HIV-1 coreceptor usage in paired plasma RNA and proviral DNA from patients with acute and chronic infection never treated with antiretroviral therapy.

Although an independent evolution of viral quasispecies in different body sites might determine a differential compartmentalization of viral variants, the aim of this paper was to establish whether sequences from peripheral blood mononuclear cells (PBMCs) and plasma provide different or complementary information on HIV tropism in patients with acute or chronic infection. Tropism was predicted using genotypic testing combined with geno2pheno (coreceptor) analysis at a 10% false positive rate in paired RNA and DNA samples from 75 antiretroviral-naïve patients (divided on the basis of avidity index into patients with a recent or long-lasting infection). A high prevalence of R5 HIV strains (97%) was observed in both compartments (plasma and PBMCs) in patients infected recently. By contrast, patients with a long-lasting infection showed a quite different situation in the two compartments, revealing more (46%) X4/DM in PBMCs than patients infected recently (3%) (P = 0.008). As- a knowledge of viral strains in different biological compartments might be crucial to establish a therapeutic protocol, it could be extremely useful to detect not only viral strains in plasma, but also viruses hidden or archived in different cell compartments to better understand disease evolution and treatment efficacy in patients infected with HIV.

A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV-infected individuals with occult HBV infection.

To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV-) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV-DNA was measured by a quantitative real-time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV- patients, HBV-DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV-DNA was found in 6/7 (86%) PBMC of occult-positive and none of occult-negative LT. Significantly lower HBV-DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult-positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper-mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV-DNA detection in PBMC may offer a useful tool to identify OHB in serum-negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.

Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus 1 or gp120 mediated by endogenous transforming growth factor beta 1.

Human CD34+ hematopoietic progenitor cells, stringently purified from the peripheral blood of 20 normal donors, showed an impaired survival and clonogenic capacity after exposure to either heat-inactivated human immunodeficiency virus (HIV) 1 (strain IIIB) or cross-linked envelope gp120. Cell cycle analysis, performed at different times in serum-free liquid culture, showed an accumulation in G0/G1 in HIV-1- or gp120-treated cells and a progressive increase of cells with subdiploid DNA content, characteristic of apoptosis. In blocking experiments with anti-transforming growth factor (TGF) beta 1 neutralizing serum or TGF-beta 1 oligonucleotides, we demonstrated that the HIV-1- or gp120-mediated suppression of CD34+ cell growth was almost entirely due to an upregulation of endogenous TGF-beta 1 produced by purified hematopoietic progenitors. Moreover, by using a sensitive assay on the CCL64 cell line, increased levels of bioactive TGF-beta 1 were recovered in the culture supernatant of HIV-1/gp120-treated CD34+ cells. Anti-TGF-beta 1 neutralizing serum or TGF-beta 1 oligonucleotides were also effective in inducing a significant increase of the plating efficiency of CD34+ cells, purified from the peripheral blood of three HIV-1-seropositive individuals, suggesting that a similar mechanism may be also operative in vivo. The relevance of these findings to a better understanding of the pathogenesis of HIV-1-related cytopenias is discussed.

In vitro exposure of human chondrocytes to pulsed electromagnetic fields.

The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI)-derived cells was studied to ascertain the healing potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibro- blastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.

Human immunodeficiency virus type 1 Tat protein protects lymphoid, epithelial, and neuronal cell lines from death by apoptosis.

We report here that the tat gene product of human immunodeficiency virus type 1 was able to protect lymphoblastoid (Jurkat), epithelial (293) and neuronal (PC12) cell lines from apoptotic death induced by serum withdrawal. The rescue from apoptosis by Tat was reflected by an increased expression of Bcl-2 protein in tat-positive Jurkat cells with respect to mock-transfected Jurkat cells after 3-6 days of serum-free cultures. We propose that the ability of the regulatory human immunodeficiency virus type 1 Tat protein to suppress apoptosis might have important implications in understanding the pathogenesis of frequent neoplastic disorders observed in human immunodeficiency virus type 1-seropositive individuals.

Discordant resistance interpretations in multi-treated HIV-1 patients.

The routine determination of drug resistance has become an important part of the clinical management of HIV-1 infected patients. Plasma samples from 130 individuals treated for at least 1 year with multiple NRTIs and NNRTIs were tested for the presence of mutations correlated to drug resistance. Since interpretation criteria represent a crucial point for virologists and clinicians, often complicated by the presence of novel and/or complex mutations patterns, we analyzed results interpreted by TruGene HIV-1 (Visible Genetics, Toronto, Ontario, Canada) and VirtualPhenotype (Virco, Mechelen, Belgium). A high degree of concordance was found for NNRTIs whereas NRTIs interpretation was highly discrepant. Since different approaches to monitoring resistance reflect different interpretation of results, the prediction of drugs resistance from a given HIV sequence might be contradictory and requires accurate standardization and unique interpretative rules.

