Research on Pathomechanism and a novel treatment of HTLV-I-associated myelopathy (HAM)

通过检测大量HAM/TSP患者及无症状HTLV-I携带者外周血单个核细胞（PBMC） HTLV-I前病毒载量，探讨HTLV-I前病毒载量在HAM发病机制中的重要性。采用双标记荧光探针（ABI PRISM 7700序列检测系统），采用精确、可重复的定量PCR方法测定前病毒载量。平均值+1-平均值的标准误差（s.e.m.）在202例HAM/TSP患者中，每1 × 10(4) PBMC的HTLV-I原拷贝数为798 1- 51（中位544）；200例非ham相关（一般）无症状HTLV-I携带者（RC） 120 +1- 17例（中位数34例）；43例与HAM/TSP患者遗传相关的无症状HTLV-I携带者中有496 +/- 82例（中位321例）。一旦原病毒负荷超过1% PBMC， HAM/TSP的流行率随log（原病毒负荷）呈指数增长。女性HAM/TSP患者的HTLV-I前病毒载量显著高于男性，而RC患者的前病毒载量无显著性差异。HAM/TSP患者脑脊液中前病毒载量与新蝶呤浓度有显著相关性。这些结果表明，PBMC中HTLV-I的前病毒载量可能与脊髓病变的炎症过程有关。FA中原病毒载量的增加表明遗传因素的存在促进了HTLV-I在体内的复制。此外，为了直接阐明HTLV-I前病毒DNA的定位，我们使用免疫组织化学和PCR原位杂交（PCR- ish）进行了双重染色。尸检时采集的4例HAM患者的新鲜冷冻脊髓切片首先用pan - T细胞（UCHL-1）、巨噬细胞（KP-1）和辅助/诱导T细胞（OPD4）的抗体进行免疫染色。然后用特异性引物和探针对HTLV-I的pX区域进行PCR-ISH检测。血管周围有UCHL- 1阳性细胞，在一定程度上也可见于实质。在uchl -1阳性细胞中，HTLV-I PCR-ISH阳性细胞分别为9.4%（病例1）、9.6%（病例2）、1.1%（病例3）和6.7%（病例4）。uchl -1阴性细胞HTLV-I PCR-ISH阴性，几乎所有kp -l阳性细胞HTLV-I阴性。HTLV-I在病例2和病例4的检查病变中定位于opd4阳性细胞。这些数据直接证明了HTLV-1前病毒DNA在HAM/TSP脊髓病变中定位于浸润的T细胞。少

HTLV-I proviral load in peripheral blood mononuclear cells (PBMC) from a large number of HAM/TSP patients and asymptomatic HTLV-I carriers were measured to explored the importance of HTLV-I proviral load to the pathomechanism of HAM.The proviral load was measured by an accurate and reproducible quantitative PCR method using a dual-labeled fluorogenic probe (ABI PRISM 7700 Sequence Detection System). The mean +1- standard error of mean (s.e.m.) HTLV-I proviral copy number per 1 x 10(4) PBMC was 798 1- 51 (median 544) in 202 HAM/TSP patients ; 120 +1- 17 (median 34) in 200 non HAM-related (general) asymptomatic HTLV-I carriers (RC) ; and 496 +/- 82 (median 321) in 43 asymptomatic HTLV-I carriers genetically related to HAM/TSP patients (FA). The prevalence of HAM/TSP rises exponentially with log (proviral load) once the proviral load exceeds 1% PBMC.The HTLV-I proviral load of female patients with HAM/TSP was significantly higher than that of male patients, however there was no significan … More t difference in proviral load between sexes in RC.There was a significant correlation between the proviral load and the concentration of neopterin in CSF of HAM/TSP patients. These results indicate that the HTLV-I proviral load in PBMC may be related to the inflammatory process in the spinal cord lesion. The increased proviral load in FA suggests the existence of genetic factors contributing to the replication of HTLV-I in vivo. Furthermore in order to elucidate the localization of HTLV-I proviral DNA directly, we performed double staining using immunohistochemistry and PCR in situ hybridization (PCR-ISH). Fresh frozen sections of the spinal cord from four HAM patients taken at autopsy were first immunostained with antibodies to pan T cells (UCHL-1), macrophages (KP-1) and helper/inducer T cells (OPD4). Then PCR-ISH was carried out with specific primers and probe for the HTLV-I pX region. UCHL- 1-positive cells were noted around perivascular areas and, to some extent, in the parenchyma. Of the UCHL-1-positive cells, 9.4% (case 1), 9.6% (case 2), 1.1% (case 3) and 6.7% (case 4) became positive in HTLV-I PCR-ISH.UCHL-1-negative cells were HTLV-I PCR-ISH negative and almost all KP-l-positive cells were HTLV-I negative. HTLV-I was localized to OPD4-positive cells in examined lesions of cases 2 and 4. These data are a direct demonstration of HTLV-1 proviral DNA localizing to infiltrated T cells in HAM/TSP spinal cord lesions. Less