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Sample records for plasma hiv rna

  1. Estimating the impact of plasma HIV-1 RNA reductions on heterosexual HIV-1 transmission risk.

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    Jairam R Lingappa

    Full Text Available BACKGROUND: The risk of sexual transmission of HIV-1 is strongly associated with the level of HIV-1 RNA in plasma making reduction in HIV-1 plasma levels an important target for HIV-1 prevention interventions. A quantitative understanding of the relationship of plasma HIV-1 RNA and HIV-1 transmission risk could help predict the impact of candidate HIV-1 prevention interventions that operate by reducing plasma HIV-1 levels, such as antiretroviral therapy (ART, therapeutic vaccines, and other non-ART interventions. METHODOLOGY/PRINCIPAL FINDINGS: We use prospective data collected from 2004 to 2008 in East and Southern African HIV-1 serodiscordant couples to model the relationship of plasma HIV-1 RNA levels and heterosexual transmission risk with confirmation of HIV-1 transmission events by HIV-1 sequencing. The model is based on follow-up of 3381 HIV-1 serodiscordant couples over 5017 person-years encompassing 108 genetically-linked HIV-1 transmission events. HIV-1 transmission risk was 2.27 per 100 person-years with a log-linear relationship to log(10 plasma HIV-1 RNA. The model predicts that a decrease in average plasma HIV-1 RNA of 0.74 log(10 copies/mL (95% CI 0.60 to 0.97 reduces heterosexual transmission risk by 50%, regardless of the average starting plasma HIV-1 level in the population and independent of other HIV-1-related population characteristics. In a simulated population with a similar plasma HIV-1 RNA distribution the model estimates that 90% of overall HIV-1 infections averted by a 0.74 copies/mL reduction in plasma HIV-1 RNA could be achieved by targeting this reduction to the 58% of the cohort with plasma HIV-1 levels ≥4 log(10 copies/mL. CONCLUSIONS/SIGNIFICANCE: This log-linear model of plasma HIV-1 levels and risk of sexual HIV-1 transmission may help estimate the impact on HIV-1 transmission and infections averted from candidate interventions that reduce plasma HIV-1 RNA levels.

  2. The relative prognostic value of plasma HIV RNA levels and CD4 lymphocyte counts in advanced HIV infection

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    Cozzi-Lepri, A; Katzenstein, T L; Ullum, H

    1998-01-01

    OBJECTIVE: It has been suggested that the plasma HIV RNA level is a better predictor of AIDS and death than the CD4 lymphocyte count. We assessed whether the prognostic value of plasma virus levels was different according to the CD4 count. DESIGN: Prospective cohort study of HIV-infected patients...

  3. Significant impact of non-B HIV-1 variants genetic diversity in Gabon on plasma HIV-1 RNA quantitation.

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    Mouinga-Ondémé, Augustin; Mabika-Mabika, Arsène; Alalade, Patrick; Mongo, Arnaud Delis; Sica, Jeanne; Liégeois, Florian; Rouet, François

    2014-01-01

    Evaluations of HIV-1 RNA viral load assays are lacking in Central Africa. The main objective of our study was to assess the reliability of HIV-1 RNA results obtained with three different assays for samples collected in Gabon. A total of 137 plasma specimens were assessed for HIV-1 RNA using the Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ® version 2.0 assays. It included HIV-1 non-B samples (n = 113) representing six subtypes, 10 CRFs and 18 URFs from patients infected with HIV-1 and treated with antiretrovirals that were found HIV-1 RNA positive (≥300 copies/ml) with the Generic HIV viral load® assay; and samples (n = 24) from untreated individuals infected with HIV-1 but showing undetectable (<300 copies/ml) results with the Biocentric kit. For samples found positive with the Generic HIV viral load® test, correlation coefficients obtained between the three techniques were relatively low (R = 0.65 between Generic HIV viral load® and Abbott RealTime HIV-1®, 0.50 between Generic HIV viral load® and Nuclisens HIV-1 EasyQ®, and 0.66 between Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ®). Discrepancies by at least one log10 were obtained for 19.6%, 33.7%, and 20% of samples, respectively, irrespective of genotype. Most of samples (22/24) from untreated study patients, found negative with the Biocentric kit, were also found negative with the two other techniques. In Central Africa, HIV-1 genetic diversity remains challenging for viral load testing. Further studies are required in the same area to confirm the presence of HIV-1 strains that are not amplified with at least two different viral load assays.

  4. Seminal Plasma HIV-1 RNA Concentration Is Strongly Associated with Altered Levels of Seminal Plasma Interferon-γ, Interleukin-17, and Interleukin-5

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    Hoffman, Jennifer C.; Anton, Peter A.; Baldwin, Gayle Cocita; Elliott, Julie; Anisman-Posner, Deborah; Tanner, Karen; Grogan, Tristan; Elashoff, David; Sugar, Catherine; Yang, Otto O.

    2014-01-01

    Abstract Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (pplasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission. PMID:25209674

  5. Physician experience and rates of plasma HIV-1 RNA suppression among illicit drug users: an observational study

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    Sangsari Sassan

    2012-01-01

    Full Text Available Abstract Background Despite the availability of antiretroviral therapy (ART, suboptimal treatment outcomes have been observed among HIV-seropositive illicit drug users. As there is an urgent need to improve responses to antiretroviral therapy among this population, we undertook this study to evaluate the role of physician experience on rates of plasma HIV-1 RNA suppression following initiation of ART. Methods Using data from a community-recruited cohort of HIV-positive illicit drug users, we used Cox proportional hazards regression to model the time to plasma viral HIV RNA Results Between May 1996 and December 2008, 267 individuals initiated ART among whom 227 (85% achieved a plasma HIV RNA Conclusions In this setting of universal HIV/AIDS care, illicit drug users with more experienced physicians exhibited faster rates of plasma viral load suppression. These findings argue for specialized services to help optimize HIV treatment outcomes among this population.

  6. Distinct genetic loci control plasma HIV-RNA and cellular HIV-DNA levels in HIV-1 infection: the ANRS Genome Wide Association 01 study.

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    Cyril Dalmasso

    Full Text Available Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662 were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes and chromosome 8 (rs2575735; within the Syndecan 2 gene. Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.

  7. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

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    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    of the Aptima HIV-1 Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 (CAPCTMv2) for the quantification of HIV-1 RNA in plasma samples. STUDY DESIGN: The performance of the two tests was compared on 216 clinical plasma samples, on dilutions series in seven replicates of five clinical...

  8. Nevirapine, sodium concentration and HIV-1 RNA in breast milk and plasma among HIV-infected women receiving short-course antiretroviral prophylaxis

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    Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy G.

    2015-01-01

    Introduction Risk factors for breast milk transmission of HIV-1 from mother to child include high plasma and breast milk viral load, low maternal CD4 count and breast pathology such as mastitis. Objective To determine the impact of nevirapine and subclinical mastitis on HIV-1 RNA in maternal plasma...... and breast milk after intrapartum single-dose nevirapine combined with either 1-week tail of Combivir (zidovudine/lamivudine) or single-dose Truvada (tenofovir/emtricitabine). Methods Maternal plasma and bilateral breast milk samples were collected between April 2008 and April 2011 at 1, 4 and 6 weeks...... postpartum from HIV-infected Tanzanian women. Moreover, plasma samples were collected at delivery from mother and infant. Results HIV-1 RNA was quantified in 1,212 breast milk samples from 273 women. At delivery, 96% of the women and 99% of the infants had detectable nevirapine in plasma with a median...

  9. Prognostic value of single measurements of beta-2-microglobulin, immunoglobulin A in HIV disease after controlling for CD4 lymphocyte counts and plasma HIV RNA levels

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    Ullum, H; Lepri, A Cozzi; Katzenstein, T L

    2000-01-01

    The interrelationships between the CD4 lymphocyte count, plasma viral load [human immunodeficiency virus (HIV) RNA], beta-2-microglobulin (beta2-M) and immunoglobulin A (IgA) and the mortality risk was explored in 234 HIV-infected individuals (median CD4 count 230 cells/mm3, range 1-1,247). Produ...

  10. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification.

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    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1). A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines (2). Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3)) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2

  11. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

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    Jean Ruelle

    2014-11-01

    Full Text Available Introduction: Low or undetectable plasma viral load (VL using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1. Materials and Methods: A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA in an accredited environment (ISO15189:2012 norm. Parameters tested were in line with the digital MIQE guidelines (2. Inter-run coefficient of variation (CV was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3 using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5’FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168 were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999 and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5. Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were

  12. Plasma HIV-2 RNA According to CD4 Count Strata among HIV-2-Infected Adults in the IeDEA West Africa Collaboration.

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    Didier K Ekouévi

    Full Text Available Plasma HIV-1 RNA monitoring is one of the standard tests for the management of HIV-1 infection. While HIV-1 RNA can be quantified using several commercial tests, no test has been commercialized for HIV-2 RNA quantification. We studied the relationship between plasma HIV-2 viral load (VL and CD4 count in West African patients who were either receiving antiretroviral therapy (ART or treatment-naïve.A cross sectional survey was conducted among HIV-2-infected individuals followed in three countries in West Africa from March to December 2012. All HIV-2 infected-patients who attended one of the participating clinics were proposed a plasma HIV-2 viral load measurement. HIV-2 RNA was quantified using the new ultrasensitive in-house real-time PCR assay with a detection threshold of 10 copies/ mL (cps/mL.A total of 351 HIV-2-infected individuals participated in this study, of whom 131 (37.3% were treatment naïve and 220 (62.7% had initiated ART. Among treatment-naïve patients, 60 (46.5% had undetectable plasma HIV-2 viral load (1000 cps/mL in 6.0% of the patients. Most of the treatment-naïve patients (70.2% had CD4-T cell count ≥500 cells/mm3 and 43 (46.7% of these patients had a detectable VL (≥10 cps/mL. Among the 220 patients receiving ART, the median CD4-T cell count rose from 231 to 393 cells/mm3 (IQR [259-561] after a median follow-up duration of 38 months and 145 (66.0% patients had CD4-T cell count ≤ 500 cells/mm3 with a median viral load of 10 cps/mL (IQR [10-33]. Seventy five (34.0% patients had CD4-T cell count ≥ 500 cells/mm3, among them 14 (18.7% had a VL between 10-100 cps/mL and 2 (2.6% had VL >100 cps/mL.This study suggests that the combination of CD4-T cell count and ultrasensitive HIV-2 viral load quantification with a threshold of 10 cps/mL, could improve ART initiation among treatment naïve HIV-2-infected patients and the monitoring of ART response among patients receiving treatment.

