Serum hepatitis B surface antigen monitoring in long-term lamivudine-treated hepatitis B virus patients.

Serum hepatitis B virus surface antigen (HBsAg) levels have been suggested to predict interferon response in chronic hepatitis B. A few data are available on the role of HBsAg measurement in nucleos(t)ide analogues (NA) treatment. We retrospectively investigated the relation between HBsAg changes and main treatment outcomes during long-term lamivudine treatment in hepatitis e antigen (HBeAg)-negative chronic hepatitis B. A total of 42 HBeAg-negative patients were consecutively enrolled in an open-label study on long-term lamivudine monotherapy (150 mg/die). Serum HBsAg levels were quantified every 6 months by Architect assay (Abbott Diagnostics). HBV-DNA was quantified quarterly by real-time PCR (Roche Diagnostics). The median duration of lamivudine treatment was 66 months (20-153). One patient (2%) was a primary nonresponder, 35 (83%) developed virological breakthrough (VB) and the remaining six patients (14%) were classified as long-term on-treatment responders. During treatment, HBsAg levels decreased only in long-term on-treatment responders, while no changes were observed in resistant patients. Failure to achieve a decrease of 0.7 log(10) IU/mL in serum HBsAg at month six of lamivudine had a positive predictive value of developing VB of 90% and a negative predictive value of 100%. These high predictive values were also maintained in the subgroup of patients negative for HBV-DNA at month six. The results of this study with a small sample size suggest a role of on-treatment HBsAg quantification in the management of lamivudine-treated patients. If validated prospectively in a larger patient cohort, HBsAg measurements would be a useful adjunct to optimize antiviral therapy.

The behavior of free purine nucleotides in lymphocytes infected with HIV-1 virus.

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.

Early loss of circulating hemopoietic progenitors in HIV-1-infected subjects.

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.

Lack of compensatory megakaryocytopoiesis in HIV-1-seropositive thrombocytopenic individuals compared with immune thrombocytopenic purpura patients.

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.

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.

Uninfected haematopoietic progenitor (CD34+) cells purified from the bone marrow of AIDS patients are committed to apoptotic cell death in culture.

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.

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.

Evidence for an HIV-1 mediated suppression of in vitro growth of enriched (CD34+) hematopoietic progenitors.

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.

Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immunoelectron microscopy.

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.

Progressive and selective impairment of IL-3 and IL-4 production by peripheral blood CD4+ T-lymphocytes during the course of HIV-1 infection.

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.

High prevalence of serum antibody against human T cell leukemia virus type I (HTLV-I) among the Bismam Asmat population (Indonesian New Guinea).

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.

Hyperimmunoglobulinemia in HIV-1 infected individuals does not clearly correlate with plasma levels of IL-6.

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.

Antibodies against full-length Tat protein and some low-molecular-weight Tat-peptides correlate with low or undetectable viral load in HIV-1 seropositive patients.

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.

Viral load trend in HIV-1 seropositive patients with different CD4 cell counts before starting HAART.

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.

Patterns of antigenaemia and antibody response in patients infected with human immunodeficiency virus (HIV) according to clinical state.

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.

Effect of human immunodeficiency virus type 1 on CD34+ cells.

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.

Vertical transmission of human immunodeficiency virus type 1. Prognostic value of IgA antibody to HIV-1 polypeptides during pregnancy.

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.

Rapid detection of HIV-1 in clinical samples by co-culture with heat-shocked cells.

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.

A rapid microneutralization assay for cytomegalovirus.

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.

Development of drug resistance in HIV-1 patients receiving a combination of stavudine, lamivudine and efavirenz.

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.