An imported case of adult T cell leukemia in a HTLV-I-infected patient in Italy.

In this study we report the case of an acute form of ATL in a HTLV-I-infected Nigeria-born 27-year-old female prostitute living in Italy from February, 2001. The presence of HTLV-I infection was demonstrated by the detection of serum antibody to HTLV-I by immunoenzymatic assay and western blot analysis. In addition, the presence of HTLV-I proviral DNA was confirmed by a hemi-nested PCR in a sample of peripheral blood mononuclear cells. From an epidemiological point of view, it is important to report new cases of imported ATL, as it may explain the otherwise untraceable origin of some rare and apparently autochthonous cases of ATL in non-endemic areas.

Extracellular Tat activates c-fos promoter in low serum-starved CD4+ T cells.

The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

HIV-1 Tat protects CD4+ Jurkat T lymphoblastoid cells from apoptosis mediated by TNF-related apoptosis-inducing ligand.

We have here investigated the effect of TNF-related apoptosis-inducing ligand (TRAIL), a new member of the TNF cytokine superfamily, on the survival of Jurkat lymphoblastoid cell lines stably transfected with plasmids expressing the wild-type or mutated (Cys22) human immunodeficiency virus type 1 (HIV-1) tat gene. Jurkat cells transfected with wild-type tat were resistant to TRAIL-mediated apoptosis, while Jurkat cells mock-transfected with the control plasmid or with a mutated nonfunctional tat cDNA were highly susceptible to TRAIL-mediated apoptosis. Also, pretreatment with low concentrations (10-100 ng/ml) of extracellular synthetic Tat protein partially protected Jurkat cells from TRAIL-mediated apoptosis. Taken together, these results demonstrated that endogenously expressed tat and, to a lesser extent, extracellular Tat block TRAIL-mediated apoptosis. Since it has been shown that primary lymphoid T cells purified from HIV-1-infected individuals are more susceptible than those purified from normal individuals to TRAIL-mediated apoptosis, our findings underscore a potentially important role of Tat in protecting HIV-1-infected cells from TRAIL-mediated apoptosis.

Stroma-derived factor 1alpha induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand.

CXC chemokine receptor 4 (CXCR4), the high-affinity receptor for stroma-derived factor 1alpha (SDF-1alpha), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum-free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte colony-stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF-1alpha, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL-3 and erythropoietin, SDF-1alpha induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF-1alpha significantly reduced the number of plurifocal erythroid colonies (erythroid blast-forming units; BFU-E), whereas it did not affect granulocyte-macrophage colony-forming units (CFU-GM). We also demonstrated that the inhibitory effect of SDF-1alpha on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF-1alpha plays a role as a negative regulator of erythropoiesis.

Human T cell leukemia virus type II increases telomerase activity in uninfected CD34+ hematopoietic progenitor cells.

The aging process of long-term self-renewing hematopoietic stem/progenitor cells is not yet completely understood and recent studies on antiapoptotic cell pathways have demonstrated a close linkage between telomerase activation and Bcl-2 deregulation in human cancer cells. The present work shows that human T cell leukemia virus type II (HTLV-II) Mo virions that have originated from the T cell line (C344), but not from the B cell line (BJAB), are critically involved in mediating survival and growth effects on hematopoietic precursors (represented by both the TF-1 CD34+ cell line and by peripheral blood-derived CD34+ cells) through the maintenance or enhancement of telomerase activity and the induction of bcl-2 expression. In addition, using an interleukin-3-dependent TF-1 cell line, it was demonstrated that IL-3 deprivation was sufficient to influence the levels of telomerase activity and Bcl-2 expression in CD34+ cells. Taken together, these findings suggest that, in appropriate conditions, extended hematopoietic progenitor cell survival and proliferation following HTLV-II exposure depends on a synergistic interaction between up-regulation of Bcl-2 and activation of telomerase activity.

Modulation of CD4, CXCR-4, and CCR-5 makes human hematopoietic progenitor cell lines infected with human herpesvirus-6 susceptible to human immunodeficiency virus type 1.

Two CD34+ human hematopoietic progenitor cell (HPC) lines, KG-1 and TF-1, became susceptible to human immunodeficiency virus type 1 (HIV-1) infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have analyzed the possible mechanism(s) underlying this phenomenon in light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific coreceptors necessary, together with the high-affinity receptor CD4, for entry into target cells of T-tropic and M-tropic HIV-1 isolates, respectively. KG-1 cells show CXCR4 and CCR5 surface molecules in a large proportion of the cell population. Therefore, their susceptibility to both T-tropic and M-tropic HIV-1 strains, caused by HHV-6 infection, can be explained by the HHV-6-induced appearance of CD4 molecules in about 40% of the cell population. In TF-1 cells, 10%-15% of which are CD4+ and exhibit a consistent CCR5 presence in a large proportion of the cell population and a hardly detectable amount of CXCR4 in a very limited number of cells, HHV-6 infection does not modify the cell surface availability of HIV-1-specific high-affinity receptor or coreceptors.

In a human lymphomyeloid progenitor cell line (KG-1) HIV-1 gp120 binds to chemokine-receptors CXC-R4 and CCR5, only in the presence of CD4.

A CD34+ human hematopoietic progenitor cell lines, KG-1, became susceptible to HIV-1 infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have now analyzed the possible mechanism(s) underlying this phenomenon, in the light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific co-receptors, necessary, together with the high affinity receptor CD4, for the entry into target cells of HIV-1. Cytofluorimetric analysis demonstrated that in KG-1 cells, after HHV-6 infection, more than 40% of cell population became CD4 positive and only in KG-1 cells expressing the CD4+ phenotype, the exposure to r-gp120 masks a significant amount, not only of CD4, but also of both CXCR4 and CCR5 chemokine receptors. In fact, only when pre-infected by HHV-6, KG-1 cells, after exposure to r-gp120, exhibit a significant reduction in the percentage of CXCR4 or CCR5-positive cells.

HIV-1 gp120 induces the activation of both c-fos and c-jun immediate-early genes in HEL megakaryocytic cells.

We have previously demonstrated that the addition in culture of recombinant HIV-1 IIIB envelope gp120 affects the survival/growth of pluripotent haemopoietic progenitors, and, in particular, of those committed towards the megakaryocytic lineage. To characterize some of the molecular mechanisms involved in this phenomenon, we investigated the expression of members of the activating protein-1 (AP-1) complex in the HEL megakaryoblastic cell line. Following the treatment of HEL cells with recombinant IIIB envelope gp120, we noticed: (i) increased levels of endogenous c-fos and c-jun mRNA and proteins, (ii) activation of both c-fos and c-jun promoters, and (iii) a very rapid stimulation of a MAPK/ERK pathway.

Impaired telomerase activity in uninfected haematopoietic progenitors in HIV-1-infected patients.

BACKGROUND: Haematopoietic progenitor cells (HPC) of HIV-1-infected patients are severely compromised in their replication and clonogenic capacities, and show an enhanced propensity to apoptosis, despite the lack of productive or latent HIV-1 infection. OBJECTIVE: To investigate telomerase enzyme levels in CD34+ HPC isolated from HIV-1-infected patients, because the absence of telomerase activity has been found to be correlated with a diminished replication potential. METHODS: Telomerase levels were measured by a PCR-based telomeric repeat amplification protocol. CD34+ HPC isolated from the peripheral blood of 11 HIV-1-infected patients were compared with CD34+ HPC isolated from peripheral blood (nine subjects) or bone marrow (six subjects) from 15 healthy donors. Telomerase levels were also studied in normal HPC after exposure to either gp120 or transforming growth factor (TGF)-beta1. RESULTS: CD34+ HPC isolated from either peripheral blood or bone marrow from healthy donors expressed a high level of telomerase activity. On the contrary, CD34+ HPC isolated from HIV-1-seropositive patients did not express any detectable telomerase activity in nine patients, and a clearly reduced enzymatic activity in two patients. Furthermore, telomerase activity in normal CD34+ HPC exposed to recombinant gp120 was significantly reduced, and to a higher extent than in CD34+ HPC exposed to recombinant TGF-beta1. CONCLUSIONS: This is the first study to demonstrate severely impaired telomerase activity in uninfected CD34+ HPC isolated from HIV-1-infected patients. The mechanism underlying this impairment probably involves the interaction of HIV-1 envelope glycoprotein gp120 with the cell membrane. These results may add to our understanding of the pathogenesis of the lesion of the HPC compartment.

Human T-cell leukemia virus type II directly acts on CD34+ hematopoietic precursors by increasing their survival potential. envelope-associated HLA class II molecules reverse this effect.

The role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell-derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.

In vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF), alone and associated with zidovudine, on HIV-1 replication.

The effects of the administration of recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) on HIV-1 replication were evaluated in 15 patients with advanced HIV-1 disease and severe leukopenia, by monitoring immunocomplex dissociated p24 antigenemia, during 21 overall courses of therapy with rHuGM-CSF (lasting 2 to 27 weeks), alone or associated with zidovudine. During most treatment courses with rHuGM-CSF (17 out of 21), no significant modifications of HIV-1 antigenemia were recognized. A remarkable increase in viral replication occurred in only two courses out of 13 performed with rHuGM-CSF alone, while a significant reduction of antigenemia was observed in two courses of rHuGM-CSF out of 8 administered with zidovudine, after 10 weeks of combined treatment. Our experience is discussed on the grounds of both experimental and clinical investigations dealing with interactions between rHuGM-CSF and zidovudine during HIV-1 disease, focusing on risks of increased viral burden during treatment with rHuGM-CSF alone, and the synergistic activity of the combination with zidovudine against HIV-1 replication.

A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1.

In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection.

Enhanced nuclear factor-kappa B activation induced by tumour necrosis factor-alpha in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein.

OBJECTIVE: An enhanced nuclear factor (NF)-kappa B activation in response to tumour necrosis factor (TNF)-alpha has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-kappa B-binding activity induced by TNF-alpha in Tat-producing cells was studied by anti-Tat antibody blocking experiments. DESIGN AND METHODS: Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-alpha-induced NF-kappa B-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations. RESULTS: The enhanced production of TNF-alpha-induced NF-kappa B binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-alpha-induced NF-kappa B binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies. CONCLUSIONS: This study demonstrates that the enhanced NF-kappa B activation exhibited by stably tat-transfected cells in response to TNF-alpha, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as a bifunctional molecule which not only acts from within facilitating NF-kappa B recruitment in the viral transcription complex, but may also act from without increasing the availability of activated NF-kappa B.

Impaired survival of bone marrow GPIIb/IIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia.

Glycoproteic (GP) IIb/IIIa+ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV-1 seropositive thrombocytopenic patients, eight HIV-1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV-1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa+ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa+ cells from HIV-1 seropositive thrombocytopenic patients showed a significant (P < 0.0001) increase of apoptosis with respect to both HIV-1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa+ cells purified from HIV-1 seropositive thrombocytopenic patients was inversely (P < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV-1 disease nor a direct infection with HIV-1 seems to play a primary role in the impaired survival of BMGPIIb/IIIa+ megakaryocytic cells. These findings strengthen the notation that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV-1 related thrombocytopenia.

An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

The CD4 receptor plays essential but distinct roles in HIV-1 infection and induction of apoptosis in primary bone marrow GPIIb/IIIa+ megakaryocytes and the HEL cell line.

We investigated whether cells belonging to the megakaryocytic lineage could be infected in vitro with human immunodeficiency virus type-1 (HIV-1). Primary GPIIb/IIIa+ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV-1. Both GPIIb/IIIa+ BM and HEL cells expressed significant levels of CD4 receptor (> 50%) and were efficiently infected with HIV-1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV-1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4+ and CD4- cells. Although unfractionated and CD4+ HEL cells were productively infected with HIV-1, CD4- HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4+ HEL cells with cross-linked recombinant env gp120 or Leu3a anti-CD4 monoclonal antibody markedly (P < 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV-1 or treated with cross-linked gag p24 or anti-GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gp120 or anti-CD4 monoclonal antibody could be more relevant than a direct infection with HIV-1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV-1 seropositive thrombocytopenic patients.

In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection.

We report a methodology for detecting specific DNA sequences directly inside cells, combining in situ PCR and flow cytometry. This technique is based on in situ PCR performed in the presence of digoxigenin-labeled dUTP to obtain a digoxigenin-labeled amplicon, which is then revealed by an anti-digoxigenin polyclonal antibody directly conjugated to fluorescein. Fluorescence intensity is next evaluated by flow cytometry. Our experimental models were represented by the lymphoblastoid cell lines 8E5LAV, carrying an integrated HIV-1 DNA proviral copy per cell, and A.301, infected in vitro with HIV-1 (strain IIIB). The technique is described in detail with particular attention to the optimization of critical fixation and permeabilization steps. This method allows not only the detection but also an accurate quantification of the number of positive cells in a background of negative cells. Moreover, it has the potentiality to develop into a multiparametric method for the simultaneous study of specific DNA or RNA sequences and surface or intracellular markers.

The lack of susceptibility to HIV-1 infection in human hematopoietic progenitor TF-1 cell line correlates with the absence of LFA-1 (CD11a) surface adhesion molecule.

Although substantially unsusceptible to HIV-1 infection, human hematopoietic progenitors (CD34+ cells) express significant amounts of gp120-binding CD4 molecules. As some experimental evidence suggests the need for one or more putative cellular accessory factor(s) for efficient CD4-mediate HIV-1 infection, we analyzed the presence and distribution of various adhesion molecules on the surface of two HIV-1-susceptible cell lines (H9 and A3.01) and one HIV-1-unsusceptible, CD4-positive, human hematopoietic progenitor cell line (TF-1). The only difference observed concerned the LFA-1 CD11a protein that has already been shown to be necessary for cell fusion in HIV-1-infected T4 lymphocytes. CD11a was present in a high percentage of HIV-1-susceptible cells and absent in HIV-1-unsusceptible TF-1 cells. A significant increase in HIV-1 replication was obtained in CD11a-enriched H9 cells in comparison with an almost completely CD11a-deprived H9 cell population. The lack of susceptibility to HIV-1 infection in human CD34+ cells may depend on various factors. Nevertheless, the lack of CD11a in a CD34+, CD4+ human hematopoietic progenitor cell line suggests that this molecule may be implicated in the susceptibility to HIV-1 infection.