RESUMO
Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.
Assuntos
Citocinas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aotus trivirgatus , Sequência de Bases , Citocinas/genética , Primers do DNA , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.
Assuntos
Linfócitos B/imunologia , Integrinas/análise , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/análise , Células Produtoras de Anticorpos/fisiologia , Linfócitos B/química , Diarreia/imunologia , Humanos , Imunofenotipagem , Lactente , Integrinas/fisiologia , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/análiseRESUMO
Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.