A novel adult T cell leukemia-derived cell line (SALT-3) susceptible to human immunodeficiency virus type 1 infection

A novel human T cell line (SALT-3) was established from the pleural effusion of a patient with adult T cell leukemia (ATL) of lymphoma type. SALT-3 showed atypical T cell markers such as CD1-CD2-CD3-CD4+CD5+CD7+CD8-CD19-CD20-CD25+HLA-DR+. T cell receptor alpha/beta and gamma/delta were undetectable. Human T cell lymphotropic virus type 1 (HTLV-I) particles were seen on SALT-3 cells by electron microscopic analysis. HTLV-I gag p19, proviral DNA and mRNA of HTLV-I genes were also detected in the cells. Chromosome analysis showed abnormal karyotypes as 47, XY, partial trisomy of No.3 chromosome, and trisomy of No. 7 chromosome. Furthermore, SALT-3 were susceptible to the infection of human immunodeficiency virus type 1 (HIV-1) and the cells were rapidly killed after HIV-1 infection. This newly established HTLV-I-infected human T cell line would be a useful tool to study biological activities of atypical type of ATL cells and to examine the cytotoxic effects of HIV-1 and it's modulators.     

Human T lymphotropic virus type I (HTLV-I)-specific T helper cell responses from HTLV-I seronegative patients with chronic myelopathy and MS in Japan

Human T lymphotropic virus type I (HTLV-I) is a human retrovirus etiologically linked to Adult T cell leukemia (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although most HAM/TSP patients have high anti-HTLV-I antibody titers in their sera, HTLV-I infected but seronegative patients with neurological diseases have been reported. To clarify whether seronegative, HTLV-I related neurological disease may exist, we have developed a method that measures the production of interleukin-2 (IL-2) from HTLV-I synthetic peptide-stimulated peripheral blood lymphocytes (PBL) of HTLV-I infected persons. This method is sensitive enough to detect exposure to HTLV-I before seroconversion or even before detection by PCR. We examined 12 patients with chronic progressive myelopathy and eight patients with multiple sclerosis (MS) in central Japan, where the prevalence rate of HTLV-I is between one and four percent among asymptomatic blood donors, using the IL-2 production assay. None of them were positive by the assay, suggesting seronegative HTLV-I myelopathy is very rare among patients with chronic progressive myelopathy and MS in Japan.     

Analysis of in vivo cell proliferation of ATL using SCID mice

We made a model of in vivo cell proliferation of leukemic cells from adult T cell leukemia (ATL) patients using severe combined immunodeficient (SCID) mice. SCID mice injected with ATL cells from 6 of 8 ATL patients were found to have the tumor. DNA analysis revealed that the clone of the cells proliferating in mice was the same as that of the original leukemic cells. Histologic examination showed that the pattern of the infiltration of ATL cells in mice was similar to that of an ATL patient. Next, we examined the tumorigenicity of HTLV-I infected cell lines using SCID mice. Seven HTLV-I infected cell lines were injected into SCID mice and it was found that 4 of them were capable of proliferating in SCID mice. HTLV-I infected cell lines of non-leukemic cell origin could not engraft in SCID mice, indicating that these cells seemed not to have the enough genetic changes to acquire the tumorigenic potential. Analysis of gene expression suggested that neither IL-2 nor HTLV-I viral product was directly involved in the neoplastic cell growth of ATL. Furthermore, T cells immortalized by introduction of Tax could not engraft in SCID mice, indicating that the expression of tax gene seemed not to be sufficient for the neoplastic cell growth in vivo.     

Inhibition of plating of human T cell leukemia virus type I and syncytium-inducing types of human immunodeficiency virus type 1 by polycations

