Abstract: BACKGROUND: Efficacy and safety of interferon induction therapy alone or in combination with ribavirin or ribavirin plus amantadine were evaluated in chronic hepatitis C patients who were nonresponders to primary antiviral treatment. METHODS: The study was designed to have 225 HCV nonresponder patients, but at an interim analysis the response rate difference between groups was lower than expected and the enrollment was stopped when 75 patients had been randomized to receive interferon-alpha2a (group A, n = 26), interferon-alpha2a plus 15 mg/kg per day of ribavirin (group B, n = 24), or interferon-alpha2a plus ribavirin plus 200 mg/day of amantadine hydrochloride (group C, n = 25). Treatment duration was 48 weeks. The dose of interferon was 6 MU/day for 4 weeks followed by 3 MU/day for the remaining 44 weeks. RESULTS: On intention-to-treat, the sustained virological response at 24 weeks of follow-up was 11.5% in group A, 12.5% in group B, and 12% in group C. Therapy was discontinued because of adverse effects in three patients in group A (11.5%), three in group B (12.5%), and two in group C (8%). CONCLUSIONS: Nonresponders with chronic hepatitis C may achieve a sustained virological response rate of approximately 12% if retreated with interferon induction treatment followed by administration of a daily dose. The addition of ribavirin or amantadine did not seem to improve the response rates.

Abstract: This paper describes the development of a SYBR Green-based multiplex real time RT-PCR for the simultaneous detection of HCV and HIV-1 genomes in plasma samples. Viral genomes were identified in the same sample by their distinctive melting temperature (Tm) which are 81.6 and 86.5 degrees C for HIV-1 gag 142 bp amplicon and HCV 5'-NCR region 226 bp amplicon, respectively. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of both HIV-1 and HCV. In addition, we also assayed HIV-1 and HCV viral load in 30 co-infected patients and in 15 blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HIV-1/HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors.

Abstract: To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used.

Abstract: The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.

Abstract: Chronic hepatitis C represents a major clinical problem after liver transplantation, but factors influencing the recurrent disease have not been well characterized. We analyzed the clinical records of all the patients transplanted for hepatitis C virus (HCV)-related liver disease in our Center between 1991 and 1997. Eighty consecutive HCV-positive (+) patients (60 men, ages 28 to 64) survived more than 1 month after transplantation and were followed for a median of 45 months. Diagnosis of recurrent chronic hepatitis C was made in 38 patients (47.5%), of whom 22 had moderate/severe chronic hepatitis. Decompensated cirrhosis occurred in six patients (7.5%). No difference in patient survival was found between patients with and without hepatitis C recurrence. No association was found between recurrent hepatitis C and presumed risk factors. The method of tapering off corticosteroids was significantly associated with both hepatitis C recurrence and the severity of hepatitis. In patients receiving a higher daily prednisone dose, 12 months after transplantation, the proportion of recurrent hepatitis C was 35.7% versus 66.6% (P = .02; odds ratio [OR], 3.6; 95% confidence interval (CI): 1.25 to 10.36), and among patients receiving a higher daily prednisone dose, 6 months after transplantation, the proportion of moderate/severe chronic hepatitis C was 40% versus 89% (P = .03; OR: 0.08, 95% CI: 0.008 to 0.84). Finally, prednisone dose at month six was significantly associated with disease-free survival of the liver graft. In conclusion, our results seem to indicate that in HCV-infected liver transplant recipients, a long-term treatment with corticosteroids, slowly tapered off over time, may prevent the more aggressive forms of recurrent liver disease.

Abstract: BACKGROUND: The efficacy of a specific humoral response to transactivating Tat protein was studied in a group of HIV-1 seropositive drug addicts, who had previously received a similar course of anti-retroviral treatment with two reverse transcriptase inhibitors. OBJECTIVES: The aim of the study was to evaluate the meaning of an immune response to Tat protein in HIV-1 seropositive patients with different levels of HIV-1 RNA viremia. STUDY DESIGN: The study analyzed the presence of anti-Tat antibody reacting either with full-length Tat or with individual overlapping Tat-peptides (Tat(6-14), Tat(11-24), Tat(36-50), Tat(46-60), Tat(56-70) and Tat(65-80)), in a group of HIV-1 seropositive subjects with different peripheral blood viral loads. Plasma samples were examined by immunoenzymatic assay for the presence of anti-Tat IgG antibody and for the quantification of peripheral blood (plasma) viral load by branched DNA assay. RESULTS: The large majority of HIV-1 patients showed detectable levels of serum IgG to full-length-Tat, and the anti-Tat antibody level presented an inverse correlation with viral load magnitude. The analysis of antibody levels against individual overlapping Tat-peptides clearly showed that an undetectable viral load was significantly associated with the presence of a high antibody concentration against Tat(6-14), Tat(36-50) and Tat(46-60) (P=0.002, P=0.027 and P<0.001, respectively). CONCLUSION: In HIV-1-infected patients, a strong humoral immune response against HIV-1 Tat protein is inversely correlated to peripheral blood viral load and, in particular, a high level of antibody against Tat peptides containing amino acid residues 6-14 (Tat(6-14)), 36-50 (Tat(36-50)) and 46-60 (Tat(46-60)) is associated with an undetectable plasma viral load. These findings may help to tailor anti-HIV-1 Tat-containing vaccines.

Abstract: The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (< or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from >10(1) to > or =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.

Abstract: 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.

Abstract: BACKGROUND: The efficacy of highly active antiretroviral therapy (HAART) was prospectively monitored in HIV-1 seropositive patients by analyzing HIV-1 RNA viral load. OBJECTIVES: The aim of the study was to evaluate the viral load course in two different groups of patients (group 1: CD4>400/mm(3), group 2: CD4<200/mm(3)) during a period ranging from 9 to 25 months. STUDY DESIGN: HIV-1 viral load, at the start and during HAART, was analyzed in 117 patients who had previously been treated only with two anti-transcriptase drugs but were naive for protease inhibitors. RESULTS: The results showed that, after the beginning of therapy, high plasma HIV-1 RNA levels dropped to undetectable values (<50 copies HIV-RNA/ml) in one third of patients over a mean period of about 9 months irrespective of the initial CD4 cell count, even though a viral reduction of at least 2log(s) in a significantly shorter period of time (P<0.001) was observed only among patients who began retroviral therapy with a higher CD4 cell count. CONCLUSION: The response to HAART was not dramatically affected by the initial CD4 count. Though restricted to a small number of subjects, the data support the idea that therapeutic intervention can be effective even in an advanced stage of HIV-1 infection, when patients show a decreased number of CD4 T-lymphocytes.

Abstract: 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.

Abstract: A branched DNA method for the quantification of human immunodeficiency virus type 1 (HIV-1) RNA levels (Quantiplex HIV RNA 2.0) was compared with a reverse transcriptase-coupled polymerase chain reaction method (Amplicor HIV-1 Monitor) and a nucleic acid sequence-based assay (Nuclisens HIV-1 QT) in plasma samples from a group of HIV-1 seropositive patients. We found a high correlation between Nuclisens and Quantiplex (r = 0.89; p < 0.001) and between Amplicor and Quantiplex (r = 0.94; p < 0.001), a shift of RNA viral load to higher Nuclisens and Amplicor values compared with the Quantiplex results and a significant positive correlation (rS = 0.60; p < 0.001) between the p24 antigen level and the RNA viral load determined with the Quantiplex assay. We also found higher sensitivities of the Nuclisens and the Amplicor procedures compared with the Quantiplex assay. The total sensivity of the Quantiplex assay in our study was 70% whereas that of the p24 antigen was only 29%.

