Search

Serum antibodies against human intracisternal A-type particle (HIAP) endogenous retrovirus have been found to be associated with various autoimmune pathologies. To evaluate the presence of serum antibody reactivity to HIAP proteins in systemic sclerosis, a Western blot analysis was performed on sera from 42 patients with systemic sclerosis, in comparison with 18 sera from patients with primary biliary cirrhosis and 52 healthy subjects. A positive Western blot was found in 55.5% of serum samples from patients with primary biliary cirrhosis and in 66.0% of patients with systemic sclerosis. None of the 52 healthy subjects showed positive results. Although this difference may be attributable either to an autoimmune response to antigenically related cellular proteins or to a specific antibody response to HIAP proteins expressed as an incidental consequence of attendant pathological processes, the high prevalence of antibodies against HIAP proteins demonstrated in patients with systemic sclerosis may be considered a hallmark of this disease.

Background: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. Objective: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. Study design: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. Results/conclusions: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients (627±1068 HIV-1 proviral DNA copies per 10 6 PBMC, with a range of 30–6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

A cross-sectional study was carried out in our tertiary care hospital between January 1998 and December 2001. All 161 consecutive patients naive to nelfinavir and who had received a nelfinavir-based highly active antiretroviral therapy (HAART) of at least 24-week duration were extrapolated from the 802 adult HIV-infected subjects treated with antiretroviral therapy. All cases of virologic failure were considered and viral genotyped. Virologic failure occurred in 80 out of 161 nelfinavir-treated patients, all belonging to the experienced group. On the whole, only 11 patients (7%) developed the D30N substitution, whose 6 was in association with the N88D mutation. Among the 80 failed patients, the M184V mutation was detected in 52 (65%), while only 7 patients showed simultaneously the M184V, T215Y and K103N substitutions. In our HIV-infected population receiving a nelfinavir-based HAART, the D30N mutation has shown a low absolute frequency, while the detection of M184V substitution and the simultaneous occurrence of M184V, T215Y and K103N mutations were related to a more favorable virological response.

The interferon-inducible DNA sensor IFI16 is involved in the modulation of cellular survival, proliferation, and differentiation. In the hematopoietic system, IFI16 is consistently expressed in the CD34+ stem cells and in peripheral blood lymphocytes; however, little is known regarding its regulation during maturation of B- and T-cells. We explored the role of IFI16 in normal B-cell subsets by analysing its expression and relationship with the major transcription factors involved in germinal center (GC) development and plasma-cell (PC) maturation.IFI16mRNA was differentially expressed in B-cell subsets with significant decrease inIFI16mRNA in GC and PCs with respect to naïve and memory subsets.IFI16mRNA expression is inversely correlated with a few master regulators of B-cell differentiation such asBCL6, XBP1, POU2AF1, andBLIMP1. In contrast,IFI16expression positively correlated withSTAT3, REL, SPIB, RELA, RELB, IRF4, STAT5B, andSTAT5A. ARACNE algorithm indicated a direct regulation ofIFI16byBCL6,STAT5B, andRELB, whereas the relationship betweenIFI16and the other factors is modulated by intermediate factors. In addition, analysis of the CD40 signaling pathway showed thatIFI16gene expression directly correlated with NF-κB activation, indicating that IFI16 could be considered an upstream modulator of NF-κB in human B-cells.

A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.

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.

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 (Tat6–14, Tat11–24, Tat36–50, Tat46–60, Tat56–70 and Tat65–80), 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 Tat6–14, Tat36–50 and Tat46–60 (P=0.002, P=0.027 and P

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

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

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.

