A TEST FOR HTLV-1 MEDIATED IN VIVO T-CELL EXPANSION

该项目的主要目的是确定利用报告基因突变(次黄嘌呤-鸟嘌呤磷酸核糖基转移酶，HPRT)作为体内细胞分裂探针的替代选择方法是否可以用作检测人类嗜T淋巴细胞HTLV-1携带者亚临床克隆性T细胞增殖的诊断工具。这样的测试将允许在扩大的克隆进展到更具侵袭性和无法治疗的状态之前启动预防性治疗(最初是在HTLV-1介导的成人T细胞白血病的情况下)。这一目标将通过检测患有HTLV-1相关性脊髓病/热带痉挛性瘫痪(HAM/TSP)的HTLV-1感染者的血液样本来实现。将分离野生型和HPRT突变的T细胞克隆，并通过多重PCR和T细胞受体(TCR)可变区β链和第三互补决定区的DNA测序来确定克隆的同源性。体内扩增的克隆将被鉴定，扩增克隆的代表将与在外周血样中观察到的相比，进行单克隆性HTLV整合测试。这些研究可能为HTLV-1白血病的确切机制提供新的见解，并指导更有效的治疗方法的开发。拟议的商业应用：开发一种克隆性T细胞扩增的自动测试将有助于病毒介导的病理的诊断和治疗。这项测试还将在疾病条件下具有诊断/测温应用，其中使用的治疗剂针对的是残存的抢救途径。

The primary objective of this project is to determine if a surrogate selection approach utilizing mutation at a reporter gene (hypoxanthine- guanine phosphoribosyltransferase (hprt]) as a probe for in vivo cell division can be used as a diagnostic tool for detection of sub-clinical clonal T-cell expansion in human T lymphotropic HTLV-1 carriers. Such a test would be to allow preventive therapy (initially in the setting of HTLV-1-mediated adult T-cell leukemia) to be initiated before the expanding clone has progressed to a more aggressive and untreatable state. The objective will be achieved by testing blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Wild type and hprt mutant T-cell clones will be isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T-cell receptor (TCR) variable region beta chain and third complimentarity determining regions. In vivo- expanded clones will be identified, and representatives of the amplified clones will be tested for monoclonal HTLV integration as compared to that observed in the peripheral blood sample. These studies may provide new insights into the precise mechanism of HTLV-1 leukemogensis, and direct the development of more effective therapies. PROPOSED COMMERCIAL APPLICATIONS: Development of an automated test for clonal T-cell expansion would aid in the diagnosis and treatment of virally-mediated pathologies. The test would also have diagnostic/therametric applications in disease conditions where therapeutic agents are used that target the puring salvage pathway.