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Antibodies against three purified structural proteins of the human type-C retrovirus, HTLV, in Japanese adult T-cell leukemia patients, healthy family members, and unrelated normals.

The immunological relationship of human T-cell leukemia/lymphoma virus (HTLV) and the virus found in Japanese adult T-cell leukemia/lymphoma (ATL) was investigated in detail by testing the specific binding of serum antibodies from Japanese ATL patients and normal Japanese donors to the purified HTLV proteins p24, p19, and p15. Sera were prescreened for antibodies to p24. Of those positive, 67% of the ATL sera and 78% of the normal sera were further shown to have antibodies against p19. In both groups 17% had antibodies to p15. Generally, the average antibody titers were twice as high in ATL as in normal sera. Competition radioimmunoprecipitation assays done with various sera and involving HTLV-producing cells, virus-positive cells from a Japanese ATL patient, and virus-positive cultured T cells of one of his healthy family members as competing materials demonstrated no differences between the p24, p19, and p15 found in these cells. These results provide strong and detailed immunological evidence that the human retrovirus isolates first made from US patients with cutaneous T-cell malignancies, and those made later in Japanese ATL are either identical or very closely related strains of the same virus, HTLV, a finding verified in other detailed analyses of the HTLV genomes of the respective isolates. To date only HTLV-II, isolated from a US case of hairy-cell leukemia of a T-cell type, is a distinct additional human retrovirus class.

Adolescent↗

Methylation pattern of human T-cell leukemia virus in vivo and in vitro: pX and LTR regions are hypomethylated in vivo.

The methylation patterns of the gag, pol, env, pX and LTR regions of proviral DNA of human T-cell leukemia/lymphoma virus type I (HTLV) in fresh leukemic cells and established cell lines were examined using HpaII/MspI endonuclease. Peripheral blood lymphocytes (PBL) isolated from patients with adult T-cell leukemia/lymphoma (ATL) did not express viral antigens of HTLV, but PBL that had been cultured for 2 days did express these viral antigens. Most parts of the gag, pol and env regions of the HTLV provirus in PBL isolated from 12 ATL patients and PBL cultured for 2 days were hypermethylated as reported by others. In contrast, in 10 established cell lines that harbored HTLV genomes and expressed viral antigens, HTLV proviruses were hypomethylated. In one cell line, ATL-IK, which harbored an HTLV genome but did not produce viral antigens, the gag, pol and env regions were hypermethylated. However, two HpaII sites, one in the middle of the gag region and the other in the middle of the pol region, were not methylated even in PBL from most ATL patients. Furthermore, the pX and LTR regions were hypomethylated not only in established cell lines but also in PBL of ATL patients. The hypomethylation of the pX and LTR regions detected in fresh leukemic cells of ATL patients may have some etiological significance in cell transformation by controlling the level of transcription of these regions, or modulating the binding of some factors to these regions.

Adult↗

Effect of human serum anti-HTLV antibodies on viral antigen induction in vitro cultured peripheral lymphocytes from adult T-cell leukemia patients and healthy virus carriers.

