Acute varicella hepatitis in human T-cell lymphotrophic virus types I and II infection

The delayed-gamma neutron activation facility at Brookhaven National Laboratory was originally calibrated using an anthropomorphic hollow phantom filled with solutions containing predetermined amounts of Ca. However, 99% of the total Ca in the human body is not homogeneously distributed but contained within the skeleton. Recently, an artificial skeleton was designed, constructed, and placed in a bottle phantom to better represent the Ca distribution in the human body. Neutron activation measurements of an anthropomorphic and a bottle (with no skeleton) phantom demonstrate that the difference in size and shape between the two phantoms changes the total body calcium results by less than 1%. To test the artificial skeleton, two small polyethylene jerry-can phantoms were made, one with a femur from a cadaver and one with an artificial bone in exactly the same geometry. The femur was ashed following the neutron activation measurements for chemical analysis of Ca. Results indicate that the artificial bone closely simulates the real bone in neutron activation analysis and provides accurate calibration for Ca measurements. Therefore, the calibration of the delayed-gamma neutron activation system is now based on the new bottle phantom containing an artificial skeleton. This change has improved the accuracy of measurement for total body calcium. Also, the simple geometry of this phantom and the artificial skeleton allows us to simulate the neutron activation process using a Monte Carlo code, which enables us to calibrate the system for human subjects larger and smaller than the phantoms used as standards.     

The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

Substrates and inhibitors of human T-cell leukemia virus type I protease

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.     

Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.     

Seroepidemiology of human T-cell lymphotropic virus type I/II in northeastern Brazil

We investigated the prevalence of human T-lymphotropic virus I/II (HTLV-I/II) infection in Bahia, a state in Northeastern Brazil. Healthy individuals (n = 327) and patients (n = 337) with a variety of diseases were screened for antibodies to HTLV-I/II using an enzyme immunoassay and Western blot. The overall prevalence among healthy subjects was 1.8% (six of 327); among patients it was 18.4% (62 of 337). Patients with AIDS had the highest prevalence of HTLV-I/II infection, 22.7% (20/88), followed by randomly selected patients from an infectious disease hospital, 19.4% (25 of 129), and tuberculosis patients, 11.1% (10 of 90). Four of 14 patients with myelopathy and three of 16 patients with lymphoid leukemia or lymphoma were seropositive for HTLV-I/II. Sixty-three of 68 HTLV-I/II-positive specimens were then typed: 53 patients were HTLV-I positive, three patients were HTLV-II positive, and in seven patients the assay could not distinguish infection by HTLV-I or II. The finding among HIV-seropositive intravenous drug users in Bahia of coinfection with HTLV-I is contrasted with reports from other areas in which dual infection occurs with HTLV-II. Although high prevalence of HTLV-I infection was found in Bahia, the extent and clinical manifestations of HTLV-I/II infection in Brazil remains imprecisely defined, and further studies are needed.     

Absence of human T-lymphotropic virus type I in Japanese patients with cutaneous T-cell lymphoma

Cutaneous T-cell lymphoma (CTCL) is a disease entity characterized by a primary sporadic T-cell proliferation in the skin. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma. Recently, several authors have detected the HTLV-1 genome in genomic DNA from patients with CTCL and proposed a causal relation of HTLV-1 to CTCL. However, it remains controversial because these studies contain some problems in materials used to detect HTLV-1. We investigated both fresh and cultured T lymphocytes (128 specimens) derived from 50 Japanese patients with CTCL, where HTLV-1 is endemic, by using polymerase chain reaction with four sets of primers including gag, pol, env, and pX regions of HTLV-1 to elucidate the relationship between HTLV-1 and CTCL in Japan. However, none of the 128 DNA specimens revealed positive for HTLV-1 in contrast to the previous studies. We conclude that CTCL, which does not include HTLV-1, is present although the pathogenesis of CTCL may be different by areas or races.     

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.     

Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome

Two principles are presented that describe directional confounds associated with baseline differences: Principle 1: Change scores are confounded with baseline whenever data are skewed. Principle 2: When baseline differences are real, ANCOVA has a directional bias that magnifies differences in one direction and masks those in the other direction. Both principles involve a directional bias that is related to the direction of baseline difference and the direction of the hypothesized difference in change. Ethical dilemmas arise if decisions (whether or not to transform, whether or not to use ANCOVA) are chosen in order to maximize power by capitalizing on the directional bias and Type 1 error.     

Antinuclear autoantibodies in human T-cell leukemia/lymphoma virus type I carriers in French Guiana

To compare the rate of antinuclear antibodies (ANA) in HTLV-I carriers and negative individuals in French Guiana, 350 sera (175 HTLV-I carriers, either symptomatic or not, and 175 controls) were screened for ANA, using an immunofluorescence assay. All positive sera were tested for autoantibodies against extractable nuclear antigens, histones and double stranded DNA. ANA were detected in 9.71% of the HTLV-I carriers and 3.43% of the control group (p < 0.05). There was no difference in ANA distribution by age, sex, or ethnic group. Neither was there any difference between asymptomatic and symptomatic HTLV-I individuals. However, ANA of medical interest were significantly higher (p < 0.04) in HTLV-I seropositive Creoles than in seropositive Noir-Marrons.     

Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV-I) tax and HTLV-II tax proteins

The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone-related protein (PTHrP) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia.     

Risk factors for adult T-cell leukemia among carriers of human T-lymphotropic virus type I

The presence of circulating "flower cells" and a low prevalence of antibody to Tax regulatory protein of human T-lymphotropic virus type I (HTLV-I) are characteristics of adult T-cell leukemia (ATL). To examine the predictability of levels of HTLV-I antibodies and of flower cell-like abnormal lymphocytes (Ably) for the risk of ATL among asymptomatic HTLV-I carriers, we prospectively evaluated the levels of viral markers of five HTLV-I carriers who developed ATL and 38 age-, sex-, and screen-matched HTLV-I-positive controls in the Miyazaki Cohort Study. After accounting for matching factors, Ably level was slightly, but not significantly, higher among cases than among controls (P =.13). Anti-HTLV-I (odds ratio [OR] = 1.6 per twofold dilution; 95% confidence interval [CI] 0.94, 3.8) was associated with ATL diagnosis, but antibody to Tax regulatory protein (anti-Tax) was not (OR = 0.78; 95% CI 0.26, 1.7). Anti-Tax level was low for all ATL cases for up to 10 years preceding their diagnosis, independent of the level of anti-HTLV-I titer. HTLV-I carriers with a higher anti-HTLV-I titer and a lower anti-Tax reactivity may be at greatest risk of ATL.     

Arsenic trioxide inhibits growth of human T-cell leukaemia virus type I infected T-cell lines more effectively than retinoic acids

Adult T-cell leukaemia (ATL) is difficult to cure using conventional therapies. Recently the therapeutic possibility of retinoic acids (RA) has been reported. In this study, suppression of in vitro growth of human T-cell leukaemia virus type I (HTLV-I) infected T-cell lines and fresh ATL cells by arsenic trioxide (As2O3) were evaluated by comparison with a series of RA derivatives. Proliferation of four HTLV-I-infected T-cell lines was significantly reduced within 72 h by 1.0 micromol/l As2O3. Growth of two out of four HTLV-I-infected T-cell lines was also inhibited by 1.0 micromol/l RA, but to a lesser extent than by As2O3. The mechanism of this growth inhibition was due to the induction of apoptosis. Apoptosis was also induced in fresh ATL cells from patients by AS2O3, but far less by RA. As described in patients with acute promyelocytic leukaemia, 1.0 micromol/l of As2O3 can be safely achieved in the serum of patients; however, it is difficult to maintain this concentration of RA. In conclusion, As2O3 has therapeutic potential for the treatment of ATL and may be far more clinically beneficial than RA.     

Enhanced human T-cell lymphotropic virus type I expression following induction of the cellular stress response

Human T-cell lymphotropic virus type I (HTLV-I) infection is typically associated with long incubation periods between virus exposure and disease manifestation. Although viral protein expression is considered to play an important role in the pathogenesis of HTLV-I-associated diseases, limited information is known regarding host cell mechanisms that control viral gene expression. This study was designed to evaluate modulation of HTLV-I gene expression following induction of the cellular stress response in HTLV-I-infected lymphocytes. The cellular stress response was elicited by treatment with either Na arsenite or thermal stress and was monitored by demonstrating increased expression of the 72-kDa heat shock protein. Induction of the cellular stress response in HTLV-I-infected lymphocytes resulted in significantly increased HTLV-I-mediated syncytia formation due to enhanced HTLV-I envelope (gp46) expression. Intracellular viral proteins and released p24 capsid protein were increased in stressed infected lymphocytes as compared to nonstressed infected lymphocytes. Furthermore, HTLV-I-LTR reporter gene constructs had increased activity (three- to sixfold) in a transiently transfected, uninfected lymphocyte cell line following induction of the cellular stress response. Quantitation of HTLV-I RNA expression by slot blot analysis of infected lymphocytes suggested variable increases in RNA accumulation. Northern blot analysis demonstrated no qualitative changes in expression of RNA species. These data suggest a relationship between modulation of viral replication and a basic cellular response to stress and have important implications for understanding host cell control mechanisms of HTLV-I expression.