LATENCY-ASSOCIATED PEPTIDE AS AN INHIBITOR OF TGFB-INDUC

系统性硬化症/硬皮病是一种病因不明的慢性自身免疫性疾病，其特征是体液免疫和细胞免疫功能改变，内脏和皮肤成纤维细胞过度沉积胶原。皮肤单核细胞进入、黏附、激活和分化为TGFbeta1产生细胞被认为是(Scl)GVHD。假设：作为转化生长因子β的天然拮抗剂，潜伏期相关肽(LAP)作为重组蛋白或通过转导的单核细胞体内高表达的LAP基因产物在体内进行基因治疗时，可以抑制SCL GVHD动物的纤维化。通过形态学研究、单核细胞的免疫染色和流式细胞术研究、转化生长因子-β1的逆转录/聚合酶链式反应以及前α1(I)胶原的核糖核酸酶保护分析，可以对皮肤的体内干预措施进行评估。我们的初步数据是有希望的，表明重组LAP肽可以预防SCL GVHD小鼠的皮肤纤维化。目的I：外源性重组LAP多肽在体内最佳抑制皮肤纤维化的参数和条件是什么？目的II：小鼠骨髓干细胞能否用含有绿色荧光蛋白的逆转录病毒载体在体外进行检测和LAP检测，然后在体外诱导分化为有功能的单核细胞？目的III：将这些转导的单核细胞移植到受体小鼠体内后，这些单核细胞能否在可检测到的数量中存活并回到皮肤中？它们能否在体内过度表达潜伏期相关肽，并抑制SCL GVHD动物的纤维化过程？这些拟议的实验对我们理解基本的单核细胞生物学、动物模型中转化生长因子β驱动的纤维化和自身免疫性疾病硬皮病，以及潜在的硬皮病和人类移植物抗宿主病的治疗具有很高的相关性。这种新的单核细胞递送免疫疗法的开发也可以用于治疗其他炎症性疾病和癌症。

Systemic sclerosis/scleroderma is a chronic autoimmune disease of unknown etiology characterized by altered humoral and cell-mediated immunity, and excessive deposition of collagen by fibroblasts in viscerae and skin. Cutaneous monocyte influx, adhesion, activation and differentiation into TGFbeta1-producing cells are thought to be (Scl GVHD). Hypothesis: A naturally occurring antagonist to TGFbeta, latency associated peptide (LAP) can inhibit fibrosis in animals with Scl GVHD when administered exogenously as recombinant protein, or as gene therapy with over-expressed LAP gene product delivered in vivo by transduced monocytes. Focused in vivo interventions can be evaluate din skin by morphologic studies, immunostaining and flow cytometry studies for monocytes, RT/PCR for TGF-beta1, and RNase protection assays for proalpha1(I) collagen mRNA. Our preliminary data are promising, showing recombinant LAP peptide prevents cutaneous fibrosis in mice with SCL GVHD. Aim I: What are the parameters and conditions for optimum in vivo inhibition of skin fibrosis by exogenous recombinant LAP peptide? Aim II: Can mouse bone marrow stem cells be transduced in vitro with a retroviral construct containing green fluorescent protein for detection and LAP and then driven in vitro to differentiate into functional monocytes? Aim III: After transfer to recipient mice, can these transduced monocytes survive in detectable numbers and home to skin? Can they over-express latency associated peptide in vivo, and inhibit the fibrosing process in animals with Scl GVHD? These proposed experiments have high relevance to our understanding of the basic monocyte biology, to TGFbeta-driven fibrosis in the animal model and in the autoimmune disease scleroderma, and potentially to the treatment of scleroderma and human graft versus host disease. Development of this novel monocyte- delivered immuno therapy can also be treated to treatment of other inflammatory disorders and of cancers.