Yorio Hinuma

A Retrospective Study on Transmission of Adult T Cell Leukemia Virus by Blood Transfusion: Seroconversion in Recipients

Possible transmission of adult T cell leukemia virus (ATLV) by blood transfusion was studied retrospectively. Of 41 recipients of whole blood or blood components containing cells from donors having antibodies to antigen that is associated with ATLV (anti‐ATLA), 26 (63.4%) produced anti‐ATLA. However, no anti‐ATLA was detected in all 14 recipients of fresh‐frozen plasma prepared from anti‐ATLA‐positive donors. None of 252 recipients of blood with cell components from anti‐ATLA‐negative donors produced anti‐ATLA. The development of anti‐ATLA in the recipients may correspond to antibody production after establishment of primary infection with ATLV that is associated with cells in blood from anti‐ATLA‐positive donors who are ATLV carriers.

HIGH INCIDENCE OF HTLV ANTIBODY IN CARRIERS OF STRONGYLOIDES STERCORALIS

SIR,-A high incidence of carriage of human T-cell leukaemia virus (HTLV) has been observed in south-western Japan, including the Okinawa islands, where adult T-cell leukaemia (ATL) is endemic. A survey by the Okinawa Haematological Association has revealed about 50 cases of ATL in the past three years in Okinawa. Okinawa is also an endemic area for strongy10idiasis,2,3 though carriers of Strongyloides stercoralis are rare in other south-

Sera from adult T-cell leukemia patients react with envelope and core polypeptides of adult T-cell leukemia virus.

Sera from five Japanese patients with adult T-cell leukemia (ATL) showed in the immunofluorescence test for ATL-associated antigen (ATLA) titers ranging from 320 to 1280. Control sera from three healthy adults were negative. These eight sera were used to immunoprecipitate radiolabeled polypeptides from three cell lines infected with adult T-cell leukemia virus (ATLV) and two noninfected human cell lines. Cells were labeled either metabolically with [35S]cysteine, [35S]methionine, or [3H]glucosamine, or chemically with 125-iodine. Immunoprecipitates from cells, virus, and concanavalin A-enriched supernatants were analyzed by polyacrylamide gel electrophoresis. Cells producing ATLV contain gp68, the putative precursor to ATLV envelope polypeptides, gp46, and possibly p15, in addition to several nonglycosylated polypeptides between 40 to 70 kDa. Gp46 is shed into the culture medium and appears to be loosely attached to the viral and cellular surface. After purification on density gradients viral particles contain immunoreactive p24, p19, p15, and small amounts of gp46. Kinetics of synthesis, distribution, size, biochemical characteristics, and immunoreactivity of these polypeptides strongly suggest that most of them are structural components of ATLV or their precursors. Apparently, the intracellular ATLA complex predominantly represents precursors of viral structural polypeptides and gp46 is the viral envelope glycopolypeptide.

Primary infection of Japanese infants with adult T-cell leukaemia-associated retrovirus (ATLV): Evidence for viral transmission from mothers to children

Primary infection with adult T-cell leukemia virus (ATLV) was investigated by follow-up studies on 16 ATLV-seropositive mothers and their breastfed infants in an ATLV-endemic area of Japan. Maternal antibody to ATLV decreased in all the infants, and was detectable in only three of 12 infants tested 6 months after birth. Reappearance of the antibody 9-18 months after birth was observed in only four of the 16 infants. The ATLV-bearing cells in peripheral blood were detected in all 16 mothers after delivery. None of the 16 infants showed ATLV-bearing cells in peripheral or cord blood sampled at birth, or 1, 3 or 6 months after birth. However, virus-bearing cells in the blood became detectable 9-18 months after birth in 13 of the 16 infants. Maternal antibody and virus-bearing cells were never detected in a control group of seven infants of ATLV-seronegative mothers. These findings provide evidence for the high incidence of primary ATLV infection during early infancy among infants born to ATLV-seropositive mothers and suggest maternal viral transmission. Furthermore, samples of breast milk from all 12 seropositive mothers examined contained cell-associated ATLV capable of being transmitted to peripheral leucocytes of neonates. This finding suggests that one of the possible maternal transmission routes of ATLV is via breast milk.

Cloning, characterization and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factor

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N‐terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S‐transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.

Prevalence of a human retrovirus in native Japanese: Evidence for a possible ancient origin

The origin of a human retrovirus (ATLV or HTLV-I) is, at present, unknown although carriers of the virus have been found in Japan, the Caribbean basin and Africa. By means of a sero-epidemiological study, the Ainu people of Hokkaido, located in the northernmost island of Japan, were shown to have antibody to the virus in high frequency. Since the Ainu are regarded as descendants of the pre-agriculture native population of northern Japan, this finding appears to indicate that the retrovirus was already present in the aboriginal Japanese of prehistoric times.

Identification of two distinct elements in the long terminal repeat of HTLV-I responsible for maximum gene expression.

Human T‐cell leukemia virus type I has a unique sequence, pX, between env and the 3′ long terminal repeat (LTR). One of its products, p40, activates gene expression directed by the LTR in a trans‐acting manner. We have analysed the mechanism of this trans‐activation mediated by p40 in human T cells co‐transfected with a plasmid expressing p40 using the transient CAT gene expression. We identified two distinct elements in the LTR which are involved in maximum gene expression. The first was present in a 230‐bp fragment upstream from TATA box in the U3 region and behaved as a classical enhancer. This region was also shown to be responsible for trans‐activation by p40. This element alone together with functional p40 could direct the gene expression at only approximately 10% of the level achieved by the complete LTR and p40. The second element was present within a 300‐bp fragment downstream from the RNA start site and profoundly enhanced the gene expression in a way independent from trans‐activation mechanism. This enhancement was observed only when the element was located immediately downstream from the RNA start site without orientation preference. These two elements participate independently in the enhancement of gene expression.