All-trans retinoic acid potentiates megakaryocyte colony formation: in vitro and in vivo effects after administration to acute promyelocytic leukemia patients.

In this study, we evaluated the in vitro growth of normal hematopoietic progenitors (CFU-GM, BFU-E, CFU-GEMM, CFU-meg) stimulated by optimal sources of colony stimulating activity in the absence or presence of 10(-6) M all-trans retinoic acid (ATRA). ATRA alone did not show any colony-stimulating ability when added in culture to partially purified bone marrow populations. On the other hand, it significantly increased the number of CFU-GM (p = 0.003) and both the number (p = 0.009) and size (p = 0.002) of CFU-meg in the presence of appropriate colony-stimulating activity. Since ATRA had only modest stimulatory effects on purified CD34+ cells, the megakaryocyte colony-stimulating activity of ATRA was mainly due to an increased production of endogenous cytokines by bone marrow accessory cells. In parallel experiments, the in vitro growth of the different hematopoietic progenitors was evaluated in 28 patients affected by acute non-lymphoid leukemia (ANLL), mainly acute promyelocytic leukemia (APL). Bone marrow cells were harvested after remission induction obtained: (i) in ten APL patients treated with ATRA followed by one chemotherapy cycle (CHT) (3/7: Daunorubicin+Ara-C): group A ('ATRA/CHT'); (ii) eight APL patients treated with one CHT cycle alone (3/7 as above): group B ('APL-CHT'); (iii) in ten ANLL-non-APL patients after one CHT cycle (3/7 as above): group C ('ANLL-CHT'). The number of the different hematopoietic progenitors, and in particular CFU-GM and CFU-meg, was significantly higher in APL patients treated with ATRA plus CHT (group A) compared to APL (group B) or ANLL-non-APL (group C) patients treated with CHT alone (CFU-GM: p = 0.01; CFU-meg: p = 0.03). Our data demonstrate that ATRA is able to potentiate both normal and APL megakaryocytopoiesis and suggest that the in vivo administration of ATRA could be beneficial in other pathological conditions, where the megakaryocyte progenitor cell compartment is impaired.

HIV-1 Tat induces tyrosine phosphorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells.

OBJECTIVE: To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. METHODS: The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA. RESULTS: Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK. CONCLUSION: HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

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.

Upregulation of urokinase-type plasminogen activator by endogenous and exogenous HIV-1 Tat protein in tumour cell lines derived from BK virus/tat-transgenic mice.

OBJECTIVE: To demonstrate that Tat modulates the plasminogen-dependent proteolytic activity of tumour cell lines derived from BK virus (BKV)/tat-transgenic mice by affecting the production of plasminogen activators (PA) and the PA inhibitor (PAI)-1 and to demonstrate that this occurs through mechanism(s) that are distinct from those responsible for transactivating activity of extracellular Tat. DESIGN AND METHODS: To assess whether endogenous Tat is responsible for PA activity in T53 adenocarcinoma cells, cell cultures were transfected with antisense Tat cDNA and evaluated for cell-associated PA activity by a plasmin chromogenic assay. The assay was also used to evaluate PA activity in T53 cells and T111 leiomyosarcoma cells stimulated by extracellular Tat. The type(s) of PA produced were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. The levels of PAI-1 were evaluated by Western blotting. Tat transactivating activity was measured by a chloramphenicol acetyltransferase (CAT) enzyme-linked immunosorbent assay in HL3T1 cells containing integrated copies of an HIV-1 long terminal repeat (LTR)-CAT plasmid. RESULTS: Transfection of T53 cells with antisense Tat cDNA results in the decrease of Tat production and PA activity. Exogenously added Tat increases PA levels in T53 and in T111 cells. PA activity was identified as urokinase-type PA (uPA). Tat also increases the production of PAI-1 in T111 but not in T53 cells. Chloroquine and heparin have different affects on the LTR-CAT-transactivating and the PA-inducing activities of Tat. The fusion protein glutathione-S-transferase-Tat and the mutant Tat-1e, lacking the second Tat exon, cause LTR-CAT transactivation without stimulating uPA upregulation. CONCLUSIONS: Tat affects the fibrinolytic activity of tumour cell lines derived from BKV/tat-transgenic mice by modulating the production of both uPA and PAI-1 via autocrine and paracrine mechanisms of action. The capacity of Tat to modulate the plasminogen-dependent proteolytic activity of these tumour cell lines may contribute to their metastatic potential. The uPA-inducing activity of Tat depends upon specific biological and structural features of the Tat protein that are distinct from those responsible for its LTR-CAT-transactivating activity, suggesting distinct mechanisms of induction for the two biological responses.