  13. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in Plasma Preparation Tubes (PPT).

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    Griffith, Brigitte P; Mayo, Donald R

    2006-02-01

    The accurate and reliable quantification of HIV RNA is an essential part of the management of HIV infected individuals, and elucidation of factors that may affect HIV RNA measurements, such as the use of Vacutainer Plasma Preparation Tubes (PPT), is crucial. The objective of this study was to determine if plasma samples with viral loads close to the lower limit of the dynamic range of the assay collected in PPT tubes had increased levels of HIV RNA as compared to samples collected in standard EDTA tubes. HIV RNA levels were compared in 112 paired plasma samples collected in PPT and standard EDTA tubes. All samples had been frozen prior to testing. Discrepancies between PPT and EDTA tubes did not occur for samples with high viral loads. However, in samples with viral loads close to the lower limit of the dynamic range, levels of HIV RNA detected were higher in a large proportion of PPT as compared to the corresponding EDTA plasma samples. Forty percent of plasma pairs had no detectable HIV RNA in the EDTA aliquot, but had low levels of HIV RNA in the corresponding PPT aliquot. This prospective study underlines the need for cautious interpretation of small transient viral load changes in samples with values close to the detection limit.

  14. Schistosomiasis and HIV-1 infection in rural Zimbabwe: effect of treatment of schistosomiasis on CD4 cell count and plasma HIV-1 RNA load

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    Kallestrup, Per; Zinyama, Rutendo; Gomo, Exnevia;

    2005-01-01

    To determine whether treatment of schistosomiasis has an effect on the course of human immunodeficiency virus type 1 (HIV-1) infection, individuals with schistosomiasis and with or without HIV-1 infection were randomized to receive praziquantel treatment at inclusion or after a delay of 3 months......; 287 participants were included in the study, and 227 (79%) were followed up. Among the 130 participants who were coinfected, those who received early treatment (n=64) had a significantly lower increase in plasma HIV-1 RNA load than did those who received delayed treatment (n=66) (P...

  15. Analysis of correlation between cerebrospinal fluid and plasma HIV-1 RNA levels in patients with neurological opportunistic diseases

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    Paulo Pereira Christo

    2011-08-01

    Full Text Available The question of whether HIV-1 RNA in cerebrospinal fluid (CSF is derived from viral replication in the central nervous system or simply reflects the transit of infected lymphocytes from the blood compartment has long been a matter of debate. Some studies found no correlation between CSF and plasma viral load, whereas others did. The lack of a correlation between the two compartments suggests that the presence of HIV-1 RNA is not simply due to the passive passage of the virus from blood to CSF but rather due to intrathecal replication. To evaluate the correlation between plasma and CSF HIV-1 RNA levels and to identify situations in which there is no correlation between the two compartments, seventy patients were prospectively studied. The association between CSF and plasma viral load was evaluated in the total population and in subgroups of patients with similar characteristics. A correlation between the CSF and plasma compartments was observed for patients undergoing highly active antiretroviral therapy (HAART, those with a CD4 T lymphocyte count lower than 200 cells/mm³, and those with increased CSF protein content. On the other hand, no correlation was observed for patients without adequate virological control, who had a CD4 count higher than 200 cells/mm³ and who did not use HAART. The correlation between the two compartments observed in some patients suggests that CSF HIV-1 RNA levels may reflect plasma levels in these subjects. In contrast, the lack of a correlation between the two compartments in patients who were not on HAART and who had normal CSF proteins and a poor virological control possibly indicates compartmentalization of the virus in CSF and, consequently, plasma-independent intrathecal viral replication.

  16. Factors influencing cerebrospinal fluid and plasma HIV-1 RNA detection rate in patients with and without opportunistic neurological disease during the HAART era

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    Aleixo Agdemir W

    2007-12-01

    Full Text Available Abstract Background In the central nervous system, HIV replication can occur relatively independent of systemic infection, and intrathecal replication of HIV-1 has been observed in patients with HIV-related and opportunistic neurological diseases. The clinical usefulness of HIV-1 RNA detection in the cerebrospinal fluid (CSF of patients with opportunistic neurological diseases, or the effect of opportunistic diseases on CSF HIV levels in patients under HAART has not been well defined. We quantified CSF and plasma viral load in HIV-infected patients with and without different active opportunistic neurological diseases, determined the characteristics that led to a higher detection rate of HIV RNA in CSF, and compared these two compartments. Methods A prospective study was conducted on 90 HIV-infected patients submitted to lumbar puncture as part of a work-up for suspected neurological disease. Seventy-one patients had active neurological diseases while the remaining 19 did not. Results HIV-1 RNA was quantified in 90 CSF and 70 plasma samples. The HIV-1 RNA detection rate in CSF was higher in patients with neurological diseases, in those with a CD4 count lower than 200 cells/mm3, and in those not receiving antiretroviral therapy, as well as in patients with detectable plasma HIV-1 RNA. Median viral load was lower in CSF than in plasma in the total population, in patients without neurological diseases, and in patients with toxoplasmic encephalitis, while no significant difference between the two compartments was observed for patients with cryptococcal meningitis and HIV-associated dementia. CSF viral load was lower in patients with cryptococcal meningitis and neurotoxoplasmosis under HAART than in those not receiving HAART. Conclusion Detection of HIV-1 RNA in CSF was more frequent in patients with neurological disease, a CD4 count lower than 200 cells/mm3 and detectable plasma HIV-1. Median HIV-1 RNA levels were generally lower in CSF than in

  17. Epstein-Barr and cytomegalovirus DNA salivary shedding correlate with long-term plasma HIV RNA detection in HIV-infected men who have sex with men.

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    Scaggiante, Renzo; Andreis, Samantha; Basso, Monica; Franchin, Elisa; Franzetti, Marzia; Del Vecchio, Claudia; Torti, Carlo; Mengoli, Carlo; Cruciani, Mario; Sarmati, Loredana; Palù, Giorgio; Parisi, Saverio Giuseppe

    2016-07-01

    The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA salivary shedding in HIV-positive men who have sex with men (MSM) and to determine whether viro-immunological parameters and long-term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV-positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm(3) and mean nadir value 333 cells/mm(3) ); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses-related cancers.

  18. Differences in HIV type 1 RNA plasma load profile of closely related cocirculating Ethiopian subtype C strains: C and C'.

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    Ayele, Workenesh; Mekonnen, Yared; Messele, Tsehaynesh; Mengistu, Yohannes; Tsegaye, Aster; Bakker, Margreet; Berkhout, Ben; Dorigo-Zetsma, Wendelien; Wolday, Dawit; Goudsmit, Jaap; Coutinho, Roel; de Baar, Michel; Paxton, William A; Pollakis, Georgios

    2010-07-01

    Two HIV-1 subtype C subclusters have been identified in Ethiopia (C and C') with little knowledge regarding their biological or clinical differences. We longitudinally monitored HIV-1 viral loads and CD4(+) T cell counts for 130 subtype C-infected individuals from Ethiopia over 5 years. The genetic subclusters C and C' were determined and comparisons were made between the groups. None of the study individuals received antiretroviral therapy. Subcluster C' was found to be the more prevalent (72.3%) genotype circulating. Individuals infected with subcluster C' harbored higher viral loads in comparison to subcluster C-infected individuals when the CD4(+) T cell counts were high (500-900 cells/mm(3)), whereas at low CD4(+) T cell counts (0-150 cells/mm(3)) individuals infected with subcluster C viruses showed higher viral loads. We identified a greater number of deaths among individuals infected with subcluster C viruses in comparison to C'. Our results indicate that infection with subcluster C viruses leads to a more rapid onset of disease, despite the initial lower HIV-1 RNA plasma loads. Additionally, the higher viral loads seen for HIV-1 subcluster C' infections at higher CD4(+) T cell counts can help explain the higher prevalence of this subtype in Ethiopia.

  19. Novel Sensitive Real-Time PCR for Quantification of Bacterial 16S rRNA Genes in Plasma of HIV-Infected Patients as a Marker for Microbial Translocation▿

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    Kramski, M.; Gaeguta, A. J.; Lichtfuss, G. F.; Rajasuriar, R.; Crowe, S M; French, M A; Lewin, S.R.; Center, R. J.; Purcell, D.F.J.

    2011-01-01

    We developed a real-time PCR to quantify 16S rRNA gene levels in plasma from HIV-infected patients as a marker of microbial translocation. The assay uses shrimp nuclease (SNuc) to eliminate DNA contamination, giving high sensitivity and low variability. The 16S rRNA gene levels measured in plasma from HIV patients correlated significantly with lipopolysaccharide levels.

  20. Predicting the duration of antiviral treatment needed to suppress plasma HIV-1 RNA

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    Rizzardi, G.P. (Paolo); Halkic, Nermin; Boer, R.J. de; Hoover, Shelley; Tambussi, Giuseppe; Chapuis, Aude; Bart, P.A.; Miller, Veronica; Staszewski, Schlomo; Notermans, D.W. (Daan)

    2000-01-01

    Effective therapeutic interventions and clinical care of adults infected with HIV-1 require an understanding of factors that influence time of response to antiretroviral therapy. We have studied a cohort of 118 HIV-1-infected subjects naive to antiretroviral therapy and have correlated the time of r

  1. HIV RNA and proviral HIV DNA can be detected in semen after 6 months of antiretroviral therapy although HIV RNA is undetectable in blood.

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    Du, Peiwei; Liu, An; Jiao, Yanmei; Liu, Cuie; Jiang, Taiyi; Zhu, Weijun; Zhu, Yunxia; Wu, Hao; Sun, Lijun

    2016-03-01

    The risk of sexual transmission of HIV is strongly correlated with amounts of genital HIV RNA. Few studies have reported amounts of HIV RNA and HIV DNA in semen in HIV-infected Chinese patients undergoing antiviral treatment (ART). In this observational study, the amounts of HIV RNA and HIV DNA in semen were assessed after six months of ART in HIV-infected Chinese individuals, when HIV RNA was undetectable in blood . This study included 19 HIV-infected Chinese men undergoing ART for six months. Amounts of HIV in paired semen and blood samples were assessed using real-time PCR. The C2-V5 region of the HIV envelope (env) genes was cloned and sequenced and genotype and co-receptor usage predicted based on the sequence. It was found that HIV RNA was undetectable in the plasma of most patients (17/19), whereas HIV RNA could be detected in the semen of most patients (16/19). HIV DNA could be detected in both semen and blood. Genetic diversity of HIV between the seminal and blood compartments was identified. Thus, amounts of HIV RNA and HIV DNA remain high in semen of HIV-infected Chinese patients after six months of ART treatment, even when HIV RNA was undetectable in blood.