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

In order to evaluate clinical efficacy and safety of combination antiretroviral therapy with protease inhibitors in children with congenital HIV disease a pilot study was performed at our department. After informed consent was obtained from parents seven children aged 8-13 y (five at disease stage A2 and two at stage C3) were treated with indinavir (four cases) at 1600-2000 mg/m2/d in three daily doses or with ritonavir (three patients) at 800-1000 mg/ m2/d in two daily doses in association with one or two reverse transcriptase inhibitors (zidovudine didanosine and zalcitabine in three cases each and zidovudine plus zalcitabine in the remaining patient). After 2-4 months stavudine plus lamivudine was introduced in all children instead of previously administered reverse transcriptase inhibitors since our patients have been receiving zidovudine and/or didanosine and/or zalcitabine for > 15 months. (excerpt)

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.

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.

The factors influencing virological response to first-line combined antiretroviral therapy (cART) in an Italian cohort of HIV-1-infected patients were examined. Eligible patients were those enrolled in a national prospective observational cohort (Antiretroviral Resistance Cohort Analysis), starting first-line cART between 2001 and 2011 and who had at least one follow-up of HIV-1 RNA. The primary endpoint was virological success, defined as the first viral load50 copies/ml. Time to events were analyzed by Kaplan-Meier analysis and Cox proportional hazard model. One thousand three hundred five patients met the study inclusion criteria. In a multivariable model adjusting for transmission mode, presence of transmitted drug resistance, baseline CD4(+) cell count, viral subtype, and type of NRTI backbone employed, independent predictors of virological success were higher baseline viral load (≥500,000 vs.100,000 HR 0.52; P 0.001), a weighted genotypic susceptibility score (wGSS)3 (HR 0.58; P = 0.003), male sex (HR 0.76 P = 0.001), and type of initial third drug employed (integrase inhibitor vs. boosted protease inhibitors HR 3.23; P 0.001). In the subset with HIV-1 RNA100,000 copies/ml, virologic success was only associated with the use of integrase inhibitors in the first cART regimen. Independent predictors of immunological success were baseline CD4(+) cell count and wGSS3. High baseline HIV-1 RNA, predicted activity of the first-line regimen based on genotypic resistance testing, gender, and use of new agents were found to predict time to achieve virological success. The type of initial nucleoside analog backbone was not found to predict virological response.

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.

HIV-infected patients have a significantly higher risk of developing cardiovascular events during the progression of HIV disease. Atherosclerosis, myocardial infarction, cerebrovascular injury, pulmonary hypertension and thrombosis are consistently described in both combined antiretroviral therapy (cART)-treated and naive HIV-positive patients as major clinical complications. Recent studies indicate that the pathogenesis of cardiovascular lesions in HIV-positive patients is related to direct and indirect effects of HIV infection on vessel structures, independently of traditional risk factors. HIV infection strongly interferes with the biology of several cellular targets such as macrophage and endothelial cells. Moreover, HIV induces a profound derangement of lipid metabolism and inflammatory cytokine networks that are directly involved in atherogenesis and progressive impairment of the cardiovascular system.In this review, we discuss these major HIV-related mechanisms able to promote atherosclerosis and cardiovascular diseases in HIV-positive patients.

Genotypic tests are the most common methods to identify patients eligible for CCR5 inhibitors administration in Europe. Among the available tools geno2pheno coreceptor (G2P) is the most used online system in routine diagnostics. This study was conceived to assess if the combination of G2P prediction with V3 peptide net charge (NC) value could improve the accuracy of tropism prediction. Sequences (129) were analyzed by G2P according to European Guidelines. NC values were calculated by the online software Peptide Property Calculator. Phenotypic assay was performed cloning the complete env gene into pcDNA 3.1 TOPO vector; infectivity of pseudotyped virions was tested on U87_CD4+CCR5+ and U87_CD4+CXCR4+ cells lines to assess viral tropism. Sequences were stratified into 3 groups according to the agreement between NC values and G2P results. Group 1: sequences assigned to the same group by both tools, group 2: sequences assigned to one group by G2P but indeterminate by NC and group 3: sequences for which G2P and NC gave discordant results. 61% of sequences predicted as X4 by G2P showed NC values higher than 5; similarly, 76% of sequences predicted as R5 by G2P had NC values below 4 (Group 1). Sequences with NC values between 4 and 5 (Group 2) were associated to different G2P predictions: 59% samples were predicted as R5-tropic and 41% sequences as X4-tropic. These data support the hypothesis that 4 to 5 NC values could be associated to the presence of dual/mixed-tropic variants (DM). Sequences identified as X4 by NC value had at least one positive residue in positions known to be involved in tropism prediction (58%) and positive residues in position 32 (39%). To further verify NC-based prediction, phenotypic assay was performed on a subset of sequences from each group. The assay confirmed the tropism prediction for group 1 sequences and demonstrated that the variants with net charge between 4 and 5 have DM tropism. Moreover, in vitro phenotyping of discordant viruses confirmed NC result, showing that this parameter is strongly associated with phenotypic assay. These results show that the combination of G2P and NC could increase the accuracy of tropism prediction and the ability to discriminate DM viruses. A more reliable identification of X4 variants would be useful for better selecting candidates for maraviroc administration, but also as a predictive marker in coreceptor switching, strongly associated to the phase of infection. (Published: 11 November 2012) Citation: Abstracts of the Eleventh International Congress on Drug Therapy in HIV Infection Montagna M et al. Journal of the International AIDS Society 2012, 15 (Suppl 4):18215 http://www.jiasociety.org/index.php/jias/article/view/18215 | http://dx.doi.org/10.7448/IAS.15.6.18215

Antiretroviral therapy has effectively tackled HIV replication and prevented the development of AIDS-related complications in the majority of HIV-positive patients. This pharmacological approach has dramatically increased the life expectancy of HIV-positive subjects transforming HIV infection into a chronic disease. Notwithstanding this major improvement in HIV disease management, several HIV-positive patients show an earlier and significant onset of aging related chronic conditions such as cardiovascular disease, osteoporosis, diabetes and neoplasias with respect to uninfected individuals. In particular, cardiovascular diseases are associated with both HIV infection and antiretroviral treatment, and represent major clinical complications in HIV-positive patients. Here, we discuss the interaction between antiretroviral therapy and cardiovascular system in HIV-positive patients focusing on the antiretroviral-related mechanisms involved in cardiovascular alterations.

Background Increased risk of fractures and osteoporosis have been associated with the use of antiretroviral drugs. There is a paucity of prospective evaluations of bone markers after the initiation of drugs currently recommended to treat HIV infection and results on the evolution of these markers are conflicting. Lastly, the effect of tenofovir on 1,25-(OH)2 vitamin D is uncertain. Methods We performed a prospective study on the evolution of bone markers, parathormone and 1,25-(OH)2 vitamin D before and after standard antiretroviral regimens. This was a sub-study of a trial conducted in antiretroviral-naïve patients randomized to tenofovir + emtricitabine in combination with either atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Follow-up lasted 48 weeks. The following bone markers were analyzed: C-terminal cross-laps (CTx), osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL). Mixed-factorial analysis of variance with random-coefficient general linear model was used to compare their trends over time and linear multivariable regression was performed with a backward selection method to assess predictors of their variations from baseline to week 48. Trends of parathormone and 1,25-(OH)2 vitamin D were also evaluated. Results Seventy-five patients were studied: 33 received EFV and 42 ATV/r. Significant increases were found for all markers except for RANKL. There was a significant direct association between CTx and OC increases. Multivariable analysis showed that higher glomerular filtration rate (estimated through cystatin C clearance) predicted greater OPG increase, while older age, higher HIV RNA at baseline and use of ATV/r predicted greater CTx increase. A significant increase of parathormone accompanied the evolution of the study markers. 1,25-(OH)2 vitamin D remained stable, though a seasonality variation was demonstrated. Conclusions These data demonstrate CTx increase (bone resorption marker) corresponding to OC increase (bone formation marker) early upon HAART initiation. Moreover, predictors of bone marker increases have been suggested, possibly indicating that a stricter monitoring of bone health and pro-active interventions are needed in older patients, those with higher HIV RNA, prescribed ATV/r rather than EFV, and with decreased renal function at baseline. Further studies are needed to clarify the mechanisms responsible for up-regulation of bone turnover markers, as well as to understand if and what markers are best correlated or predictive of pathological fractures.