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes.

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.

Flow cytometric quantification of HIV-1 Tat protein in tat-transfected Jurkat T cell lines.

The transactivator Tat protein represents a pivotal factor for the replication of human immunodeficiency virus type 1 (HIV-1). In this report, we describe a flow cytometry procedure designed to quantify the intracellular content of Tat protein in Jurkat CD4+ T lymphoblastoid cell lines, stably transfected with plasmids expressing full-length Tat protein. Various expression vectors were compared for their effectiveness to yield Tat protein in Jurkat cells, and several technical parameters were analyzed to optimize the assay. This method offers a quick and efficient approach to select stably transfected cell lines expressing different levels of specific protein.

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients.

BACKGROUND: The routine determination of drug resistance in newly HIV-1 infected individuals documents a potential increase in the transmission of drug-resistant variants. Plasma samples from twenty seven therapy naive HIV-1 infected Italian patients were analyzed by the line probe assay (LIPA) and the TruGene HIV-1 assay for the detection of mutations conferring resistance to HIV-1. RESULTS: Both tests disclosed amino-acid substitutions associated with resistance in a variable number of patients. In particular, two mutations (K70R and V118I), detectable by LIPA and by sequencing analysis respectively, revealed resistance to NRTIs in two plasma samples. At least three mutations conferring resistance to NNRTIs, not detectable by commercial LIPA, able to reveal mutations associated only with nucleoside reverse transcriptase analogues, were disclosed by viral sequence analysis. Moreover, most samples showed mutations correlated with resistance to protease inhibitors. Remarkably, a key mutation, like V82A (found as a mixture), and some "indeterminate" results (9 samples), due the absence of signal on the lines corresponding to a specific probe, was revealed only by LIPA, while a variable number of secondary mutations was detectable only by TruGene HIV-1 assay. CONCLUSION: Even if further studies are necessary to establish the impact of different tests on the evaluation of drug-resistant strains transmission, LIPA might be useful in a wide population analysis, where bulk results are needed in a short time, while sequencing analysis, able to detect mutations conferring resistance to both NRTIs and NNRTIs, might be considered a more complete assay, albeit more expensive and more technically complex.

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.

Efficient parvovirus B19 DNA purification and molecular cloning.

A simple and efficient method of purification and molecular cloning of Parvovirus B19 DNA directly from small quantities of viremic sera was developed. Purified virions were lysed in annealing conditions, then viral DNA purification in double strand (ds) DNA form was achieved using an affinity DNA binding matrix. Affinity purification yielded a consistently high recovery of viral DNA. Using affinity purified ds viral DNA, we efficiently and stably cloned the complete coding internal unique sequence of B19 DNA. In our cloning strategy AatII and BamHI restriction endonuclease sites were exploited. This permitted cleavage of the 5.0 kbp AatII fragment in two AatII-BamHI fragments which could be efficiently cloned in a directional way in pUC18 plasmid vector. The availability of the two cloned AatII-BamHI fragments thus allowed the construction of a full length clone in a single ligation reaction.

Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA.

A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.

An amplified dot immunoassay for the direct quantitation of adapted and wild strains of human cytomegalovirus.

A rapid and simple quantitative dot immunoassay for a cell-adapted reference strain of cytomegalovirus (CMV) and for wild strains of CMV present in clinical urine samples was developed. The assay was performed on nitrocellulose paper dotted with several dilutions of viral pellets free of cellular debris. Viral dilutions were treated with a monoclonal antibody to the major component of the viral capsid. To amplify the reaction, a three-dimensional complex of streptavidin and biotinylated horseradish peroxidase was used as the detector system. The dot immunoassay, which does not require cell cultures, yielded results within one day. A significant correlation was found between CMV titers obtained by dot immunoassay and CMV infectious units determined by immunoalkaline phosphatase staining of CMV-late antigen positive cells.

Detection of CMV DNA in clinical samples of AIDS patients by chemiluminescence hybridization.

A sensitive chemiluminescence dot-blot hybridization assay for the detection of CMV DNA in clinical samples of AIDS patients is described. In the chemiluminescence hybridization assay, digoxigenin-labelled CMV DNA probes were used and when hybridized they were detected by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. Adamantyl 1,2-dioxetane phenyl phosphate was used as the chemiluminescent substrate. The results were recorded by instant photographic films. The results obtained with the chemiluminescence hybridization assay were compared with the results obtained by hybridization with colourimetric detection. The chemiluminescent assay proved specific, sensitive and reliable and thus can be used as a valuable routine diagnostic test for the detection of CMV DNA in clinical samples.