We examined the effects of polycations, namely, diethylaminoethyl-dextran (DEAE-dextran) and hexadimethrine bromide (Polybrene), on infection with the retroviruses human T cell leukemia virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus type 1 (HIV-1). The plating of vesicular stomatitis virus (VSV) pseudotype bearing envelope antigens of HTLV-I [VSV(HTLV-I)] was inhibited about 2- and 10-fold by treatment with DEAE-dextran and Polybrene, respectively. The formation of HTLV-I viral DNA detected 1 day after infection was also inhibited by these polycations. In contrast, polycations enhanced the plating of the VSV (HTLV-II) pseudotype two- to threefold. The polycations did not affect the plating efficiency of HTLV-I or HTLV-II when added after virus adsorption. Infection of human T cell lines, peripheral blood lymphocytes (PBLs), or brain-derived cells with syncytium-inducing (SI) types of HIV-1 strains (GUN1 and IIIB) was inhibited 3- to 20-fold by polycations. However, infection of PBLs or monocyte-derived macrophages with the macrophage-tropic Ba-L or SF162 strain was enhanced 1.5- to twofold by polycations. On the other hand, syncytium formation in coculture induced by HTLV-I, HTLV-II, or HIV-1 was enhanced two- to threefold unanimously by DEAE-dextran or Polybrene. Although polycations have been used to potentiate human retrovirus adsorption, they inhibited infection of cell-free HTLV-I or SI-type HIV-1 strains.     

The pharmacology of impulsive behaviour in rats III: the effects of amphetamine, haloperidol, imipramine, chlordiazepoxide and ethanol on a paced fixed consecutive number schedule

Granulocyte-colony stimulating factor (G-CSF) was originally found to induce proliferation and differentiation of normal granulocyte progenitors. Recent studies demonstrated that G-CSF induces growth of some malignant cells, including lymphoid cells. G-CSF is now widely and successfully used to treat neutropenia induced by intensive chemotherapy, and the responsive growth of malignant cells becomes a major clinical issue. Adult T-cell leukemia (ATL) is a malignant lymphoid disease of T cells, etiologically associated with human T cell lymphotropic virus type I (HTLV-I). We demonstrated that primary ATL cells in about 80% of patients expressed cell surface G-CSF receptor (G-CSFR). Our recent data also show that ATL cells from a third of the patients show responsive growth to G-CSF ex vivo. Several patients whose ATL cells proliferated in response to G-CSF showed a significant increase of the ATL cell count after administration of G-CSF in vivo. These observations suggest caution for it's routine clinical use in ATL. The molecular mechanism of G-CSF responsive growth of ATL cells is obscure, however the population of G-CSFR expressing cells is larger in responsive cases than in nonresponsive cases. Expression of G-CSFR on ATL cells may relate to the expression of Tax protein encoded by HTLV-I. Precise studies on G-CSFR signaling in ATL cells are necessary for the safe use of G-CSF routinely for ATL patients.     

Human T cell lymphotropic retroviruses: association with diseases of the nervous system

In less than 1% of persistently infected individuals, human T cell lymphotropic virus type I (HTLV-I) causes a chronic inflammatory disease of the central nervous system (CNS), called HTLV-I-associated myelopathy/tropical spastic paraparesis. Important prerequisites for disease induction are oligoclonal expansion of HTLV-I-infected CD4+ T lymphocytes, their trafficking across the blood-brain barrier, expression of viral proteins in the CNS, and an increased immune response within the CNS. The HTLV-I Tax1 protein has unique abilities to transactivate viral and cellular genes in T lymphocytes, but possibly also in cells of the CNS. Thus Tax1 supports the persistence of HTLV-I, the ongoing immune responses within the CNS and the destruction of myelin and axons, most pronounced in the thoracic spinal cord.     

Reciprocal interactions between human cytomegalovirus and human T cell leukemia-lymphoma virus type I in monocyte-derived macrophages cultured in vitro

Infection of macrophages with human cytomegalovirus (HCMV) has been shown to be nonlytic and exclusively cell associated. Human T cell leukemia-lymphoma virus type I (HTLV-I) is capable of establishing productive infection in macrophages. We studied the interactions between HCMV and HTLV-I in monocyte-derived macrophages cultured in vitro. We found that coinfection of macrophages with HCMV and HTLV-I significantly enhanced HCMV replication, resulting in release of infectious HCMV from dually infected cells. On the other hand, HCMV inhibited HTLV-I replication in macrophages coinfected with both viruses. Reciprocal interactions between HCMV and HTLV-I were mediated by their trans-acting proteins. Results of transfection studies demonstrated that the tax gene product of HTLV-I alone was capable of upregulating HCMV production. In a transient gene expression assay the immediate-early 2 (IE2) protein of HCMV alone could inhibit HTLV-I replication, whereas the IE1 protein, which had no effect by itself, produced a synergistic inhibitory effect together with the IE2 protein. Results from this study suggest that in vivo double infection of macrophages with HCMV and HTLV-I may contribute to the dissemination of HCMV infection in patients suffering from HTLV-I-associated T cell leukemia-lymphoma.     

