RESUMO
OBJECTIVE: Signal transducers and activators of transcription (STATs) are crucial mediators of cytokine signalling. Constitutive activation of STATs, especially STAT3, has been reported in several diseases. Primary Sjögren's syndrome (pSS) is associated with overproduction of cytokines such as interleukin-10 (IL-10), although the mechanism by which this occurs is unknown. As STAT3 is a potent inducer of IL-10, this study focused on determining the pattern of STAT3 activation in peripheral lymphocytes from patients with pSS. METHODS: Twelve pSS patients and 12 healthy age-matched control subjects were studied. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation. Phosphorylated STAT3 (pSTAT3) and also STAT3 expression were determined by flow cytometry in gated CD3(+ )and CD19(+) lymphocytes. Similarly, pJak1 and pTyk2 were also determined in gated CD3(+) lymphocytes. RESULTS: Although the protein expression of STAT3 was similar among controls and pSS patients, we found that STAT3 was constitutively activated in CD3(+) lymphocytes from pSS patients. Neither Jak1 nor Tyk2 (the upstream activators of STAT3) was activated in pSS CD3(+) lymphocytes, suggesting that the constitutive activation of STAT3 observed in pSS patients might not depend on cytokine stimulation but instead might be the result of an abnormal inactivation of pSTAT3. CONCLUSIONS: These data provide evidence of abnormal STAT3 signalling in T cells from pSS patients.
Assuntos
Complexo CD3/análise , Fator de Transcrição STAT3/metabolismo , Síndrome de Sjogren/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Antígenos CD/análise , Linfócitos B/imunologia , Feminino , Humanos , Interleucina-10/análise , Janus Quinase 1/metabolismo , Ativação Linfocitária , Pessoa de Meia-Idade , Fosforilação , Valores de Referência , Fator de Transcrição STAT3/imunologiaRESUMO
OBJECTIVE: To assess the expression and function of the receptor for interleukin-10 (IL-10R) in immune cells from patients with systemic lupus erythematosus (SLE). METHODS: We assessed the expression and function of IL-10R in peripheral blood mononuclear cells (PBMCs) from 19 SLE patients and 15 healthy controls. The expression of IL-10R was assessed by flow cytometry, and the function of this receptor was determined by analysing both the activation of Jak-1, Tyk-2, Stat-1, and Stat-3 (Western blot) and the induction of gene expression (cDNA array test of 242 genes of cytokines, apoptosis and intracellular signalling) upon stimulation with IL-10. RESULTS: We found similar levels of IL-10R expression in SLE patients and controls. In addition, variable levels of Jak-1, Tyk-2, Stat-1, and Stat-3 activation were induced by IL-10 in PBMCs from SLE patients and controls, with no significant differences in protein phosphorylation or kinetics of activation. However, clear-cut differences in the gene expression induced through IL-10R were observed in SLE patients and controls, mainly in the genes involved in apoptosis and those encoding for cytokines and their receptors. CONCLUSIONS: Our data suggest that despite normal levels of IL-10R expression, and an apparent lack of abnormalities in the intracellular signals induced through this receptor, immune cells from SLE patients exhibit an aberrant pattern of gene expression induced through the IL-10R.
Assuntos
Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Receptores de Interleucina-10/metabolismo , Adolescente , Adulto , DNA/genética , Feminino , Regulação da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Interleucina-10/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/genética , TYK2 Quinase/metabolismoRESUMO
Leishmaniases represent an important public health problem in large parts of the world. In the south-east of Mexico, the major species isolated from patients is Leishmania mexicana mexicana, causing localised cutaneous leishmaniasis, and the development of a vaccine is a key objective for the control of this parasite. We thus performed a comparative study of DNA vaccines encoding L. m. mexicana gp63 and CPb, L. m. amazonensis gp46, and L. major LACK to define the best antigen(s) candidate(s). cDNAs encoding these antigens were subcloned into the VR1012 plasmid, and susceptible BALB/c mice were immunised with two i.m. injections of 100 microg of plasmid DNA. All mice immunised with VR1012-GP46, VR1012-CPb and VR1012-GP63 showed increased IgG levels against L. m. mexicana, but not those immunised with VR1012-LACK. Two to three weeks after the last immunisation, mice were challenged by the injection of 4 x 10(6) L. m. mexicana parasites in the foot pad to evaluate protection. Measurement of lesion size indicated that mice immunised with VR012-GP46, VR012-GP63 and VR1012-CPb were partially protected against infection, whereas the other plasmids had no effect. Thus, these plasmids represent good candidates for further development of DNA immunisation against L. m. mexicana.