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.

Summary 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-1 antigenaemias. Results indicate that the presence of CMV antigenaemia and the presence of HIV-1 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.

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.

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.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200–1,000 ng/ml), whereas lower Tat concentrations (1–100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients. J. Cell. Biochem. 113: 1132–1141, 2012. © 2011 Wiley Periodicals, Inc.

Understanding the determinants of virus transmission is a fundamental step for effective design of screening and intervention strategies to control viral epidemics. Phylogenetic analysis can be a valid approach for the identification of transmission chains, and very-large data sets can be analysed through parallel computation. Here we propose and validate a new methodology for the partition of large-scale phylogenies and the inference of transmission clusters. This approach, on the basis of a depth-first search algorithm, conjugates the evaluation of node reliability, tree topology and patristic distance analysis. The method has been applied to identify transmission clusters of a phylogeny of 11,541 human immunodeficiency virus-1 subtype B pol gene sequences from a large Italian cohort. Molecular transmission chains were characterized by means of different clinical/demographic factors, such as the interaction between male homosexuals and male heterosexuals. Our method takes an advantage of a flexible notion of transmission cluster and can become a general framework to analyse other epidemics. © 2011 Macmillan Publishers Limited. All rights reserved.

Background HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs). Results MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. Conclusions The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

Thiosemicarbazones display a wide antimicrobial activity by targeting bacteria, fungi, and viruses. Here, we report our studies on the antiviral activity of two thiosemicarbazone metal complexes, [bis(citronellalthiosemicarbazonato)nickel(II)] and [aqua(pyridoxalthiosemicarbazonato)copper(II)] chloride monohydrate, against the retroviruses HIV-1 and HTLV-1/-2. Both compounds exhibit antiviral properties against HIV but not against HTLVs . In particular, the copper complex shows the most potent anti-HIV activity by acting at the post-entry steps of the viral cycle.

Bone mass loss with the subsequent development of osteopenia and osteoporosis is related to HIV infection and antiretroviral treatment, even though the mechanisms involved have not yet been elucidated. In this report analyzes the early effects of some specific protease inhibitors on OPG/RANKL yielding and cell survival in osteoblast-like HOBIT cell line. None of the compounds, tested at scalar concentrations (C1, C2, C3), affected cell survival except for tipranavir that elicited a reliable induction of apoptosis at the highest concentration (C3). Atazanavir, saquinavir and indinavir did not affect OPG/RANKL in the cell surnatant in our experimental conditions. By contrast, at optimal concentration (C2), fosamprenavir induced a significant increase in OPG associated with a RANKL decrease whereas tipranavir down-regulated both OPG and RANKL (at C2 and C3) and darunavir increased RANKL only at C3 concentration. Together these data (coupled with the analysis of OPG/RANKL ratio) indicate that at early times and at optimal concentrations the PIs did not impair the OPG/RANKL system with the exception of fosamprenavir that showed a relative positive OPG/RANKL ratio regulation. Instead, cell cultures treated by the highest concentrations of tipranavir or darunavir showed a change in cell survival or an increase in RANKL, with a negative effect on the OPG/RANKL balance.

Summary Objectives To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. Methods Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. Results At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. Conclusions The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection.

The sequence and times of the transmission events was reported after a hospital accident by a phylodynamic reconstruction of transmission network among four subjects. The dated tree allowed to date the transmission events with good approximation, the time point and direction of each transmission, estimated on the basis of the phylogeny, and agreed with the presumptive time of infection on the basis of clinical history-taking.

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.

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.