Viral aetiology of adult T-cell leukaemia.

Overall conclusions Regarding the cause of leukaemias or malignant lymphomas, several advances in viral oncology and immunology have merged recently, resulting in a heightened appreciation of the associations between viral infection, immunodeficiency and genetic predisposition. In humans, EBV infection has been discussed thoroughly from these standpoints in relation to BL, X-linked lymphoproliferative syndrome, ataxia telangiectasia and malignant lymphomas after organ transplantations (Purtilo et al., 1984). In a previous section describing possible mechanisms of leukaemogenesis of ATL we discussed mainly transformation at the cellular level and not at the level of the host organism. It is apparent, however, that the fate of the transformed cell at the cellular level will be determined eventually by confrontation with the various immunological barriers of the host. Variation at the cellular level, of course, should be closely related to a changed response to the host immune mechanism. Cytogenetic alterations most probably endow transformed cells not only with a growth advantage at the cellular level but also with an ability to escape the immune surveillance of the host. The results described in this context clearly indicate that HTLV/ATLV is a prerequisite for the occurrence of ATL. It is also obvious, however, that this disease is ‘a chronic malignancy’ which requires a long period of time between viral infection and the occurrence of disease. This strongly indicates that some forces operate to select out a single cell and to initiate its monoclonal growth from the population of the cells which had been infected polyclonally with HTLV/ATLV. Although HTLV/ATLV is one of the major factors in the development of ATL, one or more additional factors seem to be necessary. Such factors can be assumed to be both exogenous and endogenous. It appears to us that ATL carcinogenesis results from the interaction of HTLV/ATLV with various biological, physical and chemical factors in the environment (Yamamoto, 1984).

Identification of the target cells in human B lymphocytes for transformation by epstein-barr virus

Abstract Lymphocytes from human umbilical cord blood were purified and then separated into rosette-forming cells (T cells) and non-rosette-forming cells (non-T cells). Non-rosette-forming cells were further divided by the technique of rosette formation, after neuraminidase treatment, into surface immunoglobulin (SIg)-carrying cells (B cells) and cells lacking SIg but carrying Fc receptors (null cells). The three cell subsets, T, B, and null cells, were examined for susceptibility to transformation by the B95-8 strain of Epstein-Barr virus (EBV) using the criteria of colony formation by transformed cells and/or transformation efficiency as judged by the days required for the first appearance of transformation. Not only the T cells but also the null cells were unsusceptible to transformation by EBV. In contrast, B cells were highly susceptible. In a study of the quantitative relationship between the target cells for viral transformation and those B cells which possessed SIg after an acid pH treatment, 10 lymphocyte preparations from three cord blood samples and seven adult peripheral blood samples were tested individually. The fraction size of transformable cells was determined by the growth-curve procedure for transformed cells while the procedure of direct membrane immunofluorescence with anti-IgM (μ-specific) serum and polyvalent anti-Ig serum was used to determine the fractions of SIg and SIg(M) cells. The actual fraction of EBV target cells was nearly equal to that of SIg(M) B cells but not to that of the total SIg B cells. Thirty-four lymphocyte preparations from eight cord and 26 adult peripheral blood samples were examined for the percentage of SIg B cells and for susceptibility to EBV transformation as assayed by the colony-formation procedure. EBV susceptibility and the fraction of SIg(M) B cells, but not of total SIg B cells, correlated nicely, suggesting that SIg(M)-bearing cells were probably the major target among the B lymphocytes for transformation by EBV.

Seroepidemiological study of antibodies to adult T-cell leukemia virus-associated antigen (ATLA) in free-ranging Japanese monkeys (Macaca fuscata).

Adult T-cell leukemia (ATL) in man is a newly described disease entity (7). A new human retrovirus named ATL-virus (ATLV) has been detected in cells derived from ATL patients, and specific antibodies to ATL associated antigen (ATLA) synthesized by ATLV-producer cells have been detected in sera from ATL patients (1). It was suggested that ATLA is ATLV-associated antigen(s) (9, 10). One of the striking characteristics of ATL is geographical clustering restricted to southwestern japan (8) and a high incidence of ATLA antibodies has been documented in adults in ATL endemic areas by seroepidemiological studies (2). We have detected the presence of antibodies to the antigens associated with Epstein-Barr virus (EBV), which is thought to be a human oncogenic herpes virus, in plasmas of free-ranging japanese monkeys, suggesting that they are susceptible to infection with EBV or EBV-like viral agents (Ishida and Yamamoto, in preparation). This result prompted us to examine whether or not these japanese monkeys are infected with another human tumor virus, ATLV. The present study shows the natural occurrence of anti-ATLA antibodies among a free-ranging japanese monkey (Macaca fuscata) population. The age dependency, sexual difference and familial-clustering of ATLA-antibody seropositive incidence were also revealed by an ethological study on the japanese monkey population. Fifty-one plasma specimens were obtained from a free-ranging japanese monkey population (5) consisting of 126 individuals in Nagano Prefecture (Honshu) where ATL in humans is rare (8). Since this japanese monkey population has been followed ethologically for the last thirty years, their age, sex, and mother-infant relations have been studied and recorded (Tokita, unpublished data). The age of the subjects (20 males and 31 females) ranged between 2 and 25 years old (mean age: 7.0). The ATLA antibodies and their titers were detected and determined using ATLA-bearing MT-I cells by means of the indirect immunofluorescence test described previously (I). The acetone-fixed MT-I cells were first treated with test