Abstract: 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.

Abstract: In order to compare HIV-1 p24 antigenemia and plasma HIV-1 RNA levels as markers of viral replication, 3,129 paired determinations of alkaline immunocomplex-dissociated serum HIV-1 p24 antigenemia (performed with an immunoenzymatic assay), and plasma HIV-1 RNA levels (carried out with a branched DNA method, a reverse transcriptase-coupled polymerase chain reaction, and a nucleic acid sequence-based assay) were assessed over a two-year-period. When excluding samples with undetectable plasma HIV-1 RNA levels (which tested negative or borderline positive at serum p24 antigen assay in 97.9% of cases), immunocomplex-dissociated p24 antigenemia proved significantly less sensitive than viral load at all considered HIV-1 RNA reference levels, although the profile of positive serum p24 antigen assays (values above 10 pg/ml) paralleled the trend of plasma HIV-1 viral load, especially at higher levels. However, serum HIV-1 p24 antigenemia (even after immunocomplex dissociation) can be longer suggested as a the sole virological tool in the laboratory management of HIV-1 infection, due to its significantly lower sensitivity levels compared with viral load assessment.

Abstract: 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.

Abstract: Peripheral blood lymphocytes (PBLs) from 51 HIV-1-seropositive subjects with different levels of HIV-1 replication and 20 healthy blood donors were examined for the expression of the antiapoptotic Bcl-2 protein. All the plasma samples from HIV-1 patients were characterized for the presence of HIV-1 p24 and HIV, RNA viral load. Bcl-2 protein expression in fresh peripheral blood lymphocytes was studied by different tests, including Western blot and indirect immunofluorescence techniques. Direct immunofluorescence staining, revealed by flow cytometry, was applied to quantify the number of specific anti-Bcl-2 antibody epitope binding sites, thus extrapolating the relative number of Bcl-2 into the cells. The results indicate that the expression of Bcl-2 protein is significantly lower in peripheral blood lymphocytes of HIV-1-seropositive patients showing high levels of viral replication, detected by means of HIV-1 p24 and RNA viral load, with respect to HIV-1 patients with low levels of virus replication and healthy blood donors. The clear-cut inverse correlation between viral replication and Bcl-2 expression reinforces the view that HIV-1-mediated apoptosis probably represents a key mechanism in AIDS pathogenesis.

Abstract: 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.

Abstract:

Abstract: In situ polymerase chain reaction represents an intriguing method to couple the sensitivity of PCR technology with the preservation of cell morphology. The target of this technique is the detection of specific DNA or retrotranscribed RNA inside the cell. In this brief report, we describe an in situ polymerase chain reaction technique revealed by flow cytometry in order to reveal the human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. We also discuss the key steps and parameters of our assay in comparison with current opinion on in situ polymerase chain reaction.

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Abstract: 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.

Abstract: Retrospective analysis of serum samples from a group of hemophiliac patients who became infected with human immunodeficiency virus type 1 (HIV-1) between 1984 and 1985 has shown that, at variance with other HIV-1-infected patients, at the onset, or at least at a very early phase of HIV-1 infection, they constantly have elevated levels of antibodies against HIV-1-transactivating Tat protein and an absent or barely detectable p24 antigenemia. Anti-Tat antibodies in initial serum samples from hemophiliac patients were probably the consequence of the passive administration of immunoglobulins present in low- or intermediate-purity clotting factor concentrates prepared from HIV-1-infected blood. Furthermore, the analysis of serial serum samples obtained during the course of the disease, in which passively acquired anti-Tat antibodies were substituted by actively produced antibodies, demonstrated an inverse relationship between anti-Tat antibody and p24 anti-genemia levels throughout the observation period. These data seem to suggest that anti-Tat antibody may have some influence on the course of HIV-1 infection.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LC from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LC (10-25% of CD1a+/CD4+ cells), deprived of contaminating T cells and then incubated with HIV-1IIIB. After 24 hr, purified LC and LC-depleted EC fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PCR) analysis showed the presence of HIV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LC, LC-depleted EC or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1IIIB for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Co-cultures of C8166 cells with purified LC or LC-enriched EC previously exposed to HIV-1IIIB exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.

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Abstract: 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.

Abstract: In HIV-1-infected cell cultures, a relatively low concentration (5 micrograms/ml) of monoclonal antibody (mAb) against HIV-1-transactivating Tat protein was an efficient inhibitor of HIV-1 replication both in HIV-1(IIIB)-infected Jurkat cell and peripheral blood mononuclear cell (PBMC) cultures and significantly reduced the expression of a Tat-responsive CAT-reporter construct in HIV-1(IIIB)-infected Jurkat cells. Anti-Tat mAb also caused a significant reduction and a consistent delay in HIV-1 replication when added to PBMCs from HIV-1-infected patients cocultivated with phytohemagglutinin (PHA)-stimulated normal PBMCs. These data indicate that an autocrine-paracrine loop sustained by extracellular Tat protein, which is actively released by HIV-1-infected cells, may affect HIV-1 replication in cell cultures in vitro. An inverse relationship between natural anti-Tat antibody levels and p24 antigenemia was demonstrated by retrospective analysis of serial serum samples obtained from 10 HIV-1-seropositive hemophiliac patients followed over a 7-9-year period. This datum points to a possible influence of anti-Tat antibody on the progression of HIV-1 disease in vivo. These findings have strong implications for Tat protein as a possible target for specific immunotherapy in HIV-1-infected patients.

Abstract: 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.

Abstract: Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.

Abstract: In the last few years a growing body of experimental evidence has indicated that the interaction of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein (gp120) with the membrane of CD4+ cells may deliver negative signals, eventually leading to programmed cell death (apoptosis) of either mature CD4+ lymphocytes or CD34+ haematopoietic progenitor cells, in the absence of cell infection with HIV-1. However, information on the possible activation of the classical signal transduction pathway through gp120 engagement of cell surface CD4 is contradictory. Heat shock proteins (hsp) or 'stress' proteins' are involved in protecting cells from the deleterious effects of heat and other stresses and perform various cell roles. In mammalian cells there is evidence that hsp70 is involved in the transport of proteins to lysosomes, mitochondria and the nucleus. The results obtained in our study demonstrate that early (3 h) after the exposure of permissive CD4+ cells to HIV-1 (or to purified recombinant gp120) a peak of increased synthesis and nuclear translocation of a 70K hsp (and possibly other proteins) is observed. These data indicate that gp120 possesses the capacity to trigger a cascade of events through a transmembrane signalling activity.

Abstract: In this study, we evaluated the effect of a short-term exposure (2 hours) to two different lymphocytotropic strains of human immunodeficiency virus type 1 (HIV-1; HIVIIIB and ICR-3) on the survival of a factor-dependent CD34+ hematopoietic progenitor cell line (TF-1). At flow cytometry analysis, a significant (P < .05) increase in the frequency of apoptotic cell death was observed in HIV-1-treated TF-1 cells, supplemented with low doses of either interleukin-3 (IL-3; 0.02 to 1 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 0.02 to 0.2 ng/mL) with respect to mock-treated cells. On the other hand, higher doses of both cytokines or combinations of suboptimal concentrations of IL-3 plus GM-CSF (eg, 0.2, plus 0.2 ng/mL) completely reversed the HIV-1-induced increase of apoptosis. Remarkably, no signs of productive or latent virus replication were ever observed in HIV-1-treated TF-1 cells up to 16 days of liquid culture. In parallel experiments, the in vitro exposure to HIVIIIB induced a significant and progressive increase of apoptotic death in purified bone marrow CD34+ cells, seeded in liquid cultures in the presence of 1 ng/mL IL-3. The HIV-1-induced apoptosis of TF-1 cells was likely triggered by the simple interaction of HIV-1 envelope glycoprotein gp120 with CD4 receptor, which was expressed at a low level on the surface of TF-1 cells. In fact, treatment of TF-1 cells with recombinant gp120 plus a polyclonal anti-gp120 antibody or with anti-CD4 monoclonal antibody plus rabbit antimouse IgG significantly increased the percentage of apoptotic death. These data suggest that HIV-1, and perhaps also free gp120 in the presence of anti-gp120 antibody; could play a direct role in the pathogenesis of peripheral blood cytopenias in acquired immunodeficiency syndrome patients by inducing apoptotic death of hematopoietic progenitor cells without the need of a direct infection.

