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

为探讨HTLV-I前病毒载量在HAM/TSP发病机制中的重要性，采用ABI PRISM 7700序列检测系统，以双标记荧光探针为定量PCR方法，检测了大量HAM/TSP患者和无症状HTLV-I携带者外周血单个核细胞（PBMC）中HTLV-I前病毒载量。平均值+1-平均值的标准误差（s.e.m.）HTLV-I前病毒拷贝数/1 × 10（4）PBMC为798 1- 51 202例HAM/TSP患者（中位数544）; 120 ± 1 - 17（中位数34）在200例非HAM相关的（一般）无症状HTLV-Ⅰ携带者（RC）;在43名与HAM/TSP患者遗传相关的无症状HTLV-1携带者中，496 +/- 82（中位数321）。HAM/TSP的患病率随PBMC前病毒载量的增加而呈指数增加，女性HAM/TSP患者HTLV-Ⅰ前病毒载量显著高于男性，而女性HAM/TSP患者HTLV-Ⅰ前病毒载量与男性HAM/TSP患者相比无显著性差异。 ...更多信息 RC中前病毒载量在性别间无显著性差异，HAM/TSP患者脑脊液中新喋呤浓度与前病毒载量呈显著相关。这些结果表明，PBMC中HTLV-Ⅰ前病毒负荷可能与脊髓损伤的炎症过程有关。FA中增加的前病毒载量表明存在有助于HTLV-I在体内复制的遗传因素。此外，为了直接阐明HTLV-Ⅰ前病毒DNA的定位，我们用免疫组织化学和PCR原位杂交（PCR-ISH）进行了双重染色。首先用抗泛T细胞（UCHL-1）、巨噬细胞（KP-1）和辅助/诱导T细胞（OPD 4）的抗体对尸检时采集的4名HAM患者的脊髓新鲜冷冻切片进行免疫染色。然后用HTLV-Ⅰ pX区特异性引物和探针进行PCR-ISH。UCHL- 1阳性细胞在血管周围区域，并在一定程度上，在实质中。在UCHL-1阳性细胞中，9.4%（例1），9.6%（例2），1.1%（例3）和6.7%（例4）HTLV-Ⅰ PCR-ISH阳性，UCHL-1阴性细胞HTLV-Ⅰ PCR-ISH阴性，KP-1阳性细胞几乎全部HTLV-Ⅰ阴性。HTLV-I定位于OPD 4阳性细胞在检查病变的情况下2和4。这些数据是HTLV-1前病毒DNA定位于HAM/TSP脊髓病变中浸润的T细胞的直接证明。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