Biochemical measures of thiamine deficiency

Human T cell lymphotropic virus type I (HTLV-I)-induced immunosuppression has been suggested to explain the occurrence of crusted scabies in HTLV-I-infected patients. HTLV-I is the etiologic agent of adult T cell leukemia/lymphoma (ATL). Crusted scabies diagnosed in 6 HTLV-I-seropositive patients was studied to look for an association with ATL. Four of the 6 either had concomitant ATL when crusted scabies was diagnosed or developed ATL a few months later. These findings suggest that the occurrence of crusted scabies in patients seropositive for HTLV-I could represent a sign of marked immunosuppression related to ATL.     

Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities.     

Human T cell lymphotropic virus type I does not increase human immunodeficiency virus viral load in vivo

Human T lymphotropic virus type I (HTLV-I) can increase human immunodeficiency virus (HIV) replication in vitro, and several studies suggest that HTLV-I accelerates the progression of HIV infection. To determine whether HTLV-I enhances HIV replication in vivo, a case-control study was done of serum HIV viral load, using polymerase chain reaction, in 23 subjects with HTLV-I/HIV coinfection and 92 control subjects with HIV single infection. The geometric mean serum RNA level was 11,482 copies/mL in the coinfected group and 13,804 in the single-infection group (P = .57), a result that did not change after adjustment for zidovudine use and CD4 cell count. Among subjects with advanced HIV infection, there was a trend toward higher viral load among singly infected subjects. HTLV-I did not appear to increase HIV plasma RNA levels in subjects with coinfection. These results do not provide a biologic basis for the hypothesis that HTLV-I accelerates the course of HIV infection.     

Cell-mediated HTLV-I infection of a cervix-derived epithelial cell line

There is considerable evidence that sexual transmission of human T-cell leukemia virus-I (HTLV-I) is mediated by virus-infected lymphocytes in genital tract secretions. However, it is not clear whether infection occurs through lesions in the genital tract epithelium or takes place via an intact epithelium. We have carried out experiments to test the hypothesis that sexual transmission of HTLV-I is initiated by lymphocyte-mediated infection of intact genital tract epithelia. To examine this question we added either free virus or HTLV-I producing MT-2 cells to cultures of a cervix-derived epithelial cell line, MS751. Although free virus did not infect MS751 cells, MS751 cells which had been coincubated with MT-2 cells became infected. These cultures produced about 50 pg/ml of HTLV-I p24 antigen per 10(6) cells over a 24 h period on the sixth day following exposure to donor T-cells. Proviral DNA could be detected in target MS751 epithelial cells by PCR. Infection of epithelia could be blocked, in a dose-dependent manner, by the sulfated polysaccharides dextran sulfate, heparin, and fucoidan, and by the enzymes fucosidase and mannosidase, but not by a number of other agents that were tested. Since MT-2 cells were observed to attach to the epithelial monolayer, we examined the ability of agents to inhibit adhesion. Adherence was inhibited by the same agents that inhibited infection. Based on these findings, we hypothesize that sexual transmission of HTLV-I may involve lymphocyte-mediated infection of genital tract epithelia and that lymphocyte adhesion to the epithelium is a critical event in transmission of HTLV-I. We speculate that a sugar moiety on the epithelium, possibly mannose or fucose, may be involved in adhesion of T-cells to epithelial cells. As sulfated polysaccharides block both adhesion and productive infection of the epithelium, these compounds might be used as active ingredients in a vaginal formulation to help prevent HTLV-I transmission.     