Abstract: T cell functional defects are a common aspect of human immunodeficiency virus (HIV) infection. Moreover, it has been suggested that indirect mechanisms are involved in CD4+ cell depletion. Unresponsiveness to proliferative stimuli of lymphocytes incubated with HIV particles or with viral proteins is well documented. Nevertheless, drawing a clear picture of the anergy phenomenon is difficult because of several unresolved and controversial questions. Here we report that recombinant gp120 induces anergy in T helper lymphocytes cultured with different stimuli. The proliferative responses to interleukin (IL)-2, IL-4, IL-6, anti-CD2, anti-CD3 and phorbol 12-myristate 13-acetate are inhibited. Moreover, anergic cells show a different distribution in cell cycle phases as compared to control cells, leading us to suggest that the progression in the cell cycle is hampered and that a pre-mitotic block takes place. Furthermore, since chimpanzees are susceptible to HIV-1 infection without showing immunodeficiency signs, we analyzed the proliferation of chimpanzee lymphocytes without observing anergy in cells preincubated with gp120. Taken together, these results support the hypothesis that anergy plays an important role in HIV infection in vivo.

Abstract: Human cytomegalovirus (HCMV) infection of a CD34+ hematopoietic progenitor cell line (TF1) was studied before and after TPA differentiation. TF1 cells were found to be infected but the virus does not replicate, while differentiated TF1 cells can be infected and allow HCMV complete replication. In the same system we studied the interaction between HCMV and HIV and found that while contact between HIV gp 120 and the HCMV-infected cell has an inhibitory effect, exogenous Tat protein stimulates HCMV replication. The interaction between HCMV and HIV in hematopoietic progenitor cells is complex and depends on several factors that can have opposite effects.

Abstract: The presence of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells (PBMC) of three groups (group 1, more than 500 CD4+ T cells per microliter; group 2, between 200 and 499 CD4+ T cells per microliter; group 3, fewer than 200 CD4+ T cells per microliter) of HIV-1-infected patients, in different stages of the disease, was determined by using a newly developed flow cytometry analysis of the product of in situ PCR assay and compared with other markers of viral replication (HIV-1 p24 antigenemia and viral isolation). Results showed varied percentages of HIV-1-infected PBMC, ranging from 0.6 to 20%. Patients with more than 500 CD4+ T cells per microliter showed the lowest percentage of HIV-1-infected PBMC (2.1 +/- 1.7), compared with patients with CD4+ T-cell counts of between 200 and 499 per microliter (6.5% +/- 4.1%; P < 0.001) and patients with fewer than 200 CD4+ T cells per microliter (4.9% +/- 4.7%; P < 0.05). The difference in the percentage of HIV-1-infected PBMC between group 2 and group 3 patients may in part reflect the loss of CD4+ T lymphocytes in more advanced stages of the disease. However, the results clearly indicate a striking coincidence between the fall of the CD4+ T-cell count below 400/microliter and the sharp increase in PBMC virus loading and p24 antigenemia. Since the procedure is relatively easy to perform, it could be used to monitor the evolution of HIV-1 infection and may prove a useful adjunct in tailoring therapeutic strategies.

Abstract: Starting from our previous observations that the HIV-1-mediated engagement of CD4 induced apoptotic death of TF-1 hematopoietic progenitor cells, in this study we evaluated PKC activity and intracellular Ca2+ levels in TF-1 cells treated with viable and heat-inactivated HIV-1 (strain IIIB) or anti-CD4 Leu3a monoclonal antibody (mAb). Both viable and heat-inactivated HIV-1 or anti-CD4 mAb, but not anti-human cytomegalovirus (HCMV) 66kD protein or anti-CD8 mAb induced a rapid (5-10 min) increase in PKC activity under both serum-containing and serum-free conditions. The same treatment also induced both a transient and a long-lasting (48 hours) decrease (p < 0.05) in intracellular Ca2+ levels in serum-containing cultures. We propose that the observed changes in PKC activity and intracellular Ca2+ levels might be involved in the HIV-1 mediated apoptosis of hematopoietic progenitor cells.

Abstract: We investigated the expression of CD4 antigen in normal bone marrow (BM) samples, enriched in CD34+ hematopoietic progenitor cells. At flow cytometry, a significant fraction (ranging from 25% to 65%) of CD34+ cells also showed low levels of CD4 antigen on their surface. The CD4 receptor densities on the surface of hematopoietic progenitors was approximately 50% that of peripheral blood monocytes and 5% of peripheral blood T lymphocytes. In immunoprecipitation experiments, the CD4 antigen expressed by BM hematopoietic progenitors appeared to be the same form expressed by mature peripheral blood CD4+ cells and appeared to be a potentially functional receptor for human immunodeficiency virus-type 1, because it specifically bound recombinant envelope gp120. Moreover, BM samples, highly enriched in CD34+ cells, showed the presence of CD4 mRNA at reverse transcription-polymerase chain reaction examination. In experiments of complement-mediated cytotoxicity with Leu3-a+Leu3b anti-CD4 monoclonal antibody, a significant reduction in the number of both classes of megakaryocyte (burst-forming unit-meg [BFU-meg] and colony-forming unit-meg [CFU-meg]) and granulocyte/macrophage (CFU-GM) progenitors was observed, whereas erythroid (BFU-E) progenitors were only slightly affected. Moreover, purified CD4+ BM cells obtained by immunomagnetic selection, using high concentrations of Leu3a+Leu3b, showed a colony-forming ability of megakaryocyte and granulocyte/macrophage progenitors comparable with that of CD4- BM cells. In conclusion, the present data show that immature hematopoietic progenitor cells express low levels of CD4, the high-affinity receptor of human immunodeficiency virus-type 1.

Abstract: In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant Tat protein on mRNA expression and protein synthesis of two inflammatory cytokines-interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-beta 1)-by peripheral blood (PB) monocytes. Whereas maximal levels of IL-6 protein were recovered in PB monocyte culture supernatants after 24-48 h from the addition of 1 micrograms/ml of recombinant Tat, TGF-beta 1 showed a slower and progressive increase, reaching maximal levels only after 72-96 h of culture. Consistently, the analysis of the steady-state levels of mRNA showed a sharp increase of IL-6 mRNA expression after 24h of culture, with a slow decline thereafter. On the other hand, TGF-beta 1 mRNA expression showed a slow increase only after 72-96 h of culture. Moreover, IL-6 appeared involved in the up-regulation of TGF-beta 1, because the addition of a neutralizing anti-IL-6 antibody to Tat-treated PB monocyte cultures significantly reduced the amounts of TGF-beta 1 recovered in the culture supernatants after 96 h. The present demonstration that HIV-1 Tat protein directly up-regulates IL-6 expression and stimulates TGF-beta 1 production both directly and indirectly, through early IL-6 production, could have important implications in the pathogenesis of HIV-1 disease.