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.

The most important purine nucleotides (NAD, AMP, IMP, GMP, XMP, ADP, ATP, GDP, GTP) were analyzed by HPLC in the lymphocytes of healthy subjects and HIV-1 seropositive patients at different stages of the disease (ARC-AIDS). Several differences, which focus attention on the behaviour of purine nucleotide metabolism in the lymphocytes of these patients, were observed.

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 (p0.01) 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.

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.

Dried blood spot (DBS) is a reliable method of blood collection used for the diagnosis of several human diseases. DBS is particularly useful for diagnosing children and for the screening of high-risk populations especially in countries where health facilities are not readily accessible. This report describes a qualitative SYBR Green-based real-time multiplex RT-PCR for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genomes in DBS. Specific viral amplicons were identified in the same sample by their distinctive melting temperatures. The analysis of scalar concentrations of the reference samples indicated that this multiplex procedure detects at least 2500 copies/ml of HCV and 400 copies/ml of HIV-1. HIV-1 and HCV viral loads in 20 patients infected with HIV-1 and/or HCV and in 5 healthy blood donors were also tested, confirming the sensitivity and specificity of the assay. This method may represent a reliable alternative for the detection of HIV-1/HCV co-infection, in rapid and relatively inexpensive screening programmes.

Current knowledge of HIV-primary resistance indicates that the prevalence of transmitted resistant strains has increased to substantial levels over the past few years, with a wide variation depending upon a number of factors. New infections with a virus strain already resistant to antiretroviral drugs, namely non-nucleoside reverse transcriptase inhibitor (NNRTI), have a negative impact on initial treatment response and also shorten the time to first virologic failure. The aim of this study was to determine the prevalence of antiretroviral drug resistance by a genotypic test in a population with newly diagnosed HIV-1 infection at a clinical centre in Bologna between June 2006 and September 2007.

HIV mainly replicates in CD4þ T lymphocytes andmonocyte/macrophages causing severe immunologicalimpairment. In addition to the immune system, HIVinfection affects tissues and organs such as kidney, liver,the central nervous system, heart and bone showing acomplex pathogenesis [1].The advent and widespread use of highly activeantiretroviral therapy (HAART) in the last two decadeshasled toa markedimprovementinthe treatmentofHIVdisease even though viral infection cannot be eradicatedbecause HAART does not completely eliminate the viralreservoirs [2]. HAART has dramatically changed thecourseofHIVinfection froma fatalinfectiontoachronicand relatively manageable disease. The increased lifeexpectancyofHIVpatientsandtheeffectsofHAARThavechanged the management of HIV infection. Nowadaysmedical treatment is no longer focused solely on HIVinfection, opportunistic diseases and monitoring immunederangement, but also includes the control of metabolic,cardiovascular, liver, bone and kidney complications. Inparticular, bone alterations have been observed in thecourse of HIV disease representing a pivotal clinicalprobleminthemanagementofHIVpatientsespeciallyforapossible development of bone fractures [3]. The majorbonelesionsdetectableinHIVpatientsarerelatedtobonedemineralization (osteopenia/osteoporosis and osteoma-lacia) and osteonecrosis ([4] for a review).This report will discuss the pathogenesis, diagnosis andtreatment of major bone complications represented bybone demineralization diseases during HIV infection andHAART treatment.

Background HIV infection of healthcare workers by injury is an important issue in the management and prophylaxis of HIV-related disease. Objectives To describe a case where a nurse has been HIV-1 infected by needle-stick whilst taking blood from a newborn with an unknown HIV infection. Study design Virological, immunological and clinical analysis of a peculiar case of HIV transmission from newborn to nurse has been reported. Results The nurse has been infected by needle-stick injury whilst taking blood from a newborn with an unknown HIV infection. The delayed declaration of accident by nurse and the inaccurate medical management of pregnant woman determined the subsequent absence of correct prophylaxis measures and then the impossibility to tackle the HIV transmission. Conclusion This case indicates that HIV serological screening of pregnant women and prompt accident notification by health-care workers represent basic preventive measures that should effectively tackle the spread of HIV infection.