Simple assay system for detecting human T cell leukemia virus type I-binding cells and its application in titrating binding inhibitory antibodies

BACKGROUND: A simple and rapid assay system for the detection of human T cell leukemia virus type I (HTLV-I) binding cells was developed to assess the virus specific receptor and titrate the antibodies to block the virus binding. EXPERIMENTAL DESIGN: Cells (5 x 10(5)) were incubated with 100 microliters of the concentrated HTLV-I at 37 degrees C for 1 hour. After washing, the cells were reacted with anti-HTLV-I envelope monoclonal antibody (rat) for 30 minutes on ice and then stained with fluorescein-isothiocyanate-conjugated anti-rat immunoglobulin. The stained samples were analyzed on FACScan. Antibody-titration of the virus-binding inhibition was carried out by pretreatment of the virus with serially diluted sera. RESULTS: The specificity of the virus-binding was shown by dose-response relationship, kinetics of the binding, and temperature dependency. HTLV-I was absorbed onto a wide range of human cell lines and peripheral blood lymphocytes at various levels. Antibodies to inhibit the virus-binding were also quantitatively detected in sera from HTLV-I infected individuals, including asymptomatic carriers and patients with adult T cell leukemia or HTLV-I-associated myelopathy, but not from healthy seronegative controls. CONCLUSIONS: This assay system would be useful in screening the virus-specific receptor and the neutralizing antibodies to HTLV-I. Thus, the assay could be applied to further studies on HTLV-I-related diseases.     

Accumulation of human T lymphotropic virus type I-infected T cells in the salivary glands of patients with human T lymphotropic virus type I-associated Sj?gren's syndrome

OBJECTIVE: To clarify the involvement of human T lymphotropic virus type I (HTLV-I) in the pathogenesis of Sjogren's syndrome (SS). METHODS: In HTLV-I-seropositive patients with SS, HTLV-I proviral DNA in the labial salivary glands (SG) was detected by polymerase chain reaction (PCR) amplification of the extracted cellular DNA, and the localization in the SG was examined by in situ PCR hybridization. RESULTS: The cellular DNA extracted from the SG contained full HTLV-I proviral DNA, which was present in the nucleus of the infiltrating T cells, but not in either the SG epithelial cells or the acinar cells. Furthermore, the viral loads in the SG were approximately 8 times to 9 x 10(3) times higher than those in the peripheral blood mononuclear cells. CONCLUSION: Accumulation of HTLV-I-infected T cells in the SG suggests that HTLV-I likely causes the self-reactive T cells to proliferate, which, as a result, induces SS.     

Features of the cytokines secreted by adult T cell leukemia (ATL) cells

Adult T cell leukemia (ATL) cells show a mature helper-inducer T cell phenotype and are thought to secrete many kinds of cytokines in vivo, complicating the clinical features in these patients. In an attempt to specify the cytokines produced by ATL cells, we measured the cytokine concentration in the culture supernatants of three ATL cell lines, all of which were confirmed to be true peripheral blood ATL cell in origin. All these cell lines showed the same cytokine production profile, secreting IL1-alpha, IL1-beta, LD78(MIP-l alpha), TNF-alpha, IFN-gamma, and GM-CSF, but not secreting IL-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1 Ra), IL-4, IFN-alpha, and G-CSF irrespective of the stimulatory agents used. Such limited cytokine production may indicate the specific origin of ATL cells within the helper-inducer T cell subtypes. Moreover, these results explain some of the unusual clinical features of ATL patients.     

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.     

Analysis of 309 cases of esophageal atresia for associated congenital malformations

We describe a 50-year-old man with adult T-cell leukemia complicated by laryngeal tuberculosis whose tumor cells proliferate in response to IL-2 in a paracrine manner. On admission, the patient's white blood cell count was 17,900/mm3; 73% were abnormal lymphocytes with convoluted nuclei. FACS analysis showed that the tumor cells were CD4-negative, CD8-positive T cells. Southern blot analysis of tumor cells revealed integration of a defective HTLV-I genome lacking gag and pol genes. He was diagnosed with chronic ATL complicated by laryngeal tuberculosis. The primary leukemic cells expressed IL-2R alpha and IL-2R beta detected by FACS and Northern blot analysis and showed marked growth in response to exogenously added recombinant IL-2 in short-term cultures. Northern blot analysis did not show any IL-2 mRNA. We have previously demonstrated that primary leukemic cells from some ATL patients grow in response to IL-2 in an autocrine or paracrine manner. These results suggest that in CD8 ATL, IL-2 may be involved in a paracrine manner.     

Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1

It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4(+) T cells were treated with cell-free supernatants from HTLV-I-infected MT-2 cell cultures. The primary CD4+ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease.