (PMPs) increased in a linear fashion with the presence of fever at the time of blood collection (ß=0.06, p<0.0001) and length of acute symptoms (ß=0.36, p<0.0001). Finally, the results suggest that plasma levels of PMPs diminish as patient experience more episodes of clinical malaria (ß=0.07, p<0.003). CONCLUSIONS: Abundant circulating MPs are present during acute P. vivax infection, and platelet derived-MPs may play a role on the acute inflammatory symptoms of malaria vivax.
Assuntos
Micropartículas Derivadas de Células/química , Malária Vivax/patologia , Plasma/química , Plasma/parasitologia , Adolescente , Adulto , Idoso , Anexinas/análise , Brasil , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of clusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.