Leukemic cells of adult T-cell leukemia (ATL) patients contain human T-cell leukemia virus type I (HTLV-I) genomes. However, viral antigens (HTLV-A) are not usually expressed in vivo. It has been shown that HTLV-I antigens are spontaneously induced after culture of peripheral blood lymphocytes (PBL) from patients with ATL and healthy carriers (HC), who are viral antibody-positive. In the present communication, we show the suppressive effect of human serum antibodies against HTLV-I on viral antigen expression in PBL derived from ATL patients and HC. Viral antigen synthesis in various PBL specimens is variable in terms of frequencies and kinetics. (1) Low concentrations (2%) of antibody-positive but not of antibody-negative sera efficiently suppressed HTLV antigen induction in lymphocytes from both ATL patients and HC. (2) An inhibitory effect was exerted by HTLV-I-specific antibodies which originated from either ATL, HC or even HTLV-I-positive monkeys. (3) Sera showed a fairly wide range of suppressive activity in view of their concentrations. (4) At the late stage of cultures even continuous presence of antibodies frequently failed to suppress antigen synthesis in lymphocytes. (5) Antibodies were less effective in suppressing the increase of antigens if they were added to cultures which had already HTLV-A-positive lymphocytes in antibody-free media. (6) Pretreatment with protein A completely abolished the activities of the suppressive plasmas. (7) Antibodies did not affect the inhibition of spontaneous or chemical induction of HTLV-A in 2 HTLV-I-positive cell lines. These results suggest that HTLV-I antigen expression in leukemic cells from patients with ATL in vivo and in vitro is partly regulated by antibodies to HTLV-I. Furthermore, at least 2 different pathways, one antibody-sensitive and the other -insensitive, might exist to express HTLV-A.

Antibodies, Viral↗

Sequence variations in LTR and env regions of HTLV-I do not discriminate between the virus from patients with HTLV-I-associated myelopathy and adult T-cell leukemia.

For detailed comparison of human T-cell leukemia virus type I (HTLV-I) in adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy (HAM), the nucleotide sequences of parts of the long terminal repeat (LTR) and env regions of the HTLV-I proviruses from 12 patients with HAM, 8 patients with ATL and one with both diseases were analyzed. About 340 bp of the LTR U3 region, about 450 bp of the 5' region and about 280 bp of the 3' region of env were sequenced directly in DNAs amplified by the polymerase chain reaction (PCR) with 2 or 3 sets of primers for each region. Nucleotide insertions, deletions or point mutations were observed at 50 positions in these regions of about 1,000 nucleotides length. None of these changes was specific to either HAM or ATL, and some changes were observed in proviruses from both cases of HAM and ATL. Moreover, the sequences of proviruses isolated from pairs of cell lines established from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) of the 4 patients with HAM also had different sequences. These results indicate that the proviruses from HAM and ATL are indistinguishable in these sequenced regions, suggesting that these 2 diseases are caused by infection with genetically indistinguishable HTLV-I. Therefore, the reason why these two distinct diseases, HAM and ATL, develop in HTLV-I carriers may be based on a host factor(s) or some other factor(s) rather than variation in the virus itself.

Base Sequence↗

Differences in immune functions between human T-lymphotropic virus type I carriers and patients with adult T-cell leukemia/lymphoma.

A variety of immunologic tests were compared between human T-lymphotropic virus type I (HTLV-I) carriers and patients with adult T-cell leukemia/lymphoma (ATL). The mitogenic responses of the lymphocytes to concanavalin A and phytohemagglutinin were depressed in the ATL patients but not in the carriers, while the response to pokeweed mitogen in the carriers and ATL patients was depressed compared to the HTLV-I-seronegative controls. A positive tuberculin reaction in the carriers was as frequent as in the controls, but less frequent in the ATL patients. A marked inhibition of the intracellular multiplication of Legionella pneumophila was observed within the monocytes isolated from the carriers, but not in the ATL patients or the normal controls. These results indicate that the ATL patients have severe immunodeficiency, while the HTLV-I carriers have some activation of anti-microbial activity in monocytes although they are somewhat immunosuppressed.

Adult↗

Ocular manifestations in adult T-cell leukemia/lymphoma.