  2. HIV-1 Viral RNA Dynamics at the Plasma Membrane May Provide Insight into Viral Assembly | Poster

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    Many aspects of how infectious viruses assemble in cells have yet to be completely deciphered. However, as reported in a recent Journal of Virology paper, researchers may be one step closer to understanding how HIV-1, the virus that causes AIDS, assembles and replicates.

  3. The HIV RNA setpoint theory revisited

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    Hubert Jean-Baptiste

    2007-09-01

    Full Text Available Abstract Background The evolution of plasma viral load after HIV infection has been described as reaching a setpoint, only to start rising again shortly before AIDS diagnosis. In contrast, CD4 T-cell count is considered to show a stable decrease. However, characteristics of marker evolution over time depend on the scale that is used to visualize trends. In reconsidering the setpoint theory for HIV RNA, we analyzed the evolution of CD4 T-cell count and HIV-1 RNA level from HIV seroconversion to AIDS diagnosis. Follow-up data were used from two cohort studies among homosexual men (N = 400, restricting to the period before highly active antiretroviral therapy became widely available (1984 until 1996. Individual trajectories of both markers were fitted and averaged, both from seroconversion onwards and in the four years preceding AIDS diagnosis, using a bivariate random effects model. Both markers were evaluated on a scale that is directly related to AIDS risk. Results Individuals with faster AIDS progression had higher HIV RNA level six months after seroconversion. For CD4 T-cell count, this ordering was less clearly present. However, HIV RNA level and CD4 T-cell count showed qualitatively similar evolution over time after seroconversion, also when stratified by rate of progression to AIDS. In the four years preceding AIDS diagnosis, a non-significant change in HIV RNA increase was seen, whereas a significant biphasic pattern was present for CD4 T-cell decline. Conclusion HIV RNA level has more setpoint behaviour than CD4 T-cell count as far as the level shortly after seroconversion is concerned. However, with respect to the, clinically more relevant, marker evolution over time after seroconversion, a setpoint theory holds as much for CD4 T-cell count as for HIV RNA level.

  4. Cellular Levels of HIV Unspliced RNA from Patients on Combination Antiretroviral Therapy with Undetectable Plasma Viremia Predict the Therapy Outcome

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    Pasternak, A.O.; Jurriaans, S.; Bakker, M.; Prins, J.M.; Berkhout, B.; Lukashov, V.V.

    2009-01-01

    Background: Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laborator

  5. Impact of HIV-1 genetic diversity on plasma HIV-1 RNA Quantification: usefulness of the Agence Nationale de Recherches sur le SIDA second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test.

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    Rouet, François; Chaix, Marie-Laure; Nerrienet, Eric; Ngo-Giang-Huong, Nicole; Plantier, Jean-Christophe; Burgard, Marianne; Peeters, Martine; Damond, Florence; Ekouevi, Didier Koumavi; Msellati, Philippe; Ferradini, Laurent; Rukobo, Sandra; Maréchal, Valérie; Schvachsa, Nilda; Wakrim, Lahcen; Rafalimanana, Christian; Rakotoambinina, Benjamin; Viard, Jean-Paul; Seigneurin, Jean-Marie; Rouzioux, Christine

    2007-08-01

    The high genetic diversity of HIV-1 has a major impact on the quantification of plasma HIV-1 RNA, representing an increasingly difficult challenge. A total of 898 plasma specimens positive for HIV-1 RNA by commercial assays (Amplicor v1.5; Roche Diagnostic Systems, Alameda, CA or Versant v3.0; Bayer Diagnostics, Emeryville, CA) were tested using the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test: 518 samples containing HIV-1 of known subtype, including 88 from 2 subtype panels and 430 harboring B (n = 266) and non-B (n = 164) group M HIV-1 subtypes from patients followed up in 2002 through 2005 at Necker Hospital (Paris, France), and 380 samples from 10 different countries (Argentina, Cambodia, Cameroon, Central African Republic, France, Ivory Coast, Madagascar, Morocco, Thailand, and Zimbabwe). HIV-1 RNA values obtained by G2 real-time PCR were highly correlated with those obtained by the Amplicor v1.5 for B and non-B subtypes (R = 0.892 and 0.892, respectively) and for samples from diverse countries (R = 0.867 and 0.893 for real-time PCR vs. Amplicor v1.5 and real-time PCR vs. Versant v3.0, respectively). Approximately 30% of specimens harboring non-B subtypes were underquantified by at least -0.51 log10 in Amplicor v1.5 versus 5% underquantified in G2 real-time PCR. Discrepant results were also obtained with subtype B samples (14% underquantified by Amplicor v1.5 vs. 7% by G2 real-time PCR). Similar percentages were observed when comparing results obtained with the G2 real-time PCR assay with those obtained using the Versant assay. Addressing HIV-1 diversity, continual monitoring of HIV-1 RNA assays, together with molecular epidemiology studies, is required to improve the accuracy of all HIV RNA assays.

  6. The effect of tenidap on cytokines, acute-phase proteins, and virus load in human immunodeficiency virus (HIV)-infected patients: correlation between plasma HIV-1 RNA and proinflammatory cytokine levels.

    Science.gov (United States)

    Dezube, B J; Lederman, M M; Chapman, B; Georges, D L; Dogon, A L; Mudido, P; Reis-Lishing, J; Cheng, S L; Silberman, S L; Crumpacker, C S

    1997-09-01

    Proinflammatory cytokines may be important in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) disease. Tenidap decreases interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF) production by peripheral blood mononuclear cells and decreases IL-6 plasma levels in rheumatoid arthritis patients. In this randomized double-blind study, 43 HIV-1-infected patients received tenidap (120 mg) or placebo daily for 6 weeks and then crossed over to the alternative therapy for an additional 6 weeks. Mean entry CD4 cell count was 140/microL. Analyses were performed on cytokines, acute-phase proteins, virus load, and CD4 cell counts. With the exception of small differences in plasma TNF levels, tenidap had no significant effect on these indices. Significant correlations of plasma IL-6 and TNF levels with HIV-1 RNA were noted. Six patients discontinued tenidap due to rash. The effects of tenidap in HIV-1 infection contrast to results in arthritis patients, in whom tenidap decreased plasma levels of IL-6 and acute-phase proteins.

  7. Comparison of the artus HIV-1 QS-RGQ and VERSANT HIV-1 RNA 1.0 assays for quantitative detection of human immunodeficiency virus type 1 in plasma samples.

    Science.gov (United States)

    Dvir, Roee; Canducci, Filippo; Racca, Sara; Rolla, Serena; Stucchi, Sonia; Clementi, Massimo

    2015-07-01

    Several integrated diagnostic platforms to quantify human immunodeficiency virus type-1 viremia have been developed in recent years. We evaluated the performances of the Artus HIV-1 QS-RGQ assay, using the complete QIAsymphony RGQ workflow. 192 clinical plasma specimens and external control panel samples were analyzed, using the Artus assay and the routine Siemens VERSANT HIV-1 RNA 1.0 assay. Three samples were excluded due to amplification inhibition. Among the remaining 189 specimens, 130 samples were detected as positive (above the limit of detection by both assays; median log10 difference: 0.01) and 18 samples were detected as negative. Eight samples (4.2%), all slightly above the limit of detection of the Versant assay, were negative with the Artus assay. The remaining 33 samples (beside 3 negative by Artus assay) were positive by both assays, but below the limit of detection at least in one of them. Results from the external panel samples showed a mean Log10 variation of -0.18 and -0.45 for the Versant and the Artus assays, respectively. As both assays showed highly correlated results, the QIAsymphony RGQ system, using the Artus HIV-1 QS-RGQ assay, could be considered a potential platform for HIV-1 RNA quantification in plasma.

  8. Development of an RNA Assay to Assess HIV I Latency

    Science.gov (United States)

    1993-01-10

    current study is limited to examining cellular RNAs rather than free genomic RNA in the plasma. Ottmann and colleagues demonstrated HIV-1 genomic RNA in 95...aberrant pattern of viral RNA expression: a molecular model for latency. Cell 1990; 61:1271-1276. 25 Ottmann M, Innocenti P, Tenadey M, Micoud M

  9. Difference of progression to AIDS according to CD4 cell count, plasma HIV RNA level and the use of antiretroviral therapy among HIV patients infected through blood products in japan.

    Science.gov (United States)

    Kawado, Miyuki; Hashimoto, Shuji; Yamaguchi, Takuhiro; Oka, Shin-ichi; Yoshizaki, Kazuyuki; Kimura, Satoshi; Fukutake, Katsuyuki; Higasa, Satoshi; Shirasaka, Takuma

    2006-05-01

    It is important to examine progression to acquired immunodeficiency syndrome (AIDS) or death and its predictors among human immunodeficiency virus (HIV) infected persons before and after the introduction of the highly active antiretroviral therapy (HAART) available in Japan since 1997. The data used were from a survey of persons with HIV infected through blood coagulation factor products in Japan. Progression to AIDS or death during two periods, between January 1994 and March 1997, and between April 1997 and March 2002, were observed. The AIDS-free proportion after 3 years was 74% among 417 participants for the earlier period and 94% among 605 participants in the later one. The hazard ratio of low CD4 cell count (less than 200 cells/microL) was 50.8 for the earlier period and 4.7 for the later one compared with that of 500 cells/microL or more. After adjustment by plasma HIV RNA levels and use of antiretroviral therapy, the hazard ratios of the low CD4 cell count for the later period were still significant. The AIDS-free proportion among people with HIV infected through blood products in Japan largely increased after the introduction of HAART. The CD4 cell count remains an important predictor of future progression, but its importance might be less because of HAART.

  10. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy.

    Science.gov (United States)

    Imamichi, Hiromi; Dewar, Robin L; Adelsberger, Joseph W; Rehm, Catherine A; O'Doherty, Una; Paxinos, Ellen E; Fauci, Anthony S; Lane, H Clifford

    2016-08-02

    Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

  11. HIV migration between blood plasma and cellular subsets before and after HIV therapy.

    Science.gov (United States)

    Choi, Jun Yong; Chaillon, Antoine; Oh, Jin Ok; Ahn, Jin Young; Ann, Hae Won; Jung, In Young; Ahn, Mi-Young; Jeon, Yong Duk; Ku, Nam Su; Smith, Davey M; Kim, June Myung

    2016-04-01

    The cellular source of HIV RNA circulating in blood plasma remains unclear. Here, we investigated whether sequence analysis of HIV RNA populations circulating before combination antiretroviral therapy (cART) and HIV DNA populations in cellular subsets (CS) after cART could identify the cellular sources of circulating HIV RNA. Blood was collected from five subjects at cART initiation and again 6 months later. Naïve CD4+ T cells, resting central memory and effector memory CD4+ T cells, activated CD4+ T cells, monocytes, and natural killer cells were sorted using a fluorescence-activated cell sorter. HIV-1 env C2V3 sequences from HIV RNA in blood plasma and HIV DNA in CSs were generated using single genome sequencing. Sequences were evaluated for viral compartmentalization (Fst test) and migration events (MEs; Slatkin Maddison and cladistic measures) between blood plasma and each CS. Viral compartmentalization was observed in 88% of all cellular subset comparisons (range: 77-100% for each subject). Most observed MEs were directed from blood plasma to CSs (52 MEs, 85.2%). In particular, there was only viral movement from plasma to NK cells (15 MEs), monocytes (seven MEs), and naïve cells (five ME). We observed a total of nine MEs from activated CD4 cells (2/9 MEs), central memory T cells (3/9 MEs), and effector memory T cells (4/9 MEs) to blood plasma. Our results revealed that the HIV RNA population in blood plasma plays an important role in seeding various cellular reservoirs and that the cellular source of the HIV RNA population is activated central memory and effector memory T cells.

  12. Switch to raltegravir-based regimens and HIV DNA decrease in patients with suppressed HIV RNA

    Directory of Open Access Journals (Sweden)

    Claudia Bianco

    2014-11-01

    Full Text Available Introduction: Raltegravir intensification is associated with an increase in 2-LTR episomal HIV DNA= circles, indicating a persistent low-level replication, in some individuals in ART with suppressed HIV RNA. We aimed at monitoring residual plasma HIV RNA and cellular HIV DNA in virologically suppressed patients switching to a raltegravir-based regimen. Materials and Methods: Forty-six HIV-infected subjects on PI or NNRTI based-regimens, with plasma HIV RNA level 200 cells/µL for ≥12 months were enrolled. Thirty-four patients switched to raltegravir-based regimen (RASTA study group and 12 continued a PI or NNRTI based-regimen (control group. Ultrasensitive HIV residual viremia and total PBMC HIV DNA were assessed at baseline (W0, 24 (W24 and 48 (W48 weeks. HIV RNA levels were determined by an ultrasensitive test derived from a commercial real time PCR (limit of detection 5 copies/ml. A real time PCR was used to quantify HIV DNA copy numbers in PBMCs. Results: At W0, HIV DNA was detected in all patients while at W48 it was detectable in 82.3% of RASTA group vs 100% of controls (p=0.01. The difference between the average values of HIV DNA log10 copies/10°6 CD4 at W0 (median 3.11, IQR 2.70–3.45 and W48 (median 2.87, IQR 2.24–3.38 was statistically significant for RASTA group (p=0.035. Male gender (mean difference −0.37 log10 copies/10°6 PBMC, p=0.023 and previous PI based-ART (mean difference +0.39 log10 copies/10°6 PBMC, p=0.036 were predictive of HIV DNA level at W0. After adjusting for previous PI based-ART, male gender was the only variable independently associated with HIV DNA size at W0 (mean difference −0.326 log10 copies/10°6 PBMC, 95% CI −0.641, −0.011 p=0.043. Ultrasensitive HIV-1 RNA was detectable at W0 in 50% of RASTA group versus 66.7% of controls and at W48 in 32.4% versus 45.5%, respectively. No differences were found between HIV RNA levels at W0 and W48 within and between the two groups. Conclusions: Switching to

  13. Overcoming HIV-1 resistance to RNA interference.

    Science.gov (United States)

    Boden, Daniel; Pusch, Oliver; Ramratnam, Bharat

    2007-05-01

    RNAi refers to the sequence-specific degradation of RNA that follows the cellular introduction of homologous short interfering (si) RNA. RNAi has emerged as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Advances in vector design permit the effective expression of siRNA in human cells. Numerous recent investigations have described the ability of RNAi to decrease the replication of human immunodeficiency virus type 1 (HIV-1) in lymphocytic cells using siRNA targeting viral (e.g. tat, gag, rev) and host (e.g. CCR5, CD4) proteins. Can RNAi be used as a form of genetic therapy for HIV-1 infection? Recent data indicate that the dynamic replication kinetics of HIV-1 pose a considerable barrier to achieving durable virus suppression by RNAi with the rapid emergence of HIV-1 mutants resistant to siRNA. This review summarizes recent work on HIV-1 specific RNAi with a focus on potential strategies to overcome HIV-1 resistance to RNAi.

  14. Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

    Directory of Open Access Journals (Sweden)

    Iris Ricardo Rossin

    2011-12-01

    Full Text Available INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1 in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

  15. Incidence and correlates of HIV-1 RNA detection in the breast milk of women receiving HAART for the prevention of HIV-1 transmission.

    Directory of Open Access Journals (Sweden)

    Jennifer A Slyker

    Full Text Available BACKGROUND: The incidence and correlates of breast milk HIV-1 RNA detection were determined in intensively sampled women receiving highly active antiretroviral therapy (HAART for the prevention of mother-to-child HIV-1 transmission. METHODS: Women initiated HAART at 34 weeks of pregnancy. Breast milk was collected every 2-5 days during 1 month postpartum for measurements of cell-associated HIV DNA and cell-free HIV RNA. Plasma and breast milk were also collected at 2 weeks, 1, 3 and 6 months for concurrent HIV-1 RNA and DNA measurements. Regression was used to identify cofactors for breast milk HIV-1 RNA detection. RESULTS: Of 259 breast milk specimens from 25 women receiving HAART, 34 had detectable HIV-1 RNA (13%, incidence 1.4 episodes/100 person-days 95% CI = 0.97-1.9. Fourteen of 25 (56% women had detectable breast milk HIV-1 RNA [mean 2.5 log(10 copies/ml (range 2.0-3.9] at least once. HIV-1 DNA was consistently detected in breast milk cells despite HAART, and increased slowly over time, at a rate of approximately 1 copy/10(6 cells per day (p = 0.02. Baseline CD4, plasma viral load, HAART duration, and frequency of breast problems were similar in women with and without detectable breast milk HIV-1 RNA. Women with detectable breast milk HIV-1 RNA were more likely to be primiparous than women without (36% vs 0%, p = 0.05. Plasma HIV-1 RNA detection (OR = 9.0, 95%CI = 1.8-44 and plasma HIV-1 RNA levels (OR = 12, 95% CI = 2.5-56 were strongly associated with concurrent detection of breast milk HIV-1 RNA. However, no association was found between breast milk HIV-1 DNA level and concurrent breast milk HIV-1 RNA detection (OR = 0.96, 95%CI = 0.54-1.7. CONCLUSIONS: The majority of women on HAART had episodic detection of breast milk HIV-1 RNA. Breast milk HIV-1 RNA detection was associated with systemic viral burden rather than breast milk HIV-1 DNA.

  16. Plasma virus load evaluation in relation to disease progression in HIV-infected children.

    Science.gov (United States)

    Tetali, S; Bakshi, S; Than, S; Pahwa, S; Abrams, E; Romano, J; Pahwa, S G

    1998-05-01

    The objective of this study was to investigate the relationship of plasma HIV RNA load with survival and disease progression in HIV-infected children and to determine its correlation with cellular HIV DNA. Virus load (VL, HIV RNA copies/ml) was determined retrospectively by nucleic acid sequence-based amplification (NASBA) assay in 144 stored plasma samples between birth and 48 months in 50 children of whom 40 are alive (age range, 2-13 years). On the basis of clinical and immunologic status children were classified as rapid progressors (RPs), or nonrapid progressors (NRPs). Proviral HIV DNA quantitated by QC-PCR (quantitative competitive polymerase chain reaction) in 24 children was compared with plasma HIV RNA. At age 2 years (p or =750,000 copies/ml. Increasing mortality was observed with increasing plasma HIV RNA levels at ages 3-24 months and baseline VL of infants who died before age 24 months was significantly higher (p = 0.004) than baseline VL of those who survived beyond 24 months. Although baseline VL in infants classified as RPs was higher than that of NRPs, the difference was not statistically significant. Among surviving children 2-13 years of age, the baseline VL obtained at 80%. We conclude that high plasma HIV RNA in infancy is associated with increased mortality.

  17. MicroRNA-155 Reinforces HIV Latency*

    Science.gov (United States)

    Ruelas, Debbie S.; Chan, Jonathan K.; Oh, Eugene; Heidersbach, Amy J.; Hebbeler, Andrew M.; Chavez, Leonard; Verdin, Eric; Rape, Michael; Greene, Warner C.

    2015-01-01

    The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation. PMID:25873391

  18. The HIV RNA setpoint theory revisited

    NARCIS (Netherlands)

    Geskus, Ronald B.; Prins, Maria; Hubert, Jean-Baptiste; Miedema, Frank; Berkhout, Ben; Rouzioux, Christine; Delfraissy, Jean-Francois; Meyer, Laurence

    2007-01-01

    Background: The evolution of plasma viral load after HIV infection has been described as reaching a setpoint, only to start rising again shortly before AIDS diagnosis. In contrast, CD4 T-cell count is considered to show a stable decrease. However, characteristics of marker evolution over time depend

  19. HIV DNA loads, plasma residual viraemia and risk of virological rebound in heavily treated, virologically suppressed HIV-infected patients.

    Science.gov (United States)

    Gianotti, N; Canducci, F; Galli, L; Cossarini, F; Salpietro, S; Poli, A; Nozza, S; Spagnuolo, V; Clementi, M; Sampaolo, M; Ceresola, E R; Racca, S; Lazzarin, A; Castagna, A

    2015-01-01

    In this single-centre, retrospective study, we analyzed data of 194 patients receiving antiretroviral therapy with <50 human immunodeficiency virus (HIV) RNA copies/mL in plasma and 318 HIV RNA/DNA paired samples. By kinetic polymerase chain reaction (kPCR) molecular system analysis, 104 (54%) subjects had undetectable HIV RNA and 90 (46%) had residual viraemia. Median (interquartile range) HIV DNA load was 780 (380-1930) copies/10(6) peripheral blood lymphocytes (PBL), and HIV DNA loads were independently associated with residual viraemia (p 0.002). Virological rebound occurred in 29/194 (15%) patients over a median (interquartile range) follow-up of 17.5 (13.5-31.5) months. Residual viraemia (p 0.002), but not HIV DNA load, was independently associated with virological rebound.

  20. Impact of collection method on assessment of semen HIV RNA viral load.

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    Brendan J W Osborne

    Full Text Available BACKGROUND: The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load. METHODOLOGY/PRINCIPAL FINDINGS: Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM either undiluted (Visit 1 or directly into transport medium (Visit 2. Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples. CONCLUSIONS/SIGNIFICANCE: The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.

  1. Bacterial vaginosis, human papilloma virus and herpes viridae do not predict vaginal HIV RNA shedding in women living with HIV in Denmark

    DEFF Research Database (Denmark)

    Wessman, Maria; Thorsteinsson, Kristina; Jensen, Jørgen S.

    2017-01-01

    viridae, and vaginal HIV viral load. RESULTS: Median age of the 150 included women was 41 years; ethnicity was predominantly White (35%) or Black (47%). The majority (96%) was on ART and had undetectable (85%) plasma HIV RNA (...-51 years were recruited from six Departments of Infectious Diseases in Denmark during enrolment in the SHADE cohort; a prospective cohort study of WLWH attending regular outpatient care. BV was diagnosed by microscopy of vaginal swabs and PCR was used for detection of BV-associated bacteria, HPV, herpes...... RNA. Both before and after adjustment for BV, age, ethnicity, plasma HIV RNA, CD4 cell count, herpes viridae and HPV, we found no significant predictors of HIV RNA vaginal shedding. CONCLUSION: In well-treated WLWH, BV, herpes viridae or HPV do not predict vaginal HIV RNA shedding. This implies...

  2. Genital Tract HIV RNA Levels and Their Associations with Human Papillomavirus Infection and Risk of Cervical Pre-Cancer

    Science.gov (United States)

    GHARTEY, Jeny; KOVACS, Andrea; BURK, Robert D.; MASSAD, L. Stewart; MINKOFF, Howard; XIE, Xianhong; D’SOUZA, Gypsyamber; XUE, Xiaonan; WATTS, D. Heather; LEVINE, Alexandra M.; EINSTEIN, Mark H.; COLIE, Christine; ANASTOS, Kathryn; ELTOUM, Isam-Eldin; HEROLD, Betsy C.; PALEFSKY, Joel M.; STRICKLER, Howard D.

    2014-01-01

    Objective Plasma HIV RNA levels have been associated with risk of human papillomavirus (HPV) and cervical neoplasia in HIV-seropositive women. However, little is known regarding local genital tract HIV RNA levels and their relation with cervical HPV and neoplasia. Design/Methods In an HIV-seropositive women’s cohort with semi-annual follow-up, we conducted a nested case-control study of genital tract HIV RNA levels and their relation with incident high-grade squamous intraepithelial lesions sub-classified as severe (severe HSIL), as provided for under the Bethesda 2001 classification system. Specifically, 66 incident severe HSIL were matched to 130 controls by age, CD4+ count, HAART use, and other factors. We also studied HPV prevalence, incident detection, and persistence in a random sample of 250 subjects. Results Risk of severe HSIL was associated with genital tract HIV RNA levels (odds ratio comparing HIV RNA ≥ the median among women with detectable levels versus undetectable [ORVL] 2.96; 95% CI: 0.99–8.84; Ptrend=0.03). However, this association became non-significant (Ptrend=0.51) following adjustment for plasma HIV RNA levels. There was also no association between genital tract HIV RNA levels and the prevalence of any HPV or oncogenic HPV. However, the incident detection of any HPV (Ptrend=0.02) and persistence of oncogenic HPV (Ptrend=0.04) were associated with genital tract HIV RNA levels, after controlling plasma HIV RNA levels. Conclusion These prospective data suggest that genital tract HIV RNA levels are not a significant independent risk factor for cervical pre-cancer in HIV-seropositive women, but leave open the possibility that they may modestly influence HPV infection, an early stage of cervical tumoriogenesis. PMID:24694931

  3. Bacterial vaginosis, human papilloma virus and herpes viridae do not predict vaginal HIV RNA shedding in women living with HIV in Denmark.

    Science.gov (United States)

    Wessman, Maria; Thorsteinsson, Kristina; Jensen, Jørgen S; Storgaard, Merete; Rönsholt, Frederikke F; Johansen, Isik S; Pedersen, Gitte; Nørregård Nielsen, Lars; Bonde, Jesper; Katzenstein, Terese L; Weis, Nina; Lebech, Anne-Mette

    2017-05-31

    Bacterial vaginosis (BV) has been found to be associated with HIV acquisition and transmission. This is suggested to be due to higher HIV RNA levels in cervicovaginal fluids in women living with HIV (WLWH) with BV, as bacteria associated with BV may induce viral replication and shedding in the genital tract despite undetectable HIV RNA plasma viral load. We examined the prevalence and diagnostic predictors of BV and HIV-1 RNA vaginal shedding in women living with HIV (WLWH) in Denmark, taking into account the presence of human papillomavirus (HPV) and herpes viridae. WLWH between 18-51 years were recruited from six Departments of Infectious Diseases in Denmark during enrolment in the SHADE cohort; a prospective cohort study of WLWH attending regular outpatient care. BV was diagnosed by microscopy of vaginal swabs and PCR was used for detection of BV-associated bacteria, HPV, herpes viridae, and vaginal HIV viral load. Median age of the 150 included women was 41 years; ethnicity was predominantly White (35%) or Black (47%). The majority (96%) was on ART and had undetectable (85%) plasma HIV RNA (<40 copies/mL). BV was diagnosed in 32%. Overall, 11% had detectable vaginal HIV RNA. Both before and after adjustment for BV, age, ethnicity, plasma HIV RNA, CD4 cell count, herpes viridae and HPV, we found no significant predictors of HIV RNA vaginal shedding. In well-treated WLWH, BV, herpes viridae or HPV do not predict vaginal HIV RNA shedding. This implies that HIV shedding does not seem to be increased by BV.

  4. The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

    Science.gov (United States)

    Yukl, Steven A.; Shergill, Amandeep K.; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K.; Crouch, Pierre; Deeks, Steven G.; Havlir, Diane V.; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K.

    2013-01-01

    Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir. PMID:23852128

  5. Anogenital HIV RNA in Thai men who have sex with men in Bangkok during acute HIV infection and after randomization to standard vs. intensified antiretroviral regimens

    Directory of Open Access Journals (Sweden)

    Nittaya Phanuphak

    2015-05-01

    Full Text Available Introduction: HIV transmission risk is highest during acute HIV infection (AHI. We evaluated HIV RNA in the anogenital compartment in men who have sex with men (MSM during AHI and compared time to undetectable HIV RNA after three-drug versus five-drug antiretroviral therapy (ART to understand risk for onward HIV transmission. Methods: MSM with AHI (n=54 had blood, seminal plasma and anal lavage collected for HIV RNA at baseline, days 3 and 7, and weeks 2, 4, 12 and 24. Data were compared between AHI stages: 1 (fourth-generation antigen-antibody combo immunoassay [IA]–, third-generation IA–, n=15, 2 (fourth-generation IA+, third-generation IA–, n=9 and 3 (fourth-generation IA+, third-generation IA+, western blot–/indeterminate, n=30 by randomization to five-drug (tenofovir+emtricitabine+efavirenz+raltegravir+maraviroc, n=18 versus three-drug (tenofovir+emtricitabine+efavirenz, n=18 regimens. Results: Mean age was 29 years and mean duration since HIV exposure was 15.4 days. Mean baseline HIV RNA was 5.5 in blood, 3.9 in seminal plasma and 2.6 log10 copies/ml in anal lavage (p<0.001. Blood and seminal plasma HIV RNA were higher in AHI Stage 3 compared to Stage 1 (p<0.01. Median time from ART initiation to HIV RNA <50 copies/ml was 60 days in blood, 15 days in seminal plasma and three days in anal lavage. Compared with the three-drug ART, the five-drug ART had a shorter time to HIV RNA <1500 copies/ml in blood (15 vs. 29 days, p=0.005 and <50 copies/ml in seminal plasma (13 vs. 24 days, p=0.048. Conclusions: Among MSM with AHI, HIV RNA was highest in blood, followed by seminal plasma and anal lavage. ART rapidly reduced HIV RNA in all compartments, with regimen intensified by raltegravir and maraviroc showing faster HIV RNA reductions in blood and seminal plasma.

  6. HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

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    Jing Jin

    Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

  7. RNA structure. Structure of the HIV-1 RNA packaging signal.

    Science.gov (United States)

    Keane, Sarah C; Heng, Xiao; Lu, Kun; Kharytonchyk, Siarhei; Ramakrishnan, Venkateswaran; Carter, Gregory; Barton, Shawn; Hosic, Azra; Florwick, Alyssa; Santos, Justin; Bolden, Nicholas C; McCowin, Sayo; Case, David A; Johnson, Bruce A; Salemi, Marco; Telesnitsky, Alice; Summers, Michael F

    2015-05-22

    The 5' leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a (2)H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; Ψ(CES)). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

  8. MicroRNA-155 Reinforces HIV Latency.

    Science.gov (United States)

    Ruelas, Debbie S; Chan, Jonathan K; Oh, Eugene; Heidersbach, Amy J; Hebbeler, Andrew M; Chavez, Leonard; Verdin, Eric; Rape, Michael; Greene, Warner C

    2015-05-29

    The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

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    Yu Lianbo

    2011-05-01

    Full Text Available Abstract Background MicroRNA (miRNA-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured

  10. The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

    Science.gov (United States)

    Waddington, Lynne; Hijnen, Marcel; Velkov, Tony; McKinstry, William J.

    2017-01-01

    The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔGenthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. PMID:28222188

  11. Plasma proteomic profiling in HIV-1 infected methamphetamine abusers.

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    Gwenael Pottiez

    Full Text Available We wanted to determine whether methamphetamine use affects a subset of plasma proteins in HIV-infected persons. Plasma samples from two visits were identified for subjects from four groups: HIV+, ongoing, persistent METH use; HIV+, short-term METH abstinent; HIV+, long term METH abstinence; HIV negative, no history of METH use. Among 390 proteins identified, 28 showed significant changes in expression in the HIV+/persistent METH+ group over the two visits, which were not attributable to HIV itself. These proteins were involved in complement, coagulation pathways and oxidative stress. Continuous METH use is an unstable condition, altering levels of a number of plasma proteins.

  12. Micro RNA in Exosomes from HIV-Infected Macrophages.

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    Roth, William W; Huang, Ming Bo; Addae Konadu, Kateena; Powell, Michael D; Bond, Vincent C

    2015-12-22

    Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

  13. Micro RNA in Exosomes from HIV-Infected Macrophages

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    William W. Roth

    2015-12-01

    Full Text Available Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

  14. Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis

    DEFF Research Database (Denmark)

    Dilley, Kari A; Ni, Na; Nikolaitchik, Olga A;

    2011-01-01

    HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV......-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent...... proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ∼10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation...

  15. DBR1 siRNA inhibition of HIV-1 replication

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    Naidu Yathi

    2005-10-01

    Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

  16. Achieving HIV-1 Control through RNA-Directed Gene Regulation

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    Vera Klemm

    2016-12-01

    Full Text Available HIV-1 infection has been transformed by combined anti-retroviral therapy (ART, changing a universally fatal infection into a controllable infection. However, major obstacles for an HIV-1 cure exist. The HIV latent reservoir, which exists in resting CD4+ T cells, is not impacted by ART, and can reactivate when ART is interrupted or ceased. Additionally, multi-drug resistance can arise. One alternate approach to conventional HIV-1 drug treatment that is being explored involves gene therapies utilizing RNA-directed gene regulation. Commonly known as RNA interference (RNAi, short interfering RNA (siRNA induce gene silencing in conserved biological pathways, which require a high degree of sequence specificity. This review will provide an overview of the silencing pathways, the current RNAi technologies being developed for HIV-1 gene therapy, current clinical trials, and the challenges faced in progressing these treatments into clinical trials.

  17. The Life-Cycle of the HIV-1 Gag–RNA Complex

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    Elodie Mailler

    2016-09-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles.

  18. The efficacy and safety of maraviroc addition to a stable antiretroviral regimen in subjects with suppressed plasma HIV-RNA is not influenced by age.

    Science.gov (United States)

    Blanco, José-Ramón; Arroyo-Manzano, David; Rojas-Liévano, John F; Crespo, Manuel; Bravo, Isa; Pasquau, Juan; Garcia Del Toro, Miguel; Herrero, Cristina; Rivero, Antonio; Moreno, Santiago; Llibre, Josep Maria

    2015-09-01

    There are few data about the immunovirological efficacy, safety/tolerability, and durability of maraviroc (MVC) addition to aging patients on suppressive antiretroviral therapy (cART) and undetectable viral load (HIV clinical trials. This study included 80 patients aged ≥50 years and 161 aged HIV history (≥15 years) (OR 0.43; p=0.016) or having <200/mm(3) CD4(+) T cells at MVC initiation (OR 0.27; p=0.004). Meanwhile, achieving a CD4/CD8 ratio ≥0.5 at week 48 was less likely in those with CD4(+) T cell counts <200 at MVC initiation (OR 0.09; p<0.0001) or with a previous AIDS event (OR 0.43; p=0.028). In summary, the immunovirological efficacy, safety/tolerability, and durability of MVC addition in patients virologically suppressed were independent of the patient's age at treatment onset.

  19. Undetectable hepatitis C virus RNA during syphilis infection in two HIV/HCV-co-infected patients

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Knudsen, Andreas; Krarup, Henrik Bygum;

    2014-01-01

    BACKGROUND: Treponema pallidum, the causative agent of syphilis, elicits a vigorous immune response in the infected host. This study sought to describe the impact of syphilis infection on hepatitis C virus (HCV) RNA levels in patients with HIV and chronic HCV infection. METHODS: Patients...... with chronic HIV/HCV and syphilis co-infection were identified by their treating physicians from 1 October 2010 to 31 December 2013. Stored plasma samples obtained before, during, and after syphilis infection were analysed for interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF......-α), interferon gamma (IFN-γ), and IFN-γ-inducible protein 10 kDa (IP-10). RESULTS: Undetectable HCV RNA at the time of early latent syphilis infection was observed in 2 patients with HIV and chronic HCV infection. After treatment of the syphilis infection, HCV RNA levels increased again in patient 1, whereas...

  20. Recognition of HIV TAR RNA by triazole linked neomycin dimers.

    Science.gov (United States)

    Kumar, Sunil; Arya, Dev P

    2011-08-15

    A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5'-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (T(m)) of the HIV TAR RNA up to 10°C. Ethidium bromide displacement titrations revealed nanomolar IC(50) between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC(50) in the micromolar range is observed.

  1. HIV-1 tropism testing in subjects achieving undetectable HIV-1 RNA: diagnostic accuracy, viral evolution and compartmentalization.

    Directory of Open Access Journals (Sweden)

    Christian Pou

    Full Text Available BACKGROUND: Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs. However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. METHODS & RESULTS: We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. CONCLUSIONS: The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary

  2. Changes in plasma cytokines after treatment of ascaris lumbricoides infection in individuals with HIV-1 infection.

    Science.gov (United States)

    Blish, Catherine A; Sangaré, Laura; Herrin, Bradley R; Richardson, Barbra A; John-Stewart, Grace; Walson, Judd L

    2010-06-15

    Albendazole treatment of individuals with human immunodeficiency virus type 1 (HIV-1) and Ascaris lumbricoides co-infection has led to significantly improved CD4(+) cell counts and a trend for lower plasma HIV-1 RNA levels in a previous randomized placebo-controlled trial. To define mechanisms by which deworming contributed to changes in markers of HIV-1 disease progression, plasma cytokine levels were evaluated. Albendazole treatment, compared with placebo, was associated with significantly decreased plasma interleukin (IL) 10 levels (P = .01)ot associated with significant changes in levels of IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12p70, IL-13, interferon gamma, tumor necrosis factor alpha, or thymic stromal lymphopoietin. Treatment of A. lumbricoides co-infection may delay HIV-1 disease progression by reducing helminth-induced, IL-10-mediated immunosuppression.

  3. Plasma HIV-1 tropism and risk of short-term clinical progression to AIDS or death

    DEFF Research Database (Denmark)

    Fontdevila, Maria Casadellà; Cozzi-Lepri, Alessandro; Phillips, Andrew;

    2014-01-01

    INTRODUCTION: It is uncertain if plasma HIV-1 tropism is an independent predictor of short-term risk of clinical progression / death, in addition to the CD4 count and HIV RNA level. We conducted a nested case-control study within EuroSIDA to assess this question amongst people with current HIV RNA...... level >1000 copies/mL, including both people on ART and those ART naïve. METHODS: People with an AIDS diagnosis or who died from any causes for whom there was a stored plasma sample with HIV-1 RNA (VL)≥1,000 copies/mL available in the time window of 3-12 months prior to the event were identified....... At least one control was selected for each case matched for age, VL and HCV status at the time of sampling. Controls were event-free after a matched duration of time from the date of sampling. Plasma HIV tropism was estimated using 454 and population sequencing (PS). Non-R5 HIV was defined as: (a) ≥2...

  4. Short-course TLR9 Agonist Treatment Impacts Innate Immunity and Plasma Viremia in Individuals with HIV infection

    DEFF Research Database (Denmark)

    Vibholm, Line; Schleimann, Mariane H; Højen, Jesper F

    2017-01-01

    characterized plasmacytoid dendritic cell, NK -and T cell activation using flow cytometry on baseline and after 4 weeks of treatment. HIV-1 transcription was quantified by measuring plasma HIV-1 RNA during MGN1703 administration. Results: In accordance with the cell-type specific expression of TLR9, MGN1703...

  5. HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

    Science.gov (United States)

    Demontis, Maria Antonietta; Sadiq, Maaz Tahir; Golz, Simon; Taylor, Graham P

    2015-12-01

    Plasma of patients infected with HTLV-1 is considered non-infectious but detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential value in diagnosis and prognosis. RNA from 1 ml of plasma from 65 subjects infected with HTLV-1 (27 asymptomatic carriers [AC]), 17 patients with HTLV-1-associated myelopathy (HAM/TSP), 14 with adult T-cell leukemia/lymphoma (ATLL), two co-infected with HIV, and five with other HTLV-1-associated disease, was extracted and reverse transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis. HTLV-1 RNA was detected in plasma from 18 (28%) patients, always at a very low copy number (3-13 copies viral cDNA per milliliter of plasma). Mean values of HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma. HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor.

  6. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.

    Science.gov (United States)

    Pasternak, Alexander O; DeMaster, Laura K; Kootstra, Neeltje A; Reiss, Peter; O'Doherty, Una; Berkhout, Ben

    2015-11-11

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

  7. DEFINING PLASMA MICRORNAS ASSOCIATED WITH COGNITIVE IMPAIRMENT IN HIV-INFECTED PATIENTS†

    Science.gov (United States)

    Kadri, Ferdous; LaPlante, Andrea; De Luca, Mariacristina; Doyle, Lisa; Velasco-Gonzalez, Cruz; Patterson, Jonathan R.; Molina, Patricia E.; Nelson, Steve; Zea, Arnold; Parsons, Christopher H.; Peruzzi, Francesca

    2015-01-01

    Human Immunodeficiency Virus (HIV)-infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age-matched, HIV-infected (HIV+) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV+ patients with cognitive impairment from those without cognitive impairment. These results justify follow-on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV+ patients with neurocognitive impairment. PMID:26284581

  8. Association between Plasma Homocysteine Levels and Neuronal Injury in HIV Infection

    Science.gov (United States)

    Ahlgren, Erika; Hagberg, Lars; Fuchs, Dietmar; Andersson, Lars-Magnus; Nilsson, Staffan; Zetterberg, Henrik; Gisslén, Magnus

    2016-01-01

    Objective To investigate the role of homocysteine in neuronal injury in HIV infection. Methods Using a cross-sectional design and archived samples, we compared concentrations of plasma homocysteine and cerebrospinal fluid (CSF) neurofilament light protein (NFL), a sensitive marker of neuronal injury, in 83 HIV-1-infected subjects without antiretroviral treatment. We also analyzed plasma vitamin B12, serum folate, CSF, and plasma HIV RNA, the immune activation marker neopterin in CSF and serum, and albumin ratio as a marker of blood-brain barrier integrity. Twenty-two subjects provided a second sample median of 12.5 months after antiretroviral treatment initiation. Results A significant correlation was found between plasma homocysteine and CSF NFL concentrations in untreated individuals (r = 0.52, p NFL in HIV-1-infected individuals. The correlation of plasma homocysteine and CSF NFL was also present in the group receiving antiretroviral therapy (r = 0.51, p = 0.016). Conclusion A correlation between plasma homocysteine and axonal injury, as measured by CSF NFL, was found in both untreated and treated HIV. While this study is not able to prove a causal link, homocysteine and functional B12/folate deficiency appear to play a role in neural injury in HIV-infected individuals. PMID:27441551

  9. High rate of hepatitis C virus (HCV) recurrence in HIV-infected individuals with spontaneous HCV RNA clearance

    DEFF Research Database (Denmark)

    Peters, L; Mocroft, A; Soriano, V

    2014-01-01

    OBJECTIVES: Following resolution of hepatitis C virus (HCV) infection, recurrence has been shown to occur in some persons with repeated exposure to HCV. We aimed to investigate the rate and factors associated with HCV RNA recurrence among HIV-1-infected patients with prior spontaneous HCV RNA...... clearance in the EuroSIDA cohort. METHODS: All HIV-infected patients with documented prior spontaneous HCV clearance, and at least one subsequently collected plasma sample, were examined. The last sample was tested for HCV RNA and those with HCV RNA ≥ 615 IU/mL were defined as having HCV recurrence...

