Shinji Yashiki

Improved outcome of adult T cell leukemia/lymphoma with allogeneic hematopoietic stem cell transplantation.

Adult T cell leukemia/lymphoma (ATL) is a poor prognosis T cell malignancy. In order to improve the outcome, we employed allogeneic stem cell transplantation (allo-SCT) for ATL in 10 patients, nine of whom were from HLA-identical siblings and one from an unrelated donor. Conditioning regimens varied among the patients except that all received total body irradiation. The patients tolerated the regimens well with mild, if any toxicity, and engraftment occurred in all cases. Median leukemia-free survival after allo-SCT was 17.5+ months (range 3.7–34.4+). Six of the 10 patients developed acute GVHD (one case each with grade I, III or IV, and three cases with grade II) and three patients developed extensive chronic GVHD. Four patients died after allo-SCT during the study period from either acute GVHD (grade IV), pneumonitis, gastrointestinal bleeding or renal insufficiency. Two of the 10 cases with no symptoms of GVHD relapsed with clinical ATL. These results strongly suggest that allo-SCT may improve the survival in ATL if a controlled degree of GVHD develops. Bone Marrow Transplantation (2001) 27, 15–20.

Genetic study of ossification of the posterior longitudinal ligament in the cervical spine with human leukocyte antigen haplotype.

To evaluate the genetic background of ossification of the posterior longitudinal ligament, the relationship between the presence or absence of ossification and human leukocyte antigen haplotypes was studied in 33 families of patients with ossification of the posterior longitudinal ligament. The study revealed that human leukocyte antigen haplotypes formed certain types of clusters, and that some human leukocyte antigen haplotypes were very rare in the Japanese population, suggesting the involvement of human leukocyte antigen-linked factors in the pathogenesis of ossification of the posterior longitudinal ligament of the cervical spine. In the families of these patients, ossification of the posterior longitudinal ligament was demonstrated by radiography in 56% (10/18) of the siblings. Each of these siblings shared both human leukocyte antigen haplotypes with the patient. None of those who shared only one human leukocyte antigen haplotype with the patient had developed ossification of the posterior longitudinal ligament. From these findings, the presence of both pathogenic human leukocyte antigen haplotypes is considered to be necessary for the development of ossification of the posterior longitudinal ligament, and this genetic predisposition may be activated by multiple factors, including regressive degeneration due to aging and the environment.

HTLV-I proviral DNA amount correlates with infiltrating CD4+ lymphocytes in the spinal cord from patients with HTLV-I-associated myelopathy

A quantitative method utilizing polymerase chain reaction was employed to evaluate the amount of human T-cell leukemia virus type I (HTLV-I) proviral DNA in the affected spinal cords from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Central nervous system (CNS) tissues were obtained at post-mortem from five patients with HAM/TSP, who vary in the duration of illness from 2.5-10 years, and one patient with adult T-cell leukemia (ATL), who had leukemic cell infiltration in the CNS. The presence of HTLV-I pX and pol sequences in the CNS tissues were demonstrated in all patients examined. In HAM/TSP, the proviral DNA quantified in the thoracic cord was 0.002-2 copies per 100 tissue cells, and that in the peripheral blood mononuclear cells (PBMC) was 2-8 copies per 100 PBMC. The proviral DNA amount in the thoracic cord of the patient with ATL was 0.4 copies per 100 tissue cells. An apparent propensity for the amount of integrated HTLV-I in the thoracic cord to decrease with the disease duration in patients with HAM/TSP was observed. The decline in HTLV-I proviral DNA amount in the thoracic cord lesions was paralleled with the alteration of proportion of CD4+ T lymphocytes in patients with HAM/TSP. These findings suggest that preferential virus reservoir may be infiltrating CD4+ T lymphocytes in the spinal cord lesions of patients with HAM/TSP, and HTLV-I infection in the CNS of patients is declining with the disease duration in spite of the chronic course of neurological manifestations at least in some patients with HAM/TSP.

The presence of ancient human T-cell lymphotropic virus type I provirus DNA in an Andean mummy

The worldwide geographic and ethnic clustering of patients with diseases related to human T-cell lymphotropic virus type I (HTLV-I) may be explained by the natural history of HTLV-I infection. The genetic characteristics of indigenous people in the Andes are similar to those of the Japanese, and HTLV-I is generally detected in both groups. To clarify the common origin of HTLV-I in Asia and the Andes, we analyzed HTLV-I provirus DNA from Andean mummies about 1,500 years old. Two of 104 mummy bone marrow specimens yielded a band of human β-globin gene DNA 110 base pairs in length, and one of these two produced bands of HTLV-I-pX (open reading frame encoding p40x, p27x) and HTLV-I-LTR (long terminal repeat) gene DNA 159 base pairs and 157 base pairs in length, respectively. The nucleotide sequences of ancient HTLV-I-pX and HTLV-I-LTR clones isolated from mummy bone marrow were similar to those in contemporary Andeans and Japanese, although there was microheterogeneity in the sequences of some mummy DNA clones. This result provides evidence that HTLV-I was carried with ancient Mongoloids to the Andes before the Colonial era. Analysis of ancient HTLV-I sequences could be a useful tool for studying the history of human retroviral infection as well as human prehistoric migration.