Abstract: Human cytomegalovirus (CMV) is a major cause of severe disease in HIV-infected persons and some findings suggest that it may accelerate HIV disease. In this study, a total of 621 blood samples from patients with LAS-ARC and AIDS were analysed in parallel for CMV and HIV-I antigenaemias. Results indicate that the presence of CMV antigenaemia and the presence of HIV-I p24 in the blood are highly correlated statistically and encourage other studies on the role of CMV in the evolution of AIDS. In a smaller group of cases, CMV was also isolated from saliva and/or urine. The correlation with HIV replication was positive (although much lower) with CMV detected in saliva and completely negative with CMV isolated from urine.

Abstract: In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant tat-protein on the production of interleukin-6 (IL-6), granulocyte/macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) by purified peripheral blood monocytes. Whereas no effects were observed on TNF-alpha and GM-CSF production, recombinant tat-protein was able to induce the production of IL-6 by peripheral blood monocytes in a dose-dependent fashion for concentrations ranging from 1 ng/ml to 1 micrograms/ml. Pre-exposure of tat-protein with a polyclonal neutralizing anti-tat antibody (dilution 1:100) completely abrogated the tat-dependent increase in IL-6 production. The ability of tat-protein to selectively stimulate the production of IL-6 by peripheral blood monocytes could help to explain the presence of elevated levels of IL-6 in the serum of HIV-1 seropositive individuals, especially in patients in advanced stages of the disease with an active viral replication.

Abstract: In this paper we investigated the role played by human immunodeficiency virus type 1 (HIV-1) in the pathogenesis of peripheral blood (PB) cytopenias of AIDS patients. The in vitro growth of PB granulocyte/macrophage progenitors (CFU-GM) was investigated in 45 HIV-1 seropositive (+) individuals at different stages of the disease. The number of circulating CFU-GM was significantly (p < 0.01) lower in AIDS patients (stages WR V-VI) than in HIV-1(+) asymptomatic individuals (stages WR I-II). Moreover, the presence of gag p 24 in the plasma and/or viral isolation from PB mononuclear cells of HIV-1(+) individuals was inversely correlated (p < 0.01) with the number of circulating CFU-GM, irrespectively with the stage of the disease. Viral isolates obtained from one asymptomatic and four symptomatic HIV-1(+) individuals were tested on the in vitro growth of normal hematopoietic progenitor (CD34+) cells, purified from PB of healthy donors. All the different viral isolates showed a dose-dependent inhibition of CD34+ cells, in the absence of either productive or latent infection. This suppressive effect was completely reversed by preincubating the different viral isolates with a polyclonal anti-gp 120 antibody before adding to normal CD34+ cells. These findings suggest a direct involvement of active viral replication products in the progressive impairment of hematopoiesis, characteristic of HIV-1(+) individuals in spite of the lack of a productive or latent infection of CD34+ hematopoietic progenitors.

Abstract:

Abstract: In this study we evaluated the effect of recombinant interferon-alpha 2b (IFN-alpha 2b) therapy on the number of circulating platelets and interleukin-6 (IL-6) plasma levels in 12 human immunodeficiency virus type 1 (HIV-1) seropositive patients, affected by a severe and persistent thrombocytopenia. The levels of IL-6 in plasma of HIV-1 seropositive thrombocytopenic subjects before IFN-alpha therapy were similar (80 +/- 15 pg/ml) to those observed in 15 HIV-1 seropositive asymptomatic individuals (75 +/- 12 pg/ml) and 30 HIV-1 seronegative blood donors (59.5 +/- 25 pg/ml). On the other hand, IL-6 amounts (148 +/- 36 pg/ml) in plasma of HIV-1 seropositive thrombocytopenic subjects were significantly (p < 0.01) increased after 5 weeks of IFN-alpha 2b therapy, showing a good correlation (p < 0.05, chi-square test) with the levels of circulating platelets. Moreover, an increased spontaneous IL-6 production by peripheral blood monocytes was observed after IFN-alpha 2b therapy in HIV-1 seropositive thrombocytopenic patients. Our results suggest that an increased production of IL-6, one of the main factors controlling thrombocytopoiesis, may partially explain the ability of IFN-alpha 2b therapy, to restore platelet production in a subset of HIV-1 seropositive thrombocytopenic individuals.

Abstract: OBJECTIVE: To determine the mechanism underlying the poor growth in vitro of haematopoietic progenitor cells isolated from HIV-1-infected patients. METHOD: Apoptotic death in liquid culture of bone-marrow CD34+ cells obtained from 11 HIV-1-seropositive patients and 18 HIV-1-seronegative donors was quantitatively monitored by a flow cytometry procedure. RESULTS: No significant differences in the percentage of apoptotic cells were noted between the two groups immediately after purification. When CD34+ cells were placed in liquid cultures supplemented with 2 ng/ml interleukin-3, the number of apoptotic cells progressively and significantly (P < 0.05) increased in all HIV-1-seropositive patients, while it remained constant in HIV-1-seronegative individuals. Although all HIV-1-seropositive patients showed signs of active viral replication in the bone-marrow micro-environment, progenitor CD34+ cells did not show the presence of active and/or latent HIV-1 infection. CONCLUSION: Our data demonstrate that CD34+ cells isolated from AIDS patients with active HIV-1 replication in bone-marrow accessory cells are committed to apoptotic death without being directly affected by productive infection.

Abstract: The purine nucleotide content was examined in various cells before and after HIV-1 virus infection: healthy peripheral blood lymphocytes (PBL) and cultured PBL after infection; the PBL of asymptomatic, ARC and AIDS patients. In all cases, changes in purine nucleotide concentrations were observed. The pattern of purine nucleosides and nucleobases was also evaluated by HPLC in PBL of controls and patients. The analysis was integrated by following the incorporation of a labelled precursor ([14C]formate) into purine nucleotides, which was investigated as an indication of the rate of purine metabolism in these cells. Many interesting variations in the catabolic and of anabolic pathways were observed, demonstrating that the viral penetration affects purine nucleotide metabolism. These results suggest interesting perspectives in AIDS research.

Abstract: The true extent of human T-lymphotropic virus type I and II (HTLV-I/II) infection in European countries and its pathogenetic potential are still unknown. To find out more about HTLV-I/II incidence in our area we studied a group of 160 outpatients attending a sexually transmitted disease clinic over a six-month period. All patients were screened for the presence of specific antibody by means of enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) analysis, using commercially available reagents. A surprisingly high percentage of patients showed an antibody reactivity to HTLV-I/II antigens by ELISA (9.3%) and IB (6.8%), although none of the samples satisfied the internationally accepted criteria of serological positivity. All subjects, irrespective of doubtful and inconclusive serological results, were further analyzed for the presence of proviral DNA in peripheral blood mononuclear cells by polymerase chain reaction using different pairs of primers and probes. A clear cut positive result for the presence of HTLV-I provirus-related DNA sequences was obtained in peripheral blood mononuclear cells of only one patient, a 26 years old female presenting genital condylomatosis, with no history of blood transfusion and/or intravenous drug abuse. Her serum showed a borderline result at ELISA and an IB reactivity only against p21. These data are open to various possible interpretations and, among others, may represent a hint for the presence of divergent antigenic variants of HTLV-I in the geographical area investigated.

Abstract: The effects of human immunodeficiency virus type 1 (HIV-1) and recombinant envelope glycoprotein gp120 on the in vitro growth of enriched human haematopoietic progenitors (CD34+ cells) have been investigated. A 2 h exposure to HIV-1 resulted in a progressive and significant reduction of viable CD34+ cell number in liquid cultures and of granulocyte-macrophage, erythroid and megakaryocytic progenitors in semisolid cultures. In virus-treated CD34+ cells, no signs of active virus replication were observed and the possibility of latent infection was excluded by quantitative polymerase chain reaction. Recombinant HIV-1 envelope glycoprotein gp120 added to CD34+ cell cultures displayed a dose-dependent inhibitory activity on CD34+ cell viability. Neutralizing antibody against gp120 was able to block completely the inhibitory activity on CD34+ cells of either HIV-1 or recombinant gp120. These results demonstrate that HIV-1 envelope glycoprotein gp120 has a direct cytotoxic effect on CD34+ cells.

Abstract: In an attempt to better understand the role of endothelial cells during HIV-1 infection, we report a virological and ultrastructural study on isolated endothelial cells from human adipose tissue, infected by HIV-1 in vitro. Supernatants from cultures showed the presence of p24 antigen and reverse transcriptase activity starting two days after HIV inoculation. A significant decrease of viral rescue was observed in cycloheximide treated cells confirming a de novo synthesis of viral products. SEM analysis individualized several surface slender projections and interdispersed virus-like particles in the infected cells. Furthermore, transmission electron microscopy (TEM) results showed cellular aspects of HIV phagocytosis and virus budding, suggesting that endothelial cells may represent a CD4 negative cell target of HIV-1 infection.

Abstract: Seventy infants born to human immunodeficiency virus type 1 (HIV-1) seropositive mothers were studied for specific antibody (IgA, IgM and IgG) production and the presence of active infection (detectable level of virus in peripheral blood lymphocytes). Among these children, followed for up to 15-40 months after birth, 11 presented unequivocal signs of HIV-1 infection (persistent p24 antigenemia and/or positive virus isolation). Analysis of sera by immunoblotting showed that IgA antibody to HIV-1 p24 core protein, alone or associated with envelope glycoproteins (gp120, gp41), was present in the majority of infected babies (7 of 11), while IgM was found in a lower percentage of cases (4 of 11). No IgA and or IgM antibody to HIV-1 was ever found in babies, born to seropositive mothers, who seroreverted after birth or in the control group enrolled in this study. Our results indicate that immunoblotting analysis of IgA antibody to HIV-1 polypeptides may represent a useful complementary prognostic marker in children born to HIV-1 seropositive mothers.

Abstract: The amounts of interleukin 3 (IL-3), interleukin 4 (IL-4), tumor necrosis factor alpha (TNF-alpha), and tumor necrosis factor beta (TNF-beta) were evaluated by immunoenzymatic assays in the supernatant of short-term cultures of whole mononuclear cells and purified CD4+ T-lymphocytes, obtained from the peripheral blood (PB) of 35 HIV-1(+) asymptomatic individuals (stages I-II of the Walter Reed Classification), 20 HIV-1(+) symptomatic patients (WR V-VI), and 40 HIV-1(-) blood donors. TNF-alpha and TNF-beta production was similar in HIV-1(+) asymptomatic individuals, HIV-1(+) symptomatic patients, and HIV-1(-) controls. On the other hand, IL-3 and IL-4 production by either whole mononuclear cells or isolated CD4+ T-cells was decreased approximately 2-fold (p < 0.01) in HIV-1(+) asymptomatic subjects with respect to HIV-1(-) blood donors and was very low or almost absent in HIV-1(+) symptomatic individuals. The reduced IL-3 and IL-4 production in HIV-1-infected subjects correlated not only with the stage of the disease, but also with signs of active viral replication in PB cells, monitored by gag p24 antigen in plasma and viral isolation from PB mononuclear cells. This selective and progressive impairment in IL-3 and IL-4 production by CD4+ T-lymphocytes of HIV-1-infected subjects may contribute to explain the hematopoietic abnormalities and the derangement of the inflammatory/immune system characteristic of AIDS.

Abstract: In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV-1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.

Abstract: The effect of increasing concentrations (from 0.01 to 10 micrograms/ml) of HIV-1 envelope glycoproteins gp160, gp120, gp41 and core protein p24 was evaluated on the in vitro growth of enriched hematopoietic progenitors (CD34+ cells). Both gp120 and gp160, at concentrations from 0.01 to 10 micrograms/ml, caused a progressive and significant (p less than 0.05) decrease in viable CD34+ cell count in liquid cultures supplemented with 2 ng/ml of human recombinant (r) interleukin-3 (IL-3), evaluated by means of Trypan-blue exclusion and [3H]thymidine ([3H]TdR) incorporation. In the absence of rIL-3, no inhibitory effects were observed even at the highest gp160 and gp120 concentrations explored (10 micrograms/ml). On the contrary, gp41 and p24 did not affect the number of viable CD34+ cells, either in the presence or in the absence of rIL-3. Moreover, gp160 and gp120, but not gp41 and p24, significantly (p less than 0.05) inhibited the in vitro growth of granulomacrophage progenitors (CFU-GM) in a dose-dependent fashion. These data clearly demonstrate that HIV-1 envelope glycoproteins inhibit the growth of purified hematopoietic progenitors. We propose that HIV-1 can impair hematopoiesis through the interaction of gp120/gp160 with CD34+ cell surface, independently of an infectious process.

Abstract: The production of granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were evaluated in the supernatants of short-term cultures of purified CD4+ T-lymphocytes and enriched monocytes obtained from peripheral blood (PB) of 35 HIV-1 seropositive (+) asymptomatic individuals, stages I-II of the Walter Reed (WR) classification, 15 HIV (+) symptomatic patients (WR V-VI) and 40 HIV-1 seronegative normal blood donors. IL-1 beta and TNF-alpha production by either enriched monocytes or isolated CD4+ T-cells, was similar in HIV-1 (+) asymptomatic, symptomatic subjects and normal controls. GM-CSF level in enriched monocyte culture supernatants did not show any significant difference in the three groups of subjects under investigation. On the other hand, GM-CSF production by isolated CD4+ T-lymphocytes was two-fold decreased in HIV-1 (+) asymptomatic subjects and five-fold decreased in HIV-1 (+) symptomatic patients with respect to normal blood donors. The decline in GM-CSF production was clearly correlated with viral isolation from patient's PB light density mononuclear cells (r = -0.920, p less than 0.01). The selective and progressive decline in GM-CSF production by CD4+ T-lymphocytes, starting from early stages of HIV-1 infection, suggest a preferential lesion of a specific subset of CD4+ T-lymphocytes characterized by an intense production of GM-CSF and may contribute to explain the deranged inflammatory and immune responses which characterize the course of HIV-1 infection.

Abstract: In this study, we examined the potential role of the human immunodeficiency virus (HIV) tat protein in causing the hematopoietic abnormalities frequently observed in HIV-infected individuals. Recombinant tat (r-tat) protein, at concentrations up to 10 micrograms/mL, did not display any stimulatory or inhibitory effect on the survival/proliferative capacity of CD34+ hematopoietic progenitor cells, purified from normal bone marrow (BM). However, exposure of r-tat protein (at concentrations between 10 ng/mL and 10 micrograms/mL) to enriched normal BM macrophages induced the production of a factor(s) in conditioned media that inhibited the in vitro growth of CD34+ cells in liquid cultures and of immature hematopoietic progenitors (day 14 colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) in semisolid assays. Pre-exposure of r-tat protein with a monoclonal neutralizing anti-tat antibody completely abrogated the inhibitory activity present in BM macrophage culture supernatants. The main factor responsible for this suppressive activity was transforming growth factor-beta 1 (TGF-beta 1), as shown by the ability of a polyclonal anti-TGF-beta 1 neutralizing antibody to almost completely reverse the suppressive effect of BM macrophage supernatants on CD34+ cells. TGF-beta 1 bioassays showed that exposure of r-tat protein to BM macrophages significantly increased the levels of both active and latent forms of TGF-beta 1. These results indicate that the production of TGF-beta 1, one of the most potent negative regulator of hematopoiesis, is increased by HIV tat protein and that such increase could contribute to the derangement of the hematopoietic system in HIV-infected individuals.

