VITAMIN A INFLUENCE ON INTERFERON-GAMMA GENE EXPRESSION

令人惊讶的是，维生素A缺乏的小鼠受到感染，或者 用抗原免疫，有T淋巴细胞在一年内分泌干扰素 比对照组高10倍。A基因缺陷小鼠的T细胞 作为B细胞抗体反应的刺激物作用不佳； 辅助性T细胞频率低，细胞分泌淋巴因子不足 IL-4和IL-5。补充维生素A代谢物维甲酸， 大幅下调IFNGamma，恢复辅助T细胞频率，以及 在体外，抗体反应恢复到正常水平。维甲酸补充 也恢复了体内的抗体反应。克隆小鼠MD13-10 T细胞 模拟新鲜移植的免疫T细胞对干扰素-γ的作用 制作。当MD 13-10 T细胞被维甲酸耗尽和刺激时， 它们过度产生IFNGamma，而维甲酸直接下调 干扰素-γ分泌率。我们发现了一个意想不到的维甲酸受体- α(RARpha)转录本和一种新的RARpha免疫反应蛋白 MD13-10 T细胞和其他T细胞。总而言之，我们的发现引导我们 提出一个工作假说，即维甲酸通过一种 可能涉及这种新的RARAlpha异构体的未知机制， 特别是下调T细胞干扰素-γ的产生。这些实验 将通过研究生物化学来检验我们的假说 维甲酸对克隆的MD 13-10 T细胞的干扰素-γ调节作用我们 建议定义视黄醇敏感的干扰素-伽马诱导途径，以 探索类维甲酸介导的控制是否在IFNGamma转录本或 蛋白质水平，并分析调控对 大分子合成。我们建议刻画T细胞RARpha的特征 外显子特异性探针Northern印迹转录本及测序 新的5‘和/或3’端的cDNA端。我们将调查推定的RARAlpha 使用免疫化学的蛋白质。我们计划表达和提纯这部小说 RARAlpha N-末端片段，并提高抗体。我们将使用 用于开发RARAlpha亚型特异性的抗体，维甲酸结合， 和DNA结合分析，并将其应用于MD13-10 T细胞蛋白。这个 拟议的研究具有重要意义，因为它将提供新的信息 关于免疫的两个强有力的控制者之间的调节关系 功能和细胞生长。IFNGamma调节免疫，自身免疫， 造血、病毒复制和恶性细胞生长。维甲酸 也调节免疫力，以及正常和恶性细胞生长和 差异化。强大的抗癌和免疫调节活性 强调了研究维甲酸是如何 酸下调了IFNGamma基因的表达。

Surprisingly, vitamin A-deficient mice undergoing an infection, or immunized with an antigen, had T lymphocytes that secreted IFNgamma at a 10-fold higher rate than controls. The T cells from A-deficient mice functioned poorly as stimulators of B cell antibody responses; there was a low helper T cell frequency and the cells underproduced the lymphokines IL-4 and IL-5. Repletion with retinoic acid, a vitamin A metabolite, sharply downregulated IFNgamma, restored the helper T cell frequency, and returned antibody responses to normal levels in vitro. Retinoid repletion also restored antibody responses in vivo. Cloned mouse MD13-10 T cells mimic freshly explanted immune T cells with respect to IFNgamma production. When MD 13-10 T cells are retinoid depleted and stimulated, they overproduce IFNgamma, and retinoic acid directly downregulates the IFNgamma secretion rate. We found an unexpected retinoic acid receptor- alpha (RARalpha) transcript and a novel RARalpha-immunoreactive protein in MD13-10 T cells and other T cells. Together, our findings lead us to propose as a working hypothesis that retinoic acid, acting through an undefined mechanism possibly involving this novel RARalpha isoform, specifically downregulates T cell IFNgamma production. The experiments proposed here will test our hypothesis by studying the biochemistry of retinoid-mediated IFNgamma regulation in cloned MD 13- 10 T cells. We propose to define the retinoid-sensitive IFNgamma induction pathway, to explore whether retinoid-mediated control is at the IFNgamma transcript or protein level, and to analyze the dependence of regulation on macromolecular synthesis. We propose to characterize the T cell RARalpha transcripts by Northern blotting with exon-specific probes, and sequencing of novel 5' and/or 3' cDNA ends. We will investigate the putative RARalpha proteins using immunochemistry. We plan to express and purify the novel RARalpha N-terminal fragments, and raise antibodies. We will use the antibodies to develop RARalpha isoform-specific ELISA, retinoid-binding, and DNA-binding assays and apply them to MD13-10 T cell proteins. The proposed research is significant in that it will provide new information about a regulatory relationship between two potent controllers of immune function and cell growth. IFNgamma regulates immunity, autoimmunity, hemopoiesis, viral replication, and malignant cell growth. Retinoic acid also regulates immunity, and normal and malignant cell growth and differentiation. The powerful anticancer and immunoregulatory activities of both molecules emphasize the importance of investigating how retinoic acid downregulates IFNgamma gene expression.