Ocular manifestations of adult T-cell leukemia/lymphoma (ATL) are rare events. However, several ocular lesions which resulted from human T-cell leukemia virus type I (HTLV-I) infection have been reported, including direct infiltration of ATL cells, cytomegalovirus retinitis, and HTLV-I-associated uveitis (HAU). The aim of this study was to characterize ocular involvement in ATL and to correlate these lesions with HTLV-I proviral DNA integration. Three patients with acute-type ATL and ocular lesions were evaluated hematologically and ophthalmologically. Analysis of HTLV-I proviral DNA was carried out with a standard Southern blot technique using DNA from abnormal lymphocytes in peripheral blood. Two patients developed intraocular lesions located within intermediate and/or posterior segments which were caused by infiltration of ATL cells. Ocular lesions in one patient, which were localized to the anterior-intermediate segment, closely resembled those of HAU. Analysis of HTLV-I proviral DNA revealed multiple integrations in all three patients. The present study indicated heterogeneity in ocular manifestations of ATL. Multiple HTLV-I proviral DNA integrations may be associated with intraocular involvement in this disease.

Adult↗

Treatment for adult T-cell leukemia.

The purpose of this study was to clarify the clinical efficacy of multidrug chemotherapy for aggressive adult T-cell leukemia (ATL). We report the therapeutic results of treatment of patients with aggressive ATL undertaken between 1986 and 1995. A total of 120 newly diagnosed patients with a performance status of 0-3 and aged < 70 years at diagnosis were entered into the study. Clinical features, including clinical subtypes, serum levels of lactate dehydrogenase and blood urea nitrogen, the response to chemotherapy, and doses of individual chemotherapeutic agents, were evaluated. Of the 120 patients enrolled, 97 had acute-type and 23 lymphoma-type ATL. The complete response rate and median survival of these patients were 25.3% and 9 months, respectively. The 2- and 5-year survival rates were 18.4% and 8%, respectively, and five patients have been alive for > 5 years and are disease-free. These long-term survivors had good prognostic factors at diagnosis. There was no correlation between the doses of the various chemotherapeutic agents and the survival duration. These results indicate that ordinary combined chemotherapy has limited ability to improve the prognosis of aggressive ATL. Our previous study indicated that expression of P-glycoprotein in ATL cells might be involved in resistance to chemotherapeutic agents, particularly doxorubicin, vincristine, and etoposide. Therefore, new therapeutic strategies will be necessary to improve the prognosis of ATL patients.

Adult↗

Quality evaluation of ultrasound imaging in the carotid artery based on normalization and speckle reduction filtering.

Image quality is important when evaluating ultrasound images of the carotid for the assessment of the degree of atherosclerotic disease, or when transferring images through a telemedicine channel, and/or in other image processing tasks. The objective of this study was to investigate the usefulness of image quality evaluation based on image quality metrics and visual perception, in ultrasound imaging of the carotid artery after normalization and speckle reduction filtering. Image quality was evaluated based on statistical and texture features, image quality evaluation metrics, and visual perception evaluation made by two experts. These were computed on 80 longitudinal ultrasound images of the carotid bifurcation recorded from two different ultrasound scanners, the HDI ATL-3000 and the HDI ATL-5000 scanner, before (NF) and after (DS) speckle reduction filtering, after normalization (N), and after normalization and speckle reduction filtering (NDS). The results of this study showed that: (1) the normalized speckle reduction, NDS, images were rated visually better on both scanners; (2) the NDS images showed better statistical and texture analysis results on both scanners; (3) better image quality evaluation results were obtained between the original (NF) and normalized (N) images, i.e. NF-N, for both scanners, followed by the NF-DS images for the ATL HDI-5000 scanner and the NF-DS on the HDI ATL-3000 scanner; (4) the ATL HDI-5000 scanner images have considerable higher entropy than the ATL HDI-3000 scanner and thus more information content. However, based on the visual evaluation by the two experts, both scanners were rated similarly. The above findings are also in agreement with the visual perception evaluation, carried out by the two vascular experts. The results of this study showed that ultrasound image normalization and speckle reduction filtering are important preprocessing steps favoring image quality, and should be further investigated.

Carotid Arteries↗

Epstein-Barr virus (EBV) genome carrying monoclonal B-cell lymphoma in a patient with adult T-cell leukemia-lymphoma.