Fluctuation of HTLV-I proviral DNA in peripheral blood mononuclear cells of HTLV-I-associated myelopathy

To assess the immunopathological significance of the increased replication of human T-cell leukemia virus type I (HTLV-I) in HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) we investigated the dynamics of HTLV-I proviral DNA in peripheral blood mononuclear cells (PBMC) of HAM/TSP patients at different clinical stages. We compared the dynamics to those of asymptomatic HTLV-I carriers (AC). The estimation of the amount of HTLV-I proviral DNA was carried out by quantitative polymerase chain reaction of serially diluted DNA samples where it was feasible to titrate 0.04-80 copies per 100 PBMC. The proviral DNA quantified in six patients with HAM/TSP was 2-20 copies per 100 PBMC, while that in eight cases of AC was 0.04-8 copies per 100 PBMC. Thus, the amount of HTLV-I proviral DNA in HAM/TSP patients was 3-50 times as high as that of AC. When we followed up HAM/TSP patients for 1-3 years, the amount of HTLV-I proviral DNA fluctuated from 4 to 10-fold. These data suggest that the rate of HTLV-I replication increases in HAM/TSP and the amount of HTLV-I proviral DNA fluctuates in their clinical course. Fluctuation in the amount of HTLV-I proviral DNA may reflect dynamics of HTLV-I infected cell proliferation and immunological suppression in vivo in HAM/TSP patients.

Green Tea Polyphenols Induce Apoptosis in vitro in Peripheral Blood T Lymphocytes of Adult T-Cell Leukemia Patients

Green tea polyphenols (TEA) are known to exhibit antioxidative activity as well as tumor‐suppressing activity. In order to examine the tumor‐suppressing activity of TEA against adult T‐cell leukemia (ATL), we cultivated peripheral blood T lymphocytes of ATL patients (ATL PBLs), an HTLV‐I‐infected T‐cell line (KODV) and healthy controls (normal PBLs) for 3 days in the presence of TEA and its main constituent, epigallocatechin‐3‐gallate (EGCg), to measure cell proliferation and apoptosis, and to quantitate mRNAs of HTLV‐I pX and β‐actin genes of the cultured cells. Growth of ATL PBLs was significantly inhibited by 9–27 μg/ml of TEA and EGCg, in contrast to minimal growth inhibition of T cells of normal PBLs. Inhibition of KODV was intermediate between ATL PBLs and normal PBLs. The ATL PBLs and KODV treated with 27 μg/ml of either TEA or EGCg induced apoptotic DNA fragmentation, producing terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL)‐positive cells, while the normal PBLs treated with the same concentration of TEA or EGCg produced a negligibly small number of TUNEL‐positive cells, in which apoptotic DNA fragmentation was not detectable. Expression of HTLV‐I pX mRNA was suppressed more than 90% in ATL PBLs by treatment with 3–27 μg/ml of either TEA or EGCg, while expression of β‐actin mRNA was much less suppressed by treatment with the same concentration of TEA or EGCg. These results indicate that TEA and EGCg inhibit growth of ATL PBLs, as well as HTLV‐I‐infected T‐cells, by suppressing HTLV‐I pX gene expression and inducing apoptotic cell death.

HLA-A*26, HLA-B*4002, HLA-B*4006, and HLA-B*4801 Alleles Predispose to Adult T Cell Leukemia: The Limited Recognition of HTLV Type 1 Tax Peptide Anchor Motifs and Epitopes to Generate Anti-HTLV Type 1 Tax CD8+ Cytotoxic T Lymphocytes

Genetic risk for adult T cell leukemia (ATL) has been implicated by ethnic and familial segregation of ATL patients from HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the genetic risk for ATL, we characterized HLA class I alleles of ATL patients and analyzed the anchor motifs of HTLV-1 peptides binding to HLA class I molecules, using 291 lines of anti-HTLV-1 CD8(+) cytotoxic T lymphocytes (CTLs) generated in vitro with a total of 165 synthetic peptides for HTLV-1 Tax and Env proteins. Allele frequencies of HLA-A*26, B*4002, B*4006, and B*4801 were significantly higher in ATL patients than in HAM/TSP patients and asymptomatic HTLV-1 carriers in southern Japan. CD8(+) CTL analysis revealed the HTLV-1 Tax peptide sequence to completely lack anchor motifs of peptides binding to HLA-A*26,B*4002, and B*4006 molecules but to possess one anchor for HLA-B*4801, while the HTLV-1 Env peptide sequence had many anchor motifs for HLA-A*26, B*4002, B*4006, and B*4801 molecules. Most ATL patients featured heterozygous HLA class I alleles composed of HLA-A*26, B*4002, B*4006, and B*4801, with a lower number of HTLV-1 Tax peptide anchor motifs and epitopes generating anti-HTLV-1 Tax CD8(+) CTLs than individuals possessing other HLA alleles. The relationship between Tax epitope and ATL incidence was verified by the significantly decreased number of HTLV-1 Tax epitopes in ATL patients compared with asymptomatic HTLV-1 carriers (p < 0.01) as well as late onset ATL patients (p < 0.001). These results indicate that HLA-A*26, B*4002, B*4006, and B*4801 alleles predispose to ATL because of the limited recognition of HTLV-1 Tax peptide anchor motifs and epitopes capable of generating anti-HTLV-1 Tax CD8(+) CTLs.