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.

Objective The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population. Methods The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995–2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses. Results 18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification. Conclusion The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

Summary Objective To evaluate HIV-1 DNA load in PBLs and monocytes from both long-term HAART-treated and antiretroviral naive HIV-1 infected patients. Methods Cross-sectional quantitative analysis of HIV-1 DNA load was performed in PBLs and monocytes, purified from 34 long-term HAART-treated and 34 naive HIV-1 infected patients, and compared to RNA viral load and CD4+ cell count. Results HAART-treated patients showed significantly lower levels of viral DNA both in PBLs and monocytes in comparison with naive individuals. Variable levels of HIV-1 DNA amount in monocytes were detected in all naive patients but only in 12 of 34 HAART-treated individuals. PBLs HIV-1 DNA load was inversely correlated to CD4+ cell count in naive and HAART-treated patients whereas no association was detected in monocytes. Conclusions Long-term HAART decreased HIV-1 DNA load in PBLs and monocytes demonstrating a valuable inhibitor effect, especially in short-lived reservoirs. In addition, the positive correlation of DNA burden between PBLs and monocytes may suggest a dynamic relation between these reservoirs in the course of disease. HIV-1 DNA load quantitative analysis in PBLs and monocytes may be considered an important approach to study the HIV-1 reservoir and the effectiveness of HAART therapy in HIV-1 seropositive patients.

Osteopenia and osteoporosis are common in HIV-1-infected individuals and represent a challenge in clinical and therapeutic management. Since the mechanisms underlying this degenerative process are largely unsettled and it has not yet been determined whether bone dysfunction is linked to HIV-1-mediated direct and/or indirect effects on osteoblasts/osteoclasts cross-talk regulation, this brief review analyzes an array of mechanisms that could account for the dramatic bone derangement (bone loss and osteopenia/osteoporosis) during the course of HIV-1 infection.

Genotypic resistance analysis on viral DNA and plasma was performed in 56 therapy naive patients with recent and chronic infection to assess the prevalence of mutations associated with drug resistance and compare cell-free and cell-associated strains. Direct sequencing of DNA provirus disclosed key mutations to RT inhibitors more frequently than in plasma RNA. In addition, major mutations associated with drug resistance in the PR region were only found in PBMCs. Although our data are limited to a small cohort, they show a different resistance profile between plasma and PBMC compartments and may yield additional information for first-line antiretroviral regimens.

Osteopenia and osteoporosis are common in HIV-1-infected individuals and represent a challenge in clinical and therapeutic management. This report investigated osteopenia/osteoporosis in a group of 31 antiretroviral naive HIV-1-positive men and the role of specific molecules belonging to TNF and the TNF-receptor family in HIV-1-related bone mass loss. Osteoprotegerin (OPG), the receptor activator of NF-kappab-ligand (RANKL), and the TNF-related apoptosis-inducing ligand (TRAIL) were significantly increased in the plasma of antiretroviral naive HIV-1-positive patients compared to a control group of healthy blood donors. In addition, TRAIL and RANKL plasma concentrations were positively correlated to HIV-1-RNA viral load. Measurement of bone mineral density in 20 out of 31 HIV-1-positive subjects disclosed osteopenia/osteoporosis in 40% of these patients. The antiretroviral naive HIV-1-positive subjects with low bone mineral density had a decreased plasma OPG/RANKL ratio and a plasma RANKL concentration >500 pg/ml. Together, these data indicate that plasma concentrations of specific factors involved in bone homeostasis were increased during HIV-1 infection and that RANKL and OPG/RANKL ratio deregulation may be involved in osteopenia/osteoporosis occurring in antiretroviral naive HIV-1 individuals. (c) 2007 Wiley-Liss, Inc.