No correlation between statin exposure and incident diabetes mellitus in HIV-1-infected patients receiving combination antiretroviral therapy.

OBJECTIVES: Recent clinical studies and one meta-analysis have shown a modest but significant increase in the incidence of diabetes mellitus associated with statin exposure, so this correlation was investigated in a cohort of HIV-positive subjects. METHODS: A retrospective cohort study including adult HIV-1-infected patients followed at our Clinic of Infectious Diseases between 2007 and 2014 was performed. RESULTS: We assessed 3170 HIV-positive patients with a median follow-up of 5.2 years. The incidence of diabetes mellitus was 1.2 per 100 person-years and it was not significantly associated with the prescription of statins [hazard ratio (HR) 1.09 per year of statin exposure; 95% confidence interval (CI) 0.7-1.49; P = 0.067], while it was associated with older age, chronic hepatitis C, antiretroviral-naïve vs. antiretroviral experienced condition, high body mass index, and high serum concentration of triglycerides. CONCLUSIONS: In our study, a higher risk of diabetes mellitus was not associated with statin treatment, but with some traditional risk factors.

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.

Subgingival periodontal pathogens in Down syndrome children without periodontal breakdown. A case-control study on deciduous teeth.

AIM: Down syndrome is the most common form of aneuploidia compatible with a long survival. The affected subjects are more susceptible to severe early-onset periodontal disease and show a lower risk to develop dental caries than the non-affected population. This study investigated the prevalence of periodontal pathogens in the subgingival plaque of deciduous teeth in children with Down syndrome without signs of periodontal breakdown. METHODS: Thirty children suffering from Down syndrome and 46 matched healthy subjects were studied. A total of 228 subgingival plaque samples from deciduous teeth were separately collected and evaluated by polymerase chain reaction assays. CONCLUSION: In absence of periodontal impairment, Down syndrome children display a clear presence of periodontal pathogens already in the deciduous dentition. The hypothesis of an intrinsic predisposing condition is here supported.

Bloodstream infection caused by KPC-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam: epidemiology and genomic characterization.

OBJECTIVES: The aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. METHODS: We collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. RESULTS: During the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime-avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime-avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. CONCLUSIONS: Our results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

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

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

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.

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

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

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

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

Enhanced nuclear factor-kappa B activation induced by tumour necrosis factor-alpha in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein.

OBJECTIVE: An enhanced nuclear factor (NF)-kappa B activation in response to tumour necrosis factor (TNF)-alpha has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-kappa B-binding activity induced by TNF-alpha in Tat-producing cells was studied by anti-Tat antibody blocking experiments. DESIGN AND METHODS: Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-alpha-induced NF-kappa B-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations. RESULTS: The enhanced production of TNF-alpha-induced NF-kappa B binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-alpha-induced NF-kappa B binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies. CONCLUSIONS: This study demonstrates that the enhanced NF-kappa B activation exhibited by stably tat-transfected cells in response to TNF-alpha, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as a bifunctional molecule which not only acts from within facilitating NF-kappa B recruitment in the viral transcription complex, but may also act from without increasing the availability of activated NF-kappa B.

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

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

Impaired telomerase activity in uninfected haematopoietic progenitors in HIV-1-infected patients.

BACKGROUND: Haematopoietic progenitor cells (HPC) of HIV-1-infected patients are severely compromised in their replication and clonogenic capacities, and show an enhanced propensity to apoptosis, despite the lack of productive or latent HIV-1 infection. OBJECTIVE: To investigate telomerase enzyme levels in CD34+ HPC isolated from HIV-1-infected patients, because the absence of telomerase activity has been found to be correlated with a diminished replication potential. METHODS: Telomerase levels were measured by a PCR-based telomeric repeat amplification protocol. CD34+ HPC isolated from the peripheral blood of 11 HIV-1-infected patients were compared with CD34+ HPC isolated from peripheral blood (nine subjects) or bone marrow (six subjects) from 15 healthy donors. Telomerase levels were also studied in normal HPC after exposure to either gp120 or transforming growth factor (TGF)-beta1. RESULTS: CD34+ HPC isolated from either peripheral blood or bone marrow from healthy donors expressed a high level of telomerase activity. On the contrary, CD34+ HPC isolated from HIV-1-seropositive patients did not express any detectable telomerase activity in nine patients, and a clearly reduced enzymatic activity in two patients. Furthermore, telomerase activity in normal CD34+ HPC exposed to recombinant gp120 was significantly reduced, and to a higher extent than in CD34+ HPC exposed to recombinant TGF-beta1. CONCLUSIONS: This is the first study to demonstrate severely impaired telomerase activity in uninfected CD34+ HPC isolated from HIV-1-infected patients. The mechanism underlying this impairment probably involves the interaction of HIV-1 envelope glycoprotein gp120 with the cell membrane. These results may add to our understanding of the pathogenesis of the lesion of the HPC compartment.

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

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

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.

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

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

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

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

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

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

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.