Characteristic distribution of HTLV type I and HTLV type II carriers among native ethnic groups in South America.

To confirm the geographic and ethnic segregation of HTLV-I and HTLV-II carriers in native populations in South America, we have conducted a seroepidemiological study of native populations in South America, including HTLV-I carriers distributed among seven ethnic groups in the Andes highlands of Colombia, Peru, Bolivia, Argentina, and Chile, and two ethnic groups on Chiloe Island and Easter Island; and HTLV-II carriers distributed among seven ethnic groups of the lowlands along the Atlantic coast of Colombia, Orinoco, Amazon, and Patagonia, and one ethnic group on Chiloe Island. The incidence rate of HTLV-I and HTLV-II carriers varied among the ethnic groups, ranging from 0.8 to 6.8% for HTLV-I seropositivity and from 1.4 to 57.9% for HTLV-II seropositivity. A new HTLV-I focus was found among the Peruvian Aymara (1.6%), the Bolivian Aymara (5.3%) and Quechua (4.5%), the Argentine Puna (2.3%), and the Chilean Atacama (4.1%), while on HTLV-II focus was found among the Brazilian Kayapo (57.9%), the Paraguayan Chaco (16.4%), and the Chilean Alacalf (34.8%) and Yahgan (9.1%). The distribution of HTLV-I/II foci showed a geographic clustering of HTLV-I foci in the Andes highlands and of HTLV-II foci in the lowlands of South America. It was thus suggested that South American natives might be divided into two major ethnic groups by HTLV-I and HTLV-II carrier state.

Immunogenetics of HTLV-I/II and associated diseases

The ethnic background of human T-lymphotropic virus types I and II (HTLV-I/II) infections and associated diseases was investigated in association with human leukocyte antigens (HLA) (alleles) and haplotypes. Japanese HTLV-I carriers were characterized by two categories of HLA class I antigens (A24, A26, B7, B61, Cw1, and Cw7) and class II alleles (DRB1 *0101, 0803, 1403, 1501, and 1502 and DQB1 *0303, 0501, and 0601); one category was associated with adult T-cell leukemia (ATL) patients and the other with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. The ATL-associated haplotypes had unique DRB1-DQB1 alleles (0901-0303, 1501-0602, 1401-0503), which were correlated with a low immune responsiveness to HTLV-I, while the HAM/TSP haplotypes had different DRB1-DQB1 alleles (0101-0501, 0803-0601, 1502-0601), which were correlated with a high immune responsiveness to HTLV-I. Both ATL- and HAM/TSP-associated haplotypes were found among HTLV-I carriers and the patients from other ethnic groups (Jamaican blacks, Andes natives, South American mestizos, and Mashhadi Jews). HLA haplotypes of HTLV-II carriers were different from those of HTLV-I carriers among South American natives. These results suggested that HTLV-I/II infections and the associated diseases might be determined by immunogenetic factors segregated with HLA alleles and haplotypes.

HTLV-1 provirus load in peripheral blood lymphocytes of HTLV-1 carriers is diminished by green tea drinking

Human T‐cell lymphotropic virus type 1 (HTLV‐1) is causatively associated with adult T‐cell leukemia (ATL) and HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since a high level of HTLV‐1 provirus load in circulating lymphocytes is thought to be a risk for ATL and HAM/TSP, diminution of HTLV‐1 provirus load in the circulation may prevent these intractable diseases. Our previous study (Jpn J Cancer Res 2000; 91: 34–40) demonstrated that green tea polyphenols inhibit in vitro growth of ATL cells, as well as HTLV‐1‐infected T‐cells. The present study aimed to investigate the in vivo effect of green tea polyphenols on HTLV‐1 provirus load in peripheral blood lymphocytes on HTLV‐1 carriers. We recruited 83 asymptomatic HTLV‐1 carriers to examine HTLV‐1 provirus DNA with or without administration of capsulated green tea extract powder. Thirty‐seven subjects were followed up for 5 months by measuring HTLV‐1 provirus load after daily intake of 9 capsules of green tea extract powder per day (equivalent to 10 cups of regular green tea), and 46 subjects lived ad libitum without intake of any green tea capsule. The real‐time PCR quantification of HTLV‐1 DNA revealed a wide range of variation of HTLV‐1 provirus load among asymptomatic HTLV‐1 carriers (0.2‐200.2 copies of HTLV‐1 provirus load per 1000 peripheral blood lymphocytes). Daily intake of the capsulated green tea for 5 months significantly diminished the HTLV‐1 provirus load as compared with the controls (P=0.031). These results suggest that green tea drinking suppresses proliferation of HTLV‐1‐infected lymphocytes in vivo.