RESUMO
Human fetal pancreas (HFP) is a potential source of transplantable islets for the treatment of type 1 insulin-dependent diabetes mellitus (IDDM). Pretransplant culture techniques such as long-term culture, high-oxygen culture, UVB irradiation, and low-temperature culture have previously been used to reduce the immunogenicity of tissue for transplantation. In this study, we use hyperbaric oxygen culture (HOC) to modify MHC Class I expression on HFP and to reduce the immunological response of human peripheral blood mononuclear cells (PBMC) to HFP using a sponge matrix allograft model. To study the interaction of naïve PBMC with HOC-treated or untreated HFP allografts, sponges embedded with HFP tissue were implanted into the peritoneal cavity of NOD-SCID mice and injected with 1 x 10(7) freshly isolated human PBMC at the time of transplant. By day 14, human CD45 cells represented less than 2% of the cells recovered from the sponges implanted with HOC-treated HFP. In contrast, human CD45(+) cells represented nearly 15% (P =.0018) of the cells isolated from sponges implanted with conventionally cultured HFP grafts. Approximately 75% of the human CD45(+) cells from conventionally cultured HFP allografts were producing IFNgamma as determined by intracellular cytokine analysis. These data suggest that HOC treatment of HFP abrogates the activation and proliferation of PBMC. Pretransplant HOC treatment of islets is a simple technique that could be used to reduce immunogenicity and increase allograft survival while decreasing the requirement for immunosuppressive drugs.
Assuntos
Transplante de Tecido Fetal/imunologia , Transplante de Pâncreas/imunologia , Pâncreas , Animais , Antígenos CD/análise , Diabetes Mellitus Tipo 1/cirurgia , Modelos Animais de Doenças , Teste de Histocompatibilidade , Humanos , Oxigenoterapia Hiperbárica , Antígenos Comuns de Leucócito/análise , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos NOD , Técnicas de Cultura de Órgãos , Pâncreas/embriologia , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Transplante HomólogoRESUMO
We have previously shown that hyperbaric oxygen culture (HOC [95% O(2), 5% CO(2), 25 psi]) is an effective pretransplant tissue-modification technique that results in long-term allograft survival and the induction of systemic immune tolerance in a murine model. Here we address the immune modulatory effects of HOC-treatment of human immune responses using the in vitro mixed lymphocyte reaction (MLR). Pretreatment of allogeneic stimulator cells with HOC results in abrogation of cytotoxic T lymphocyte (CTL) activity, proliferative responses, and IFN gamma production in a 7-day MLR. These responses can be restored either by the addition of IFN gamma or IL-2 on day 0, or by blocking the activity of IL-4 and IL-10. The addition of IL-2 on day 4 does not restore allospecific CTL activity. The failure of HOC-treated cells to induce allospecific CTL is not due to the induction of anergy, demonstrated by the failure to restore responses after restimulation with allogeneic cells in the presence of IL-2. Removal of CD4(+) cells prior to restimulation, results in restoration of CTL activity in MLR cultures restimulated with HOC-treated allogeneic cells. These results suggest that HOC-induced immune nonresponsiveness is mediated by the development of CD4(+) regulatory cells in a Th2-type environment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Oxigenoterapia Hiperbárica , Tolerância Imunológica/imunologia , Anticorpos Bloqueadores/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Anergia Clonal/imunologia , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade/métodos , Humanos , Soros Imunes/farmacologia , Interferon gama/farmacologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/imunologia , Interleucina-4/antagonistas & inibidores , Interleucina-4/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos/métodos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The putative gene encoding acetyl-CoA carboxylase, accA, has been isolated from Aspergillus nidulans. This single-copy gene has an open reading frame (ORF) of 6864 bp and contains two small introns near the 5'-end. A short ORF upstream of the ATG start codon has been identified in this gene by RT-PCR. Based on sequence homology to acetyl-CoA carboxylases from other organisms, putative biotin-, ATP-, HCO3-- and acetyl-CoA- binding sites have been assigned. Northern data and ACC enzyme-activity measurements from A. nidulans suggested that expression of accA was higher in media containing nitrate than ammonia as a sole nitrogen source. Deletion of accA in A. nidulans was unsuccessful. The failure of A. nidulans to grow in the presence of the ACC-specific inhibitor, soraphen A, supplemented with C16-18 fatty acids suggested that ACC is an essential enzyme.