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.

Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK-. Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK- and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK-, respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated.

Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway.

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.

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.

HIV-1 DNA proviral load in treated and untreated HIV-1 seropositive patients.

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 + or - 731 to 715 + or - 673 copies/10(5) PBMC and 2-LTR HIV-1 DNA ranging from 94 + or - 105 to 65 + or - 44 copies/10(5) PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 + or - 676 to 262 + or - 174 copies/10(5) PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 + or - 55 to 26 + or - 35 copies/10(5) PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4(+) T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

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.

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.

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

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

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance.

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.

Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors.

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1 x 10(4) and 9 x 10(4) HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly (P=0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Selective up-regulation of functional CXCR4 expression in erythroid cells by HIV-1 Tat protein.

CXCR4 is the high affinity receptor for the SDF-1 alpha chemokine and represents the main coreceptor for HIV-1 T-tropic strains. The surface expression of CXCR4 was analysed in CD34+ haematopoietic progenitors, induced to differentiate along the erythroid or granulocytic lineages, in liquid cultures supplemented or not with HIV-1 Tat protein. At concentrations as low as 1-10 ng/ml, synthetic Tat protein significantly increased the surface expression of CXCR4 in erythroid but not in granulocytic cells. The Tat-mediated up-regulation of surface CXCR4 was accompanied by a concomitant increase of CXCR4 mRNA and total CXCR4 protein content in cells developing along the erythroid lineage after 6-10 days of culture. Moreover, addition of SDF-1 alpha (200 ng/ml) induced a significant higher rate of apoptosis in Tat-treated erythroid cells in comparison with control cells. These results demonstrated for the first time a direct positive role in haematopoietic gene regulation of Tat protein, and suggest the possible involvement of Tat in HIV-1-induced anaemia.

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.

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a cohort of Italian HIV-1 seropositive patients extensively treated with antiretroviral drugs.

As the emergence of highly resistant virus might compromise antiretroviral regimens in HIV-1 infected patients, a constant analysis of genotypic mutations should be performed to establish the magnitude of mutation prevalence and gauge their impact in patients treated extensively with combination therapy. The frequency of multiple dideoxynucleoside analogue resistance (MddNR) was evaluated in a group of Italian HIV-1 seropositive patients who failed to respond to therapy despite a long-lasting drug treatment. Results showed the presence of one or more mutations (A62V, V75I, F77L, F116Y and Q151M) able to confer resistance to all NRTIs in a relatively high percentage (7.9%) of patients enrolled in the study. Moreover, a significantly lower HIV-1 viral replication in patients with MddNR, suggested the importance of monitoring HIV-1 subjects not only by viral load, but also by drug resistance testing, so that a correct drug regimen may be chosen.

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.

Relationships between the presence of anti-Tat antibody, DNA and RNA viral load.

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.

Antibody to HIV-1 Tat protein, a key molecule in HIV-1 pathogenesis. A brief review.

In the last few years, literature reports have unequivocally established that the 86-101 aminoacid Tat protein, essential for an efficient viral replication, can be actively secreted by infected cells. The contribution of extracellular Tat to the progression of viral infection is underlined by the ability of neutralizing anti Tat antibody to reduce the viral load in vitro and possibly also in vivo. Considering that at least some of the effect of Tat protein seem to be the consequence of an autocrine loop and that anti Tat antibody is an efficient inhibitor of viral replication, it is reasonable to suppose that extracellular Tat play a functional role in HIV-1 infection and that HIV antibody may interfere with a possible Tat driven pathogenesis. This review explores the meaning of anti Tat antibody in vitro and in vivo and its importance to shed more light on viral pathogenesis and the recent development of Tat containing vaccine.