  10. Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA

    DEFF Research Database (Denmark)

    Jakobsen, Martin Roelsgaard; Tolstrup, M; Bertelsen, L

    2007-01-01

    BACKGROUND: Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO(+)T cell compartment. OBJECTIVE: To develop an ARMS assay for plasma virions and t...... an unequal distribution of linked-mutation populations in plasma and CD45RO(+)T cells. Furthermore, the linked 103N-184V mutation may be more fit than the single 184V mutation and this linked population emerges rapidly under inadequate drug pressure. Udgivelsesdato: 2007-Jul...

  11. Utility of Pooled HIV RNA RT-PCR Assay in Diagnosing Acute HIV Infections

    Institute of Scientific and Technical Information of China (English)

    张麒; 蒋岩; 刘全忠

    2004-01-01

    Abstract: The P24 antigen test, HIV RNA PCR test,HIV isolation/culture and fourth-generation HIV uniform Ag/Ab assay are being utilized in diagnosing acute HIV infection in different labs. Many factors limit the use of screening for acute HIV in high-risk populations, in blood donors and during voluntary HIV testing, including, cost, technique, sensitivity and specificity. In this review we explore a new NAAT method which involves HIV RNA RT-PCR on pooled samples. This technique is able to screen for acute infections in a large testing volume and may he used as a screening method in high-risk populations and blood donors.

  12. Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1–Seropositive Kenyan Men

    Science.gov (United States)

    Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L.; Sanders, Eduard J.; Peshu, Norbert M.; Krieger, John N.; Muller, Charles H.; Coombs, Robert W.; Fredricks, David N.; Graham, Susan M.

    2017-01-01

    Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission. PMID:27861240

  13. Roles of the linker region of RNA helicase A in HIV-1 RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Li Xing

    Full Text Available RNA helicase A (RHA promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNA(Lys3 to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNA(Lys3 annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNA(Lys3 annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5'-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

  14. Plasma cytokine levels in Tanzanian HIV-1-infected adults and the effect of antiretroviral treatment

    DEFF Research Database (Denmark)

    Haissman, J.M.; Vestergaard, L.S.; Sembuche, S.;

    2009-01-01

    OBJECTIVE: To evaluate the role immune activation leading to the production and circulation of cytokines has in the pathogenesis of HIV infection in sub-Saharan Africa and the effect of antiretroviral treatment (ART) on these parameters. METHODS: Plasma concentrations of tumor necrosis factor (TNF...... counts below 200 cells per microliter than individuals with CD4 cell counts above 200 cells per microliter. HIV RNA was the strongest predictor of all cytokine expression in multivariate analysis. ART leads to a decrease in all cytokines to levels close to those of HIV-uninfected individuals. CONCLUSIONS...

  15. Longitudinal analysis of HIV-1 coreceptor tropism by single and triplicate HIV-1 RNA and DNA sequencing in patients undergoing successful first-line antiretroviral therapy

    Science.gov (United States)

    Meini, Genny; Rossetti, Barbara; Bianco, Claudia; Ceccherini-Silberstein, Francesca; Di Giambenedetto, Simona; Sighinolfi, Laura; Monno, Laura; Castagna, Antonella; Rozera, Gabriella; D'Arminio Monforte, Antonella; Zazzi, Maurizio; De Luca, Andrea; Moroni, M.; Angarano, G.; Antinori, A.; Armignacco, O.; d'Arminio Monforte, A.; Castelli, F.; Cauda, R.; Di Perri, G.; Galli, M.; Iardino, R.; Ippolito, G.; Lazzarin, A.; Perno, C. F.; von Schloesser, F.; Viale, P.; d'Arminio Monforte, A.; Antinori, A.; Castagna, A.; Ceccherini-Silberstein, F.; Cozzi-Lepri, A.; Girardi, E.; Lo Caputo, S.; Mussini, C.; Puoti, M.; Andreoni, M.; Ammassari, A.; Antinori, A.; Balotta, C.; Bonfanti, P.; Bonora, S.; Borderi, M.; Capobianchi, M. R.; Castagna, A.; Ceccherini-Silberstein, F.; Cingolani, A.; Cinque, P.; Cozzi-Lepri, A.; d'Arminio Monforte, A; De Luca, A.; Di Biagio, A.; Girardi, E.; Gianotti, N.; Gori, A.; Guaraldi, G.; Lapadula, G.; Lichtner, M.; Lo Caputo, S.; Madeddu, G.; Maggiolo, F.; Marchetti, G.; Marcotullio, S.; Monno, L.; Mussini, C.; Puoti, M.; Quiros Roldan, E.; Rusconi, S.; Cozzi-Lepri, A.; Cicconi, P.; Fanti, I.; Formenti, T.; Galli, L.; Lorenzini, P.; Giacometti, A.; Costantini, A.; Angarano, G.; Monno, L.; Santoro, C.; Maggiolo, F.; Suardi, C.; Viale, P.; Vanino, E.; Verucchi, G.; Castelli, F.; Quiros Roldan, E.; Minardi, C.; Quirino, T.; Abeli, C.; Manconi, P.E.; Piano, P.; Vecchiet, J.; Falasca, K.; Sighinolfi, L.; Segala, D.; Mazzotta, F.; Lo Caputo, S.; Cassola, G.; Viscoli, G.; Alessandrini, A.; Piscopo, R.; Mazzarello, G.; Mastroianni, C.; Belvisi, V.; Bonfanti, P.; Caramma, I.; Castelli, A. P.; Galli, M.; Lazzarin, A.; Rizzardini, G.; Puoti, M.; d'Arminio Monforte, A.; Ridolfo, A. L.; Piolini, R.; Castagna, A.; Salpietro, S.; Carenzi, L.; Moioli, M. C.; Cicconi, P.; Marchetti, G.; Mussini, C.; Puzzolante, C.; Gori, A.; Lapadula, G.; Abrescia, N.; Chirianni, A.; Guida, M. G.; Gargiulo, M.; Baldelli, F.; Francisci, D.; Parruti, G.; Ursini, T.; Magnani, G.; Ursitti, M. A.; Cauda, R.; Andreoni, M.; Antinori, A.; Vullo, V.; Cingolani, A.; d'Avino, A.; Ammassari, A.; Gallo, L.; Nicastri, E.; Acinapura, R.; Capozzi, M.; Libertone, R.; Tebano, G.; Cattelan, A.; Mura, M. S.; Madeddu, G.; Caramello, P.; Di Perri, G.; Orofino, G. C.; Bonora, S.; Sciandra, M.; Pellizzer, G.; Manfrin, V.

    2014-01-01

    Objectives Maraviroc has been shown to be effective in patients harbouring CCR5-tropic HIV-1. While this CCR5 antagonist has initially been used in salvage therapy, its excellent safety profile makes it ideal for antiretroviral treatment simplification strategies in patients with suppressed plasma viraemia. The aim of this study was to compare HIV-1 tropism as detected in baseline plasma RNA and peripheral blood mononuclear cell (PBMC) DNA prior to first-line therapy and to analyse tropism evolution while on successful treatment. Methods HIV-1 tropism was determined using triplicate genotypic testing combined with geno2pheno[coreceptor] analysis at a 10% false positive rate in 42 patients. Paired pre-treatment plasma RNA and PBMC DNA and two subsequent PBMC DNA samples (the first obtained after reaching undetectable plasma HIV-1 RNA and the second after at least 2 years of suppression of plasma viraemia) were evaluated. Results Coreceptor tropism was completely concordant in paired pre-treatment RNA and DNA, with 26.2% of HIV-1 sequences predicted to be non-CCR5-tropic. During follow-up, coreceptor tropism switches were detected in 4 (9.5%) patients without any preferential direction. Although false positive rate discrepancies within triplicates were common, the rate of discordance of coreceptor tropism assignment among triplicate results in this mostly CCR5-tropic dataset was only 2.1%, questioning the added value of triplicate testing compared with single testing. Conclusions HIV-1 coreceptor tropism changes during virologically successful first-line treatment are infrequent. HIV-1 DNA analysis may thus support the choice of a CCR5 antagonist in treatment switch strategies; however, maraviroc treatment outcome data are required to confirm this option. PMID:24155059

  16. Residual viraemia in HIV-1-infected patients with plasma viral load

    DEFF Research Database (Denmark)

    Ostrowski, S.R.; Katzenstein, T.L.; Pedersen, Bente Klarlund

    2008-01-01

    antiretroviral therapy (HAART)-treated HIV-1-infected patients with plasma HIV-1 RNA or=1 episode with TMA-RV whereas 9 patients had undetectable TMA-RV throughout the study-period. Time-points with TMA-RV and PCR-RV were associated with higher circulating sTNFrII (+0.234 ng/ml, P = 0.030) and beta(2......)-microglobulin (+22 nmol/l, P = 0.016) and time-points with PCR-RV were also associated with higher IgA (+0.82 micromol/l, P = 0.035) and CD8-count (+1.18-fold, P = 0.001). Patients with TMA-RV in the study-period had higher HIV-1 RNA pre-HAART (P = 0.032). RV was not associated with proviral-HIV-1-DNA, CD4...

  17. Cell-associated HIV DNA measured early during infection has prognostic value independent of serum HIV RNA measured concomitantly

    DEFF Research Database (Denmark)

    Katzenstein, Terese L; Oliveri, Roberto S; Benfield, Thomas;

    2002-01-01

    into 3 groups, according to whether their cell-associated HIV DNA load was or = 2,500 DNA copies/10(6) peripheral blood mononuclear cells. Clinical progression rates differed significantly between the groups (p HIV DNA load had prognostic value independent......Using data from the Danish AIDS Cohort of HIV-infected homosexual men established in the 1980s, the prognostic value of early HIV DNA loads was evaluated. In addition to DNA measurements, concomitant serum HIV RNA levels, CD4 cell counts and CCR5 genotypes were determined. The patients were divided...... of serum HIV RNA (p HIV DNA, HIV RNA and CD4 cell counts were all included in a Cox model, only serum HIV RNA had independent prognostic value. Patients heterozygous for the CCR5 delta 32 allele had significantly lower HIV DNA loads than those homozygous for the normal allele (p

  18. Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

    Directory of Open Access Journals (Sweden)

    Christian Diamant Mossoro-Kpinde

    2016-01-01

    Full Text Available Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France, combining automated station extraction (Amplix station 16 Dx and real-time PCR (Amplix NG, for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL, across wide dynamic range (1.4–10 log copies/mL, 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche, with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa.