Abstract: Hematopoietic progenitor (CD34+) cells were purified from the bone marrow of 6 human immunodeficiency virus (HIV) type 1-seropositive cytopenic patients and 10 healthy donors. HIV-1-seropositive patients showed a reduced number of granulocyte/macrophage, erythroid, and megakaryocyte progenitors and also a progressive and significant decline of numbers of CD34+ cells in liquid culture, which did not result from a productive or latent HIV-1 infection of CD34+ cells. However, all HIV-1-seropositive patients showed signs of active viral replication at the bone marrow level. Moreover, virus isolates from 3 HIV-1-seropositive patients showed a dose-dependent inhibition on growth of normal CD34+ cells. This suppressive activity was almost completely reversed by incubating the virus isolates with an anti-gp120 polyclonal antibody before adding to normal CD34+ cells.

Abstract: The pathogenetic potential and the true extent of human T leukemia/lymphotropic virus type I (HTLV-I) and type II (HTLV-II) infection are unknown. To find out more about HTLV-I/II seroepidemiology and the risks of iatrogenic transmission, we performed a serological study, screening 4086 healthy blood donors. A surprisingly high percentage of serum reactivity to HTLV-I/II antigens was observed by commercial ELISA (2.08%) and immunoblotting (IB) (0.85%) analysis, although none of the samples satisfied the (IB) criteria for positivity based on detection of gag protein p24 and at least one env gene product, either gp46 or gp61/68. To clarify these inconclusive results, we performed polymerase chain reaction (PCR) analysis for HTLV-I and HTLV-II provirus detection in peripheral blood lymphocytes, obtained from individuals with an apparent pattern of seropositivity. The data obtained by PCR failed to reveal evidence of HTLV-I/II provirus integration in peripheral blood cells, ruling out the possibility of a viral infection in these cases, and pinpointing the limitations of both serological methods used. Our observations suggest that serological assays alone are not a reliable tool for blood donor screening of HTLV-I/II infection and raise the important question of interpreting inconclusive results.

Abstract: In a retrospective study of 31 pregnant women infected with human immunodeficiency virus type 1 (HIV-1), nine (29%) infants presented unequivocal signs of HIV-1 infection (persistent p24 antigenemia and/or positive virus isolation). All serum samples obtained from the others, during pregnancy and on delivery, were studied for specific antibody (IgA) production by immunoblotting analysis to establish a possible link between the presence of a defined antibody class and mother-to-child viral transmission. The majority (16 of 22) of HIV-1-seropositive mothers who delivered uninfected children showed IgA antibody to low-molecular-weight HIV-1 polypeptides during pregnancy. Among those who delivered infected babies, only one showed a weak IgA reactivity to HIV-1 during pregnancy. Thus, our results suggest that immunoblotting study of IgA may be a diagnostic adjunct to predict the risk of mother-to-child HIV-1 transmission.

Abstract: In this study we evaluated interleukin-6 (IL-6) plasma levels in 80 human immunodeficiency virus type 1 (HIV-1) seropositive (+) individuals and 51 HIV-1 seronegative (-) blood donors. Plasma IL-6, detectable only in a subset of HIV-1(+) individuals (45 of 80) and normal blood donors (28 of 51), was significantly (p less than 0.01) increased in HIV-1(+) subjects 187 +/- 20.5 vs. 86.3 +/- 14 pg/ml). Among HIV-1-infected individuals, ARC/AIDS patients showed the highest IL-6 values (243.3 +/- 43.3 pg/ml). HIV-1(+) subjects showed, at all the different stages of the disease, a significant increase in total gammaglobulins, particularly IgG (2071 +/- 101 vs 1265 +/- 34 of HIV-1 seronegative controls). Although among HIV-1-infected individuals, the group with detectable plasma levels of IL-6 shows the highest levels of IgG (2243 +/- 146 vs. 1790 +/- 105, p less than 0.05), no positive correlations were observed between plasma levels of IL-6 and total gamma globulins (r = 0.2) or IgG (0.17). IL-6 production was also examined in the endotoxin-free supernatants of peripheral blood cultured monocytes and CD4+ T lymphocytes, in the presence or absence of specific stimuli. The amount of IL-6 released in monocyte and CD4+ T-lymphocyte culture supernatants was similar in 40 HIV-1(+) individuals and 35 HIV-1(-) controls. Our data show that plasma levels of IL-6 are significantly increased in HIV-1-infected individuals, in particular in ARC/AIDS patients. However, such an increase does not strictly correlate with the degree of hypergammaglobulinemia in the same HIV-1-infected individuals.

Abstract: The persistence of anti-HIV-1 antibodies in bloodstains has been studied by ELISA and Western Blot (WB) analysis. The immunoblot technique was found to be specific and more sensitive and the antibodies could be detected in 0.7 mg of bloodstains for up to 6 months. The authors emphasize the importance of such an investigation on non-genetic markers in individual diagnosis for forensic purposes.

Abstract: The effect of HIV-1 on the in vitro growth of CD34+ cells, purified from bone marrow of normal donors, was studied. HIV-1 treated CD34+ cells exhibited a progressive and significant decrease of cell viability in liquid cultures and a reduced percentage of committed progenitors in the absence of viral infection. The same results were obtained treating CD34+ cell cultures with recombinant gp 120 alone. These results point to a direct cytotoxic activity of gp120 for CD34+ cells.

Abstract: In order to establish whether endothelial cells are involved in immunodeficiency virus type 1 (HIV-1) infection, we performed a virological study on endothelial cells isolated from human adipose tissue and infected with HIV-1 in vitro. Supernatants from cultures showed a reverse transcriptase activity starting one day after HIV inoculation. Viral rescue was significantly impaired in cycloheximide treated cells confirming a de novo synthesis of viral products.

Abstract: The effect of HIV-1 on the in vitro growth of enriched hematopoietic stem cells (CD34+ cells) obtained from normal peripheral blood samples was studied. In comparison to untreated controls, the number of viable CD34+ cells progressively and significantly decreased in liquid cultures containing interleukin-3 (IL-3, 100 U/ml) after inoculation with HIV-1. In inoculated samples there was a significant reduction of all the hematopoietic progenitors (CFU-GM, BFU-E, CFU-Meg) starting from the second day of culture, CFU-GM being the most affected. In spite of these findings, no evidence of viral replication was observed: the total amount of p24 in HIV-1-inoculated CD34+ cell cultures showed a plateau, slightly declining towards the end of the experimental observation period. Moreover, erythroid and granulomacrophage colonies harvested from inoculated CD34+ cell cultures were unable to infect susceptible cells.

Abstract: This review examines the wide spectrum of hematological abnormalities frequently observed in the peripheral blood and bone marrow of HIV-1 infected subjects. Several pathogenetic mechanisms have been implicated in the derangement of the hematopoietic system occurring during the course of HIV-1 infection: imbalance of the T-cell subpopulation (CD4/CD8 ratio), altered cytokine production by infected CD4+marrow cells, production of inhibitory factors by infected marrow stromal cells, antibody-mediated suppression of hematopoietic progenitors, direct infection of hematopoetic progenitors and/or precursors, and HIV-1 mediated suppression of CD34+ cell growth in the absence of a complete viral replication cycle. Each point is considered in the text, with particular attention to the mechanisms most likely to operate in the early stages of the syndrome.

Abstract: In this study we demonstrate that HIV-1-seropositive thrombocytopenic individuals, in contrast with immune thrombocytopenic purpura (ITP) patients, fail to have a compensatory increase of megakaryocytopoiesis. The in vitro growth of bone-marrow megakaryocyte progenitors (CFU-MK) and the production of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and IL-6 by bone-marrow mononuclear adherent cells and peripheral blood (PB) light-density mononuclear cells were studied in 12 HIV-1-seropositive thrombocytopenic individuals with respect to 12 ITP patients and 15 normal controls. In HIV-1-seropositive thrombocytopenic individuals, CFU-MK size (number of megakaryocytes per colony) was similar to normal controls but significantly lower (P less than 0.05) than in ITP patients. IL-1 and IL-6 production was similar in the three groups of subjects. On the other hand, GM-CSF production by bone-marrow mononuclear adherent cells in HIV-1-seropositive thrombocytopenic individuals was similar to normal controls but significantly (P less than 0.05) lower than in ITP patients, whereas GM-CSF production by PB light-density mononuclear cells was markedly (P less than 0.05) defective compared with both normal controls and ITP patients. The positive correlation between number and size of CFU-MK and production of GM-CSF by bone-marrow mononuclear adherent cells, observed in all three groups of subjects, demonstrates the central role of GM-CSF in the control of megakaryocytopoiesis.

Abstract: The production of granulocyte/macrophage colony-stimulating factor (GM-CSF) by peripheral blood (PB) light density mononuclear cells (LD-MNC), CD2+ T lymphocytes and purified CD4+ T lymphocytes was investigated in 20 human immunodeficiency virus type 1 (HIV-1) seropositive (WRII-III) individuals in comparison with 18 normal controls. GM-CSF in supernatants of stimulated cultures was determined by biological and immunoenzymatic assays. GM-CSF production by LD-MNC, CD2+ T lymphocytes and purified CD4+ T lymphocytes was significantly (p less than 0.01) reduced in HIV-1 infected individuals, especially in patients at the more advanced stages of the disease. Moreover, the number of circulating granulocyte/macrophage colony-forming units (CFU-gm) was significantly (p less than 0.01) reduced in HIV-1 seropositive subjects (31.5 +/- 4.4) compared with normal controls (78 +/- 10). There was a positive correlation (r = 0.720, p less than 0.01) between CFU-gm and GM-CSF production by LD-MNC in HIV-1 seropositive individuals. On the other hand, the absolute number of CD4+ lymphocytes did not correlate with GM-CSF production by LD-MNC (r = 0.158) or CD2+ T lymphocytes (r = 0.225). These data indicate that the impaired production of GM-CSF in HIV-1-infected individuals is not only due to a reduction in CD4+ T lymphocytes, but also to a qualitative impairment of these cells which may contribute to the loss of circulating hematopoietic progenitors in HIV-1-infected subjects.

Abstract: Poly(ADP-ribose)polymerase is a chromatin-bound enzyme which is activated by free DNA ends and is therefore stimulated by a variety of DNA-damaging agents. The enzyme transfers the ADP moiety of NAD to nuclear proteins to create protein-bound ADP-ribose polymers. Under conditions favouring an accelerated poly(ADP-ribose) polymer formation, the enzyme may exhaust cellular NAD pools. At the same time, or shortly thereafter ATP levels drop and cell viability eventually declines. As a series of chemical and physical agents which may play a role in activating latent HIV-1 infection or favouring HIV-1 replication, have a DNA-damaging activity, we investigated the behaviour of poly(ADP-ribose)polymerase activity in various types of HIV-1-infected cells. The results obtained show that HIV-1-infected cells to possess an increased poly(ADP-ribosol)ating activity together with an accentuated fragmentation of cellular DNA which are associated with the time course of HIV-1 replication. These data give circumstantial support to the hypothesis that a NAD-depdendent cellular suicide response to DNA damage, could play a role in the death of HIV-1 infected cells. In this respect, the impared immunocompetence of HIV-1-infected patients could bear some resemblance to immune attribution that sometimes accompanies some inborn errors affecting DNA precursor metabolism and DNA integrity.

Abstract: Rapid exposure to supra-optimal temperature (heat-shock) and a variety of other treatments are able to induce changes in cellular translational and transcriptional activity referred to as "the heat-shock response". The effect of heat-shock was investigated in H9 lymphoblastic cells and in peripheral blood lymphocytes infected with human immunodeficiency virus type 1 (HIV-1). The results showed that a mild heat-shock performed either immediately before infection or 7 days after infection consistently increased the recovery of p24 core antigen and reverse transcriptase activity in the supernatants of experimentally infected cell cultures. On the contrary, heat-shock had no effect on the HIV-1 marker recovery from persistently infected H9/HTLV-III cell cultures.

Abstract: Acute or recent Cytomegalovirus infections were serologically identified in HIV1-seropositive individuals and studied in relation to HIV1 replication as HIV1-p24 antigenemia. The results obtained indicate that active CMV infections are often observed in HIV1-seropositive individuals and they preferentially precede or are coincident with HIV1 replication.

Abstract: A virological and immunomorphological study has been performed on endomyocardial biopsy from an AIDS patient. Some data suggest the possible involvement of HIV in the pathogenesis of the cardiomyopathy.

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Abstract: We investigated the in vitro growth of circulating progenitors from mononuclear nonadherent cells (MNAC) and T-depleted MNAC (non-T-MNAC) in the peripheral blood (PB) of 20 human immunodeficiency virus type 1 (HIV-1) seropositive subjects, compared with 12 normal adult volunteers, in order to clarify whether the loss of hemopoietic progenitors described in the bone marrow (BM) of AIDS-related complex (ARC)/AIDS patients could occur in PB before the AIDS stage, only those patients at the early stages of the disease who had never undergone cytotoxic therapy were considered in the study. We found a significant reduction in the number of granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM; p less than 0.001), megakaryocytic progenitors (megakaryocyte colony-forming units, CFU-MK; p less than 0.001) and erythroid progenitors (erythroid burst-forming units, BFU-E; p less than 0.05) in non-T-MNAC cultures of PB from HIV-1 seropositive subjects compared with normal PB control cultures. Although most of our patients had an inverted CD4/CD8 ratio and a marked reduction in the absolute number of CD4+ cells, there was no correlation with the absolute number of CD4+ cells or with the CD4/CD8 ratio. The loss of hemopoietic progenitors in PB seemed to occur earlier than in BM, because the hemograms of the patients considered in the study were normal in most cases.

Abstract: Neutralization and immunoblot or immunoprecipitation assays of serum samples from seronegative or seropositive volunteers immunized with the attenuated Towne and challenged with the virulent Toledo cytomegalovirus strains were carried out. Titers of neutralizing antibodies differed as a function of the strain used for immunization. All serum samples with neutralizing activity detected a 58-kDa protein that is the abundant component of the major glycoprotein complex of the envelope, suggesting that this protein complex is involved in the induction of neutralizing antibodies. Complement-independent neutralizing activity was found to develop later than complement-dependent activity, and no correlation was observed between complement-independent titers of neutralizing antibodies and antibody to the 86-kDa protein, which bears a complement-independent neutralizing epitope. Antibodies to the 66-kDa major tegument protein were present early after infection but were not correlated with serum neutralizing activity.

