Relationship Between Traffic Of HTLV-1 Across The Blood-brain Barrier And Mechanism Of HAM/TSP

Background:Human T-cell leukemia type I-associated myelopathy (TSP/HAM) /tropical spastic paraparesis is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis and an inflammatory process in the spinal cord. Recently, more attention was paid to the pathogenetic mechanism of HAM/TSP. The current hypothesis about the pathogenesis of TSP/HAM includes cellular immunity, humoral immunity, autoimmunity and HTLV-I virus infiltrating into spinal cord. Cellular immunity is supported by most of people. HTLV-1 is a complex type C retrovirus. The proviral DNA integrates into the host cell genome if HTLV-1 infectes the patient. The presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord . Direct infection is not critical. A strong antiviral humoral and cytotoxic immune response has been reported. HTLV-1 infected T-lymphocyte in the central nervous system (CNS) seem to be the major reservoir for the virus and may play an important role in the pathogenesis of HAM/TSP. Little, however, is known about the mechanisms by which HTLV-1 crosses the blood-brain barrier. There are a few reports about HAM/TSP in China. Little was known about the pathogenetic mechanism of HAM/TSP. Shantou, a city of eastern Guangdong province on the bay of Pacific ocean, is according with the area coverage of geography distributing. Our previous study has showed that Shantou is the prevalence area of HAM/TSP. For further study of the pathogenetic mechanism and the process of HAM/TSP, we investigate the relationship between traffic of HTLV-1 across the Blood-brain barrierand mechanism of HAM/TSP.Objectives;To investigate the way of HTLV-1 proviral DNA integrating into T-lymphocyte and the traffic of HTLV-1 across the blood-brain and viral entry into the CNS in order to elucidate the mechanism of HAM/TSP.Materials and Methods:Five patients with a clinical diagnosis of HAM/TSP were from neurology department of First Affiliated Hospital of Shantou University Medical College (4 males and 1 females, age range 20-61 years, mean age 41 years), according to the criteria of Osame et al. Peripheral blood mononuclear cells (PBMC) were isolated from Heparinised blood by centrifugation on a Ficoll-Hypaque density gradient; CSF samples were centrifuged to obtain cells. Both were stored at -20 °C until analysis. To compare the amplification nucleotide sequences of HTLV-1 integration sites between CSF and PBMC by Linker-mediated polymerase chain reaction (LMPCR) and sequenceing. The amplified sequences were compared with the HTLV-1 complete genome (Genbank accession: AF033817) and positioned in the HTLV-1 complete genome. The homogeneity of nucleotide sequences was analyzed by CLUSTAL W software.Results:(1) Our datas show that the same HTLV-1 proviral integration sites were found in both the peripheral blood and the CSF of patients with HAM/TSP ,as determined by a sensitive polymerase chain reaction (PCR) method. The amplified HTLV-1 proviral flanking sequences have the same length (400bp and 196bp, separately) and the same sequence. Furthermore, when they were compared with the HTLV-1 complete genome (Genbank accession: AF033817), the accord rate is 98%(392/400)and 47%(92/196) separately. The 400bp proviral flanking sequence can be deteded in five HAM/TSPpatients, in both the CSF and the peripheral blood. The 196bp proviral flanking sequence can be deteded in five HAM/TSP patients in both the CSF and the peripheral blood, except for the CSF of patient 1. The 400bp proviral flanking sequence positions in the 5' UTR gene of the HTLV-1 complete genome (Genbank accession: AF033817). The 196bp proviral flanking sequence positions in the gpl gene of the HTLV-1 complete genome.(2) The 124bp proviral flanking sequence for the peripheral blood of patient 1 positions in the gpl gene of the HTLV-1 complete genome.Its accord rate is 51%(63/124) .(3) From 1 to 3 distinct amplified clones were obtained from the 5 patients, in both the CSF and the peripheral blood.Conclusions:(1) HTLV-1 crosses the blood-brain barrier by migration of HTLV-1-infected lymphocytes in vivo following that HTLV-1 proviral integrates into the T-lymphocyte cell genome. There is a cellular-associated transport of HTLV-1 through the BBB in HAM/TSP. The proliferation of infected cells will lead to pathogenic mechanisms of HAM/TSP.(2) During the mutual affection course between HTLV-1 and infected host, 5' UTR sequence is stable* while the gpl sequence is unstable.(3) There are multi-integrations site-specific in HTLV-1-infected lymphocytes for HAM/TSP patients, in both the CSF and the peripheral blood. Therefore, they will proliferate into different clonal HTLV-1-infected lymphocytes.