A Japanese patient with adult T-cell leukemia-lymphoma (ATL) showed a disease progression from the smoldering type to the chronic type and finally to the acute type. The patient was variously treated, including 2'-deoxycoformycin, with some beneficial effects. During the chronic type he developed a composite lymphoma consisting of T-cell lymphoma (ATL) of medium-sized cells and B-cell lymphoma of diffuse large cell type. At that time, he also suffered from miliary tuberculosis and adenovirus type 11-induced hemorrhagic cystitis, indicating that he was in a marked immunodeficient state. Southern-blot analysis revealed that the two malignancies have distinct clonal origin on the basis of the following results: (1) clonally rearranged T-cell receptor beta-chain gene (TcR-beta gene) and germline configuration of immunoglobulin heavy chain gene (IgH gene) in ATL leukemic cells, (2) clonal rearrangement of IgH gene in lymphoma cells, indicating a monoclonal B-cell lymphoma, (3) monoclonal integration of HTLV-I provirus in ATL leukemic cells, (4) definite presence and monoclonal origin of EBV genome in lymphoma cells. This is the first report of secondary EBV genome carrying monoclonal B-cell lymphoma in an ATL patient. It is suggested that the immunodeficient state in the patient with ATL allows the emergence of EBV-related B-cell lymphoma.

Adult↗

Production of interleukin-1 beta-like factor with synovial cell growth promoting activity from adult T-cell leukemia cells.

We observed a case of adult T-cell leukemia (ATL) with proliferative synovitis. Culture supernatants from ATL cells (ATL-SN) obtained from the peripheral blood constitutively produced an interleukin-1 (IL-1)-like factor in vitro, as shown by the growth inhibition factor (GIF) assay using the A375 melanoma cell line and the lymphocyte activating factor (LAF) assay using C3H/HeJ thymocytes. Neutralization studies indicated that polyclonal antibodies against IL-1 beta blocked most (80%) of the activity in ATL-SN. In addition, increased amounts of IL-1 beta mRNA were found in the ATL cells by dot blot analysis. Sephacryl S-200 chromatography showed that the molecular weight of this factor was approximately 17.5 kDa, and Western blot analysis revealed that this factor reacted with polyclonal anti-IL-1 beta antibody under the reduced condition. The isoelectric point was 7.5. Furthermore, ATL-SN showed significant activity in promoting the growth of synovial cells in parallel with IL-1 activity. These data suggest that the constitutive production of this IL-1 beta-like factor might be responsible for proliferative synovitis in this case.

Aged↗

Identification of HLA class I-restricted tumor-associated antigens in adult T cell leukemia cells by mass spectrometric analysis.

OBJECTIVE: In this study, we attempted a comprehensive analysis of MHC class I-bound peptides in adult T cell leukemia (ATL) cells in order to identify as many tumor-associated antigens (TAAs) as possible that could be used for CTL-based immunotherapy. METHODS AND RESULTS: Using mass spectrometry combined with reversed-phase liquid chromatography, we could sequence 188 HLA class I-restricted candidate peptides from three ATL-derived cell lines. In accordance with the restrained expression of HTLV-I viral RNA in these cell lines, there were no HTLV-I-encoded peptides among these candidates. Based on the differential expression between ATL cells and normal CD4+ T cells, we selected 10 novel peptides as T cell epitopes of overexpressed source proteins. RT-PCR analysis revealed that 5 source proteins including PRAME, a known tumor-testis antigen, were highly expressed in the majority of 16 ATL cases. Furthermore we could induce PRAME-specific CTLs in vitro from an HLA-B62+ healthy donor that showed specific cytotoxicity against HLA-B62+ PRAME+ ATL cells. CONCLUSION: These results demonstrate that comprehensive analysis of HLA class I-bound peptides by mass spectrometry is useful for identification of TAA-derived peptides in ATL. Considering that expression patterns of leukemia/lymphoma-associated antigens vary from case to case, this approach appears to be suitable for the tailor-made immunotherapy of hematological malignancies.

Antigens, Neoplasm↗

Methylation analysis of the adenomatous polyposis coli (APC) gene in adult T-cell leukemia/lymphoma.

We investigated methylation status of the adenomatous polyposis coli (APC) gene in adult T-cell leukemia/lymphoma (ATL). APC methylation was found in 15 of 31 (48%) primary samples, and 2 of 4 (50%) ATL cell lines. Methylation of the APC gene occurred more frequently in acute ATL (12/21) (57%) than chronic ATL (1/8) (13%) (P = 0.03). APC was not expressed in the APC-methylated ATL cell line ST1. Demethylation with 5-azacytidine treatment restored APC expression in the ST1 cell line. Our data show that hypermethylation of the APC gene is involved in the pathogenesis of ATL.

Adenomatous Polyposis Coli Protein↗

Prognosis of HTLV-I-positive renal transplant recipients.

HTLV-I is the pathogen that causes adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy (HAM). The rate of disease development is low and the latency time is a few decades. However, the possible influence of immunosuppression on this disease development is unclear. The purpose of this study was to investigate the risk of development of ATL and HAM among the large number of HTLV-I-positive renal transplant recipients in western Japan. In principle immunosuppressive drugs have the possibilities to accelerate ATL development but are thought to suppress HAM development. Of 120 renal transplant recipients, 10 HTLV-I-positive recipients were reviewed, none of whom developed ATL or HAM. There are 11,896 dialysis patients in Japan and 300 dialysis patients in Okinawa who are registered with the JOTN for cadaveric renal transplant. The numbers of HTLV-I-positive patients in these groups were 97 (0.82%) and 26 (8.67%), respectively. These numbers are thought to be sufficient for an HTLV-I-positive recipient pool for HTLV-I-positive donors. Ten cases of ATL development and two of HAM development have been previously reported. Because of low number of ATL development, renal transplantation does not appear to be a contraindication for HTLV-I-positive chronic renal failure patients. In other words, kidneys from HTLV-I carriers, which include cadaveric donors, could be used for HTLV-I-positive recipients.

Adult↗

A survey of advanced trauma life-support training for trainees in acute surgical specialties.

There have been few published data describing the demand for and amount of Advanced Trauma Life Support (ATLS) training received by trainees in acute surgical specialties. We undertook a survey on aspects of ATLS training, questioning all senior house officers in accident and emergency (A & E) medicine, general surgery and orthopaedic surgery in the west of Scotland in January 1995. More general surgical trainees were ATLS trained than orthopaedic or A & E trainees. Ninety-seven per cent of respondents felt ATLS was essential training for FRCS. There were few problems gaining study leave for the courses, but concern was expressed regarding the long waiting lists. ATLS training is therefore thought to be essential for surgical trainees, and the number of ATLS courses needs to be increased to cope with the demand.

Attitude of Health Personnel↗

Expression and phosphorylation status of retinoblastoma protein in adult T-cell leukemia/lymphoma.

The deletion or hyperphosphorylation of the retinoblastoma protein (pRB), is reported to progress various tumors. But its relevance to adult T-cell leukemia/lymphoma (ATL) remains to be elucidated. To better understand the role of pRB in ATL, we examined the expression and phosphorylation status of pRB in three ATL cell lines and 43 clinical samples, eight peripheral blood samples and 35 lymph node samples, from patients with ATL by Western blotting. In addition, 30 lymph node sections were also evaluated immunohistochemically. As a result, Western blotting analysis revealed that the pRB in the ATL cell lines was in the hyperphosphorylated, but that in 39 of 43 clinical samples, pRB was exclusively in the hypophosphorylated form. Four peripheral blood samples were negative for pRB. Immunohistochemistry revealed that the lymph nodes of all of 30 patients tested were positive for pRB at various staining levels, weak, mild, and strong. But weak expression may be essentially negative for pRB function. Patients with weak pRB expression in their lymph nodes lived significantly shorter lives than those with mild expression. Surprisingly, patients with strong expression also showed a significantly worse prognosis than those with mild expression. Although only the absence of pRB expression was considered previously to be indicative of RB functional loss, it has been reported recently that overexpression of pRB is correlated with progression of disease in patients with advanced bladder carcinoma or follicular lymphoma. These findings indicate that pRB controls tumor proliferation not only as a cell cycle regulator but also by other mechanisms, possibly through the inhibition of apoptosis, as suggested by recent findings in an osteosarcoma cell line, Saos-2. In conclusion, pRB may play an essential role in its hypophosphorylated form for progression of ATL, as well as a cell cycle promoter in hyperphosphorylated or negative/excessive reduced form.

Adult↗

HTLV-1 proviruses encoding non-functional TAX in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1). TAX, the major transactivator of HTLV-1, has been implicated in the immortalization of infected T-cells, but molecular mechanisms of in vivo malignant cell transformation induced by HTLV-1 remain unclear. To investigate the role of TAX in the monoclonal proliferation of ATL cells, we determined the nucleotide sequence of tax DNA clones obtained from 6 ATL patients and analysed the biological function of their products. We found that ATL cells from 2 of these patients possessed tax with a nonsense or frame-shift mutation resulting in the premature termination of its protein product, which was no longer functional. This strongly argued against an indispensable role of TAX for the maintenance of ATL cells in vivo. On the other hand, the frequency of nucleotide substitutions found in non-functional tax DNA clones from these patients was significantly lower than those in functional tax DNA clones from the others, suggesting a role for TAX in the genome instability of infected cells. Although mismatch repair defects in the microsatellite markers, including those in hMSH3, hMSH6, BAX, TGF-beta RII, and E2F4 genes, were infrequent, we found an increase in the number of CAG repeats of the E2F4 microsatellite marker in 1 patient. These findings indicate that while TAX may be a necessary prerequisite for malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo.

Aged↗

Poor outcome of autologous stem cell transplantation for adult T cell leukemia/lymphoma: a case report and review of the literature.

A limited number of patients with adult T cell leukemia/lymphoma (ATL) who received autologous stem cell transplantation (ASCT) have been reported. We report here a case of fatal systemic Candida krusei infection in a female patient with ATL undergoing ASCT. All of the eight patients (including seven patients in the literature) with ATL who received ASCT developed relapse of ATL or death due to ASCT complication, irrespective of subtype or remission state of ATL, source or selection of SCT or conditioning regimen. At present, ASCT appears to provide little benefit for ATL in contrast to that for other types of aggressive non-Hodgkin's lymphoma.

Adult↗

Distinct modes of ATR activation after replication stress and DNA double-strand breaks in Caenorhabditis elegans.

ATM and ATR are key components of the DNA damage checkpoint. ATR primarily responds to UV damage and replication stress, yet may also function with ATM in the checkpoint response to DNA double-strand breaks (DSBs), although this is less clear. Here, we show that atl-1 (Caenorhabditis elegans ATR) and rad-5/clk-2 prevent mitotic catastrophe, function in the S-phase checkpoint and also cooperate with atm-1 in the checkpoint response to DSBs after ionizing radiation (IR) to induce cell cycle arrest or apoptosis via the cep-1(p53)/egl-1 pathway. ATL-1 is recruited to stalled replication forks by RPA-1 and functions upstream of rad-5/clk-2 in the S-phase checkpoint. In contrast, mre-11 and atm-1 are dispensable for ATL-1 recruitment to stalled replication forks. However, mre-11 is required for RPA-1 association and ATL-1 recruitment to DSBs. Thus, DNA processing controlled by mre-11 is important for ATL-1 activation at DSBs but not following replication fork stalling. We propose that atl-1 and rad-5/clk-2 respond to single-stranded DNA generated by replication stress and function with atm-1 following DSB resection.

Animals↗