RESUMO
BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E-selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte-EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug-mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC.
Assuntos
Ciclosporina/farmacologia , Selectina E/metabolismo , Endotélio Vascular/fisiologia , Glucocorticoides/farmacologia , Imunossupressores/farmacologia , Interleucina-8/antagonistas & inibidores , Sirolimo/farmacologia , Animais , Células Cultivadas , Regulação para Baixo , Combinação de Medicamentos , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/genética , Lipopolissacarídeos/farmacologia , Ratos , Suínos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We generated a monoclonal antibody (MAb), H8.98, that recognizes an antigen shared by 50% of examined renal carcinoma cell (RCC) lines and is susceptible to lysis by a Vgamma3Vdelta1(+) T-cell clone derived from RCC tumor-infiltrating lymphocytes. H8.98 inhibited Vgamma3Vdelta1(+ )T-cell clone-mediated lysis of RCC lines. It did not stain normal kidney lines, melanomas, fibroblasts, Burkitt's lymphoma or Epstein-Barr virus-transformed B-cell lines but it did stain 2 of 4 tested breast cancer lines. Through screening of a renal carcinoma cDNA library using H8.98, we isolated a cDNA clone which, upon sequencing, was found to be cytochrome b with 2 point mutations.
Assuntos
Antígenos de Neoplasias/genética , Carcinoma de Células Renais/imunologia , Grupo dos Citocromos b/genética , Neoplasias Renais/imunologia , Mutação Puntual/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Células COS , Carcinoma de Células Renais/genética , Grupo dos Citocromos b/imunologia , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Dados de Sequência Molecular , Linfócitos T/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
In transgenic animals, genotype-specific modifiers exert a control over transgene methylation and expression that may or may not be position dependent. These factors belong to different classes, some of them possibly related to modifiers of position-effect variegation in Drosophila. The study of hepatitis B virus (HBV) gene expression in transgenic mice has revealed the existence of many factors influencing transcription, including hormones and tissue-specific transcription factors. We now report the effect of genotype-specific modifiers on HBV surface antigen (HBsAg) expression and transgene methylation. Compared with the C57BL/6 background, the DBA/2 and 129sv backgrounds cause enhancement of HBsAg expression, with little or not effect on transgene methylation or transcription. In contrast, a single cross with a BALB/c mouse is responsible for de novo methylation and silencing of the transgene in all offspring. Several modifiers appear to segregate in the progeny of a transgenic E36 male mouse crossed with (C57BL/6 x BALB/c) F1 females, with the emergence of a high-expressor group. Our observations suggest that different modifiers act cooperatively, at both the transcriptional and post-transcriptional levels, as part of a complex system regulating transgene expression. This transgenic model provides a system to genetically map new mouse strain-specific modifiers, some of them involved in epigenetic modification and transcription control.
Assuntos
Metilação de DNA , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Transgenes , Animais , Cruzamentos Genéticos , Feminino , Antígenos de Superfície da Hepatite B/biossíntese , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Especificidade da EspécieRESUMO
Expression of the Gal alpha1,3 Gal epitope on membrane glycolipids and glycoproteins is known to vary widely from one tissue to another. In the course of studying the mechanisms underlying this variability, we have isolated from pig cDNA four sequences corresponding to four isoforms of alpha1,3-galactosyltransferase (alpha1,3GT), the Golgi enzyme that links galactose in alpha1,3 on the galactose residue of N-acetyllactosamine. The isoforms differ from each other in the alternative presence of two nucleotide stretches of 36 and 63 base pairs in a segment encoding the stem region of the protein. Stable expression experiments show that all four isoenzymes can confer alpha-galactosyltransferase activity to HeLa cells, and that they are all located within the Golgi compartment, indicating that variations in length in the stem region do not affect enzyme activity or cellular localization. Analysis of RNA from different pig organs and cells shows quantitative differences between tissues in levels of alpha1,3GT, as well as qualitative differences, the four isoforms being unequally represented in different tissues.