Abstract: Five hundred and fifty six subjects, known to be homosexuals or intravenous drug abusers and seropositive for HIV antibody, were selected on the basis of their clinical state--symptom free, lymphadenopathy syndrome (LAS), AIDS related complex (ARC), and AIDS. The presence of antigenaemia and the humoral response to viral polypeptides was investigated. The prevalence of patients positive for p31 antibody was significantly increased in those with AIDS and detectable antigenaemia.

Abstract: Forty children born to mothers seropositive for the human immunodeficiency virus type 1 (HIV-1) followed-up to 15 months after birth were studied by means of serum antibody patterns to individual viral polypeptides, by the presence of detectable levels of viral core antigen (p24) and virus in serum and peripheral blood lymphocytes, and by total lymphocyte counts and T4/T8 lymphocyte ratios. The results obtained indicate that a persistent antigenemia is significantly associated with positive virus isolation from peripheral blood lymphocytes and with changes in the intensity of antibody reaction to core (p24, p17) and pol (p31) antigens. Six children (15%) presented unequivocal signs of HIV-1 infection and five also had signs of immune system involvement.

Abstract: Human immunodeficiency virus (HIV-1) can be transmitted by blood transfusions. A recent report focused on the relativey high risk of HIV-1 infection in American patients treated for leukemia and multiply transfused as a consequence of therapy. We therefore conducted a retrospective study on the presence of HIV-1 antibodies among 91 acute leukemia patients diagnosed between 1978 and 1985, before the onset of routine tests for HIV-1 contamination of blood products. The transfusion requirement (platelet units, red blood cell concentrates) involved almost 7,000 donors. We did not find any case of seropositivity in patients transfused with units from the donor pool. The only case of HIV-1 seropositivity was due to a bone marrow transplant donor, retrospectively found to be HIV-1 seropositive. These results differ from the American data previously cited. This is probably due both to differences in diffusion of the HIV-1 infection in the two countries and to differences in the selection of the two donor populations. We conclude that the risk of contracting HIV-1 infections before 1985 through multiple transfusions from registered donors in our Italian area was very low, if not absent, not only for leukemia patients but reasonably for other categories of heavily transfused groups.

Abstract: A mild heat-shock (10 min at 44 degrees C), performed just before seeding on healthy PBL used for co-cultivation, allows the detection of viral markers (p24 core antigen and reverse transcriptase) in a very short time. This suggests that heat-shocked PBL can be employed routinely for the rapid detection of HIV-1 in clinical samples.

Abstract: An unusually high prevalence (45%) of serum antibodies to human T cell leukemia virus type I (or to an antigenically related virus) in comparison with that observed against other viral pathogens (human immunodeficiency virus type 1, herpes simplex virus, human cytomegalovirus, varicella zoster virus, and respiratory syncytial virus) has been observed in a group of Bismam Asmat (Papua) subjects, living in a very limited and geographically isolated area of Indonesian New Guinea.

Abstract: Twelve homosexual males who seroconverted to human immunodeficiency virus (HIV) were followed-up for over two years. Analysis of sera by immunoblotting showed that seroconversion was characterized by the presence of specific IgM that reacted mainly with viral polypeptides of molecular weights ranging from 17 Kd to 55 Kd. Specific IgG to all HIV proteins was detected. Immunoblotting showed that antibodies to 24 Kd core protein alone or in association with 17 Kd polypeptide appeared first in some cases. Virus antigen was detected in six patients: five subjects were positive at the time of seroconversion, and one became positive afterwards. It is concluded that detection of IgG and IgM antibody against the different viral polypeptides, together with detection of viral antigen is necessary in order to determine the stage of HIV infection.

Abstract: An antibody response to human immunodeficiency virus (HIV) is described in a young woman with T-lymphoblastic leukemia, who received a bone marrow transplant from a donor retrospectively found to be HIV positive.

Abstract: Monoclonal antibodies (MAbs) that neutralized human cytomegalovirus (HCMV) were produced by ten hybrid cells lines, generated from BALB/c mice immunized with HCMV-infected human fibroblasts. By immunoblot technique six antibodies detected a set of HCMV glycosylated polypeptides which, when separated under reducing conditions, migrated with apparent molecular weights (m.wt.) of 47.5K, 51K, 54K, 58K, and 60-69K. One other antibody reacted only with the 47.5 and 51K polypeptides and the 60-69K broad band. Under nonreducing conditions, these antibodies showed no reactivity with any polypeptide. The three remaining MAbs reacted with two high-m.wt. polypeptides of approximately 200K and greater than 200K when separated under nonreducing conditions. One of these antibodies showed no clear reactivity with the polypeptides, one detected a 58K and 92-94K species and one detected a 58K and 130K species, when separated under reducing conditions.

Abstract: Using specific monoclonal antibodies, we have localized two structural proteins (p65-69 and p28) of human cytomegalovirus in infected cells and in virions and/or virus-related particles by immunoelectron microscopy using protein A-gold. Protein p65-69 is present in some roundish structures in the nuclei, often in contact with the viroplasm, and in the cytoplasm, exclusively within the dense body matrix. Protein p28 is present only in the outline of cytoplasmic capsids, and reaches the highest density in the large aggregates of virions and dense bodies which are particularly numerous during the late phases of the viral replication cycle.

Abstract: A rapid, simple and reproducible microneutralization test for human cytomegalovirus is described. The results can be read in 1-2 days and the neutralization titer detected in human and guinea pig sera and in monoclonal antibody-containing supernatants is consistent with that derived by the plaque-reduction neutralization test.

Abstract: We have produced a monoclonal antibody against human cytomegalovirus (HCMV) which recognizes a structural protein of 28 000 mol. wt. which is present in both the cytoplasm of infected cells during the late phase of the viral replication cycle and in the extracellular viral particles. This antigen was detected in all HCMV strains assayed and reacted with human sera having anti-HCMV antibodies.

Abstract: Indirect immunofluorescence and enzyme-linked immunosorbent assay to detect serum antibodies against Legionella pneumophila serotype 1 were compared. A very good agreement was found with the great majority of serum samples (85.07%). For detecting high antibody levels and rise in antibody titres, both methods seem to be suitable, however for the detection of relatively low antibody titres, very important in epidemiological studies, enzyme immunoassay could be more accurate.

Abstract: ADP ribosylation has been studied during human Cytomegalovirus (HCMV) replication in human embryo fibroblasts. The intranuclear level of this enzyme is higher during the whole viral replication cycle with respect to mock infected cells. The increase begins during the first hour ater the addition of the viral stock to the cells. Results are discussed in view of the possibility that this enhanced activity might be correlated with HCMV-induced alteration of the cell chromatin leading to an increase in host cell macromolecular synthesis.

Abstract: The primary murine cytomegalovirus infection and the subsequent persistent infection have been studied in respect to virus isolation from various organs and specific humoral immune response. The first acute phase of infection took place in parenchymal organs and it was followed by persistent infection in the salivary glands. The transition from the acute phase to the persistent infection coincided with the peak of specific humoral immune response.

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Abstract: .5 mM of novobiocin completely suppresses the replication of human cytomegalovirus (HCMV) in vitro. Based on the evidence that murine cytomegalovirus (MCMV) titres were reduced in vitro by 60% with the same concentration of the drug, the in vivo antiviral activity of novobiocin has been tested in the murine model. The results showed that if treatment started before the infection virus was not isolated from parenchymal organs during treatment. Virus could be isolated 24 h after stopping treatment, but not from kidney or spleen.