  19. Controlling HIV-1: Non-Coding RNA Gene Therapy Approaches to a Functional Cure.

    Science.gov (United States)

    Ahlenstiel, Chantelle L; Suzuki, Kazuo; Marks, Katherine; Symonds, Geoff P; Kelleher, Anthony D

    2015-01-01

    The current treatment strategy for HIV-1 involves prolonged and intensive combined antiretroviral therapy (cART), which successfully suppresses plasma viremia. It has transformed HIV-1 infection into a chronic disease. However, despite the success of cART, a latent form of HIV-1 infection persists as integrated provirus in resting memory CD4(+) T cells. Virus can reactivate from this reservoir upon cessation of treatment, and hence HIV requires lifelong therapy. The reservoir represents a major barrier to eradication. Understanding molecular mechanisms regulating HIV-1 transcription and latency are crucial to develop alternate treatment strategies, which impact upon the reservoir and provide a path toward a "functional cure" in which there is no detectable viremia in the absence of cART. Numerous reports have suggested ncRNAs are involved in regulating viral transcription and latency. This review will discuss the latest developments in ncRNAs, specifically short interfering (si)RNA and short hairpin (sh)RNA, targeting molecular mechanisms of HIV-1 transcription, which may represent potential future therapeutics. It will also briefly address animal models for testing potential therapeutics and current gene therapy clinical trials.

  20. Plasma Soluble CD163 Level Independently Predicts All-Cause Mortality in HIV-1-Infected Individuals

    DEFF Research Database (Denmark)

    Knudsen, Troels Bygum; Ertner, Gideon; Petersen, Janne

    2016-01-01

    BACKGROUND: CD163, a monocyte- and macrophage-specific scavenger receptor, is shed as soluble CD163 (sCD163) during the proinflammatory response. Here, we assessed the association between plasma sCD163 levels and progression to AIDS and all-cause mortality among individuals infected with human...... immunodeficiency virus type 1 (HIV). METHODS: Plasma sCD163 levels were measured in 933 HIV-infected individuals. Hazard ratios (HRs) with 95% confidence intervals (CIs) associated with mortality were computed by Cox proportional hazards regression. RESULTS: At baseline, 86% were receiving antiretroviral treatment......, 73% had plasma a HIV RNA level of CD163 levels were higher in nonsurvivors than in survivors (4.92 mg/L [interquartile range {IQR}, 3.29-8.65 mg/L] vs 3.16 mg/L [IQR, 2...

  1. Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples

    Science.gov (United States)

    Sollis, Kimberly A.; Smit, Pieter W.; Fiscus, Susan; Ford, Nathan; Vitoria, Marco; Essajee, Shaffiq; Barnett, David; Cheng, Ben; Crowe, Suzanne M.; Denny, Thomas; Landay, Alan; Stevens, Wendy; Habiyambere, Vincent; Perrins, Jos; Peeling, Rosanna W.

    2014-01-01

    Background Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. Methods and Findings A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10). Conclusions This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  2. Systematic review of the performance of HIV viral load technologies on plasma samples.

    Directory of Open Access Journals (Sweden)

    Kimberly A Sollis

    Full Text Available BACKGROUND: Viral load (VL monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. METHODS AND FINDINGS: A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25, Cobas TaqMan v2.0 (n = 11, Abbott RealTime HIV-1 (n = 23, Versant HIV-1 RNA bDNA 3.0 (n = 15, Versant HIV-1 RNA kPCR 1.0 (n = 2, ExaVir Load v3 (n = 2, and NucliSens EasyQ v2.0 (n = 1. All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2-26% and 9-70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0-5.1% and 5.44% (range 1.17-30.00% across the range of VL counts (2log10-7log10. CONCLUSIONS: This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  3. Translational Control of the HIV Unspliced Genomic RNA

    Directory of Open Access Journals (Sweden)

    Bárbara Rojas-Araya

    2015-08-01

    Full Text Available Post-transcriptional control in both HIV-1 and HIV-2 is a highly regulated process that commences in the nucleus of the host infected cell and finishes by the expression of viral proteins in the cytoplasm. Expression of the unspliced genomic RNA is particularly controlled at the level of RNA splicing, export, and translation. It appears increasingly obvious that all these steps are interconnected and they result in the building of a viral ribonucleoprotein complex (RNP that must be efficiently translated in the cytosolic compartment. This review summarizes our knowledge about the genesis, localization, and expression of this viral RNP.

  4. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA

    NARCIS (Netherlands)

    Pasternak, A.O.; DeMaster, L.K.; Kootstra, N.A.; Reiss, P.; O'Doherty, U.; Berkhout, B.

    2016-01-01

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent

  5. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA

    NARCIS (Netherlands)

    Pasternak, A.O.; DeMaster, L.K.; Kootstra, N.A.; Reiss, P.; O'Doherty, U.; Berkhout, B.

    2016-01-01

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent

  6. RNA Control of HIV-1 Particle Size Polydispersity

    CERN Document Server

    Faivre-Moskalenko, Cendrine; Thomas, Audrey; Tartour, Kevin; Beck, Yvonne; Iazykov, Maksym; Danial, John; Lourdin, Morgane; Muriaux, Delphine; Castelnovo, Martin

    2014-01-01

    HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP.

  7. Quantification of infectious HIV-1 plasma viral load using a boosted in vitro infection protocol.

    Science.gov (United States)

    Rusert, Peter; Fischer, Marek; Joos, Beda; Leemann, Christine; Kuster, Herbert; Flepp, Markus; Bonhoeffer, Sebastian; Günthard, Huldrych F; Trkola, Alexandra

    2004-08-15

    Methods currently used for HIV-1 viral load measurements are very sensitive, but cannot distinguish between infectious and noninfectious particles. Here we describe the development of a novel, sensitive, and highly reproducible method that allows rapid isolation and quantification of infectious particles from patient plasma. By immobilizing HIV-1 particles in human plasma to platelets using polybrene, we observed a 10- to 1000-fold increase in infectivity over infection protocols using free virus particles. Using this method, we evaluated infectivity in plasma from 52 patients at various disease stages. At plasma viral loads of 1000-10000 HIV-1 RNA copies/ml 18%, at 10,000-50,000 copies/ml 73%, at 50,000-100,000 copies/ml 90%, and above 100,000 copies 96% of cultures were positive. We found that infectious titers among patients vary distinctively but are characteristic for a patient over extended time periods. Furthermore, we demonstrate that by evaluating infectious titers in conjunction with total HIV RNA loads, subtle effects of treatment intervention on viremia levels can be detected. The immobilization procedure does not interfere with viral entry and does not restore the infectivity of neutralized virus. Therefore, this assay system can be utilized to investigate the influence of substances that specifically affect virion infectivity such as neutralizing antibodies, soluble CD4, or protease inhibitors. Measuring viral infectivity may thereby function as an additional, useful marker in monitoring disease progression and evaluating efficacy of antivirals in vivo.

  8. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Science.gov (United States)

    Beck, Zoltan; Jagodzinski, Linda L; Eller, Michael A; Thelian, Doris; Matyas, Gary R; Kunz, Anjali N; Alving, Carl R

    2013-01-01

    Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC

  9. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Directory of Open Access Journals (Sweden)

    Zoltan Beck

    Full Text Available Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC, platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV

  10. Interplay between the RNA interference machinery and HIV-1

    NARCIS (Netherlands)

    Schopman, N.C.T.

    2012-01-01

    Resistente infecties zijn lastig te behandelen. Nick Schopman onderzocht een verbeterde RNA-interferentie (RNAi)-gebaseerde anti-hiv-1 gentherapie. Dit kan in de toekomst leiden tot een nieuwe aanpak van de behandeling van resistente infecties. Schopman beschrijft een nieuw ontwerp van een RNAi-mole

  11. Association between Plasma Homocysteine Levels and Neuronal Injury in HIV Infection.

    Directory of Open Access Journals (Sweden)

    Erika Ahlgren

    Full Text Available To investigate the role of homocysteine in neuronal injury in HIV infection.Using a cross-sectional design and archived samples, we compared concentrations of plasma homocysteine and cerebrospinal fluid (CSF neurofilament light protein (NFL, a sensitive marker of neuronal injury, in 83 HIV-1-infected subjects without antiretroviral treatment. We also analyzed plasma vitamin B12, serum folate, CSF, and plasma HIV RNA, the immune activation marker neopterin in CSF and serum, and albumin ratio as a marker of blood-brain barrier integrity. Twenty-two subjects provided a second sample median of 12.5 months after antiretroviral treatment initiation.A significant correlation was found between plasma homocysteine and CSF NFL concentrations in untreated individuals (r = 0.52, p < 0.0001. As expected, there was a significant inverse correlation between homocysteine and B12 (r = -0.41, p < 0.001 and folate (r = -0.40, p = < 0.001 levels. In a multiple linear regression analysis homocysteine stood out as an independent predictor of CSF NFL in HIV-1-infected individuals. The correlation of plasma homocysteine and CSF NFL was also present in the group receiving antiretroviral therapy (r = 0.51, p = 0.016.A correlation between plasma homocysteine and axonal injury, as measured by CSF NFL, was found in both untreated and treated HIV. While this study is not able to prove a causal link, homocysteine and functional B12/folate deficiency appear to play a role in neural injury in HIV-infected individuals.

  12. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

    Directory of Open Access Journals (Sweden)

    Shephard Enid G

    2011-10-01

    Full Text Available Abstract Background HIV-1 Gag virus like particles (VLPs used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates

  13. Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

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    Sano Kouichi

    2009-07-01

    Full Text Available Abstract Background It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. Results We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1 and U1 cells (latently infected U937 cells with HIV-1 displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. Conclusion Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute

  14. Plasma Soluble CD163 Level Independently Predicts All-Cause Mortality in HIV-1-Infected Individuals.

    Science.gov (United States)

    Knudsen, Troels Bygum; Ertner, Gideon; Petersen, Janne; Møller, Holger Jon; Moestrup, Søren K; Eugen-Olