RESUMO
To define the clonal diversity of autoreactive T cells associated with the induction of type 1 diabetes, we characterized TCR expression in the earliest detectable islet infiltrates of non-obese diabetic (NOD) mice. The islets of young NOD females were examined for V beta and J beta germ-line gene usage and V(D)J beta junctional sequence diversity. The results from 7-wk-old mice corroborate prior studies demonstrating that the T cell repertoire of islet infiltrates diversifies early in the inflammatory process. In contrast, examination of 4-wk-old NOD mice showed that TCR-beta expression in the peri-islet infiltrates was restricted both in V beta and J beta gene utilization and, most significantly, in V(D)J junctional sequence diversity. Islet-infiltrating T cells from young mice included V beta 3+ T cells, despite the presence of a mammary tumor virus-3-associated superantigen that deletes the majority of immature V beta 3+ thymocytes in NOD mice. Few other TCR V beta types were repeatedly detectable in early stage infiltrates. V(D)J junctional sequence diversity was evaluated in cDNA libraries made from the islets of young NOD mice. Analysis of these clones revealed limited junctional CDR3 diversity in early-infiltrating T cells, as compared with lymph node T cell libraries. Evaluation of TCR expression in individual islets revealed CDR3 sequence conservation between animals and among islets from a single animal. These results suggest that T cells bearing limited TCR-beta-chain diversity contribute to the inductive phases of autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , Diabetes Mellitus Tipo 1/genética , Feminino , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/genéticaRESUMO
Conflicting reports exist concerning ultraviolet-B (UVB) effects on keratinocyte (KC) interleukin-1 (IL-1) expression. To clarify the modulatory effects of UVB on IL-1, the following study was undertaken. Normal human epidermal KCs cultured in a standard low Ca2+ and serum-free medium were irradiated in quiescent phase with UVB. In this study, we used semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the mRNA level of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta). After exposure to 100 or 300 J/m2 UVB, a transient increase in mRNA levels was observed within 1 hour for IL-1 alpha and 3 to 6 h for IL-1 beta. Following this transient induction, mRNA levels for both IL-1 alpha and IL-1 beta returned to steady-state levels after 100 J/m2. After 300 J/m2 irradiation, IL-1 alpha and IL-1 beta levels were downregulated compared to unirradiated cultures at 24-h post-irradiation. The half-life for IL-1 alpha and IL-1 beta was estimated using actinomycin D treatment. Both IL-1 alpha and IL-1 beta mRNAs half-lives (t1/2) decreased faster in irradiated cells (t1/2 = 30 minutes for IL-1 alpha and 2 h for IL-1 beta) compared to unirradiated cells (t1/2 = 1 h and 4 h, respectively). These results suggest that IL-1 alpha and IL-1 beta mRNA expression are differentially regulated by UVB. In contrast to down-regulation of mRNA levels, a significant increase in IL-1 alpha protein levels, measured by ELISA, was observed in culture supernatants from 6 h to 24 h after 300 J/m2 UVB irradiation. Cycloheximide treatment did not abrogate this increase in IL-1 alpha protein level. Since this dose of UVB irradiation decreased the stability of IL-1 alpha and IL-1 beta mRNA, this suggests that the release of IL-1 alpha after UVB irradiation was due to leakage from UVB-damaged cells and not from de novo protein synthesis.
Assuntos
Interleucina-1/biossíntese , Queratinócitos/efeitos da radiação , RNA Mensageiro/metabolismo , Raios Ultravioleta , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Humanos , Recém-Nascido , Interleucina-1/análise , Interleucina-1/genética , Queratinócitos/metabolismo , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Fatores de Tempo , Transcrição GênicaRESUMO
A general method to clone the HSP70 gene from any species employing polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of this protein family is described. Using this method, a clone containing the entire coding sequence for the HSP70 gene from Clostridium perfringens, has been isolated and sequenced. The HSP70s from C. perfringens as well as other Gram-positive groups of bacteria contain a large deletion of 25 amino acids, near the N-terminal end, which is not seen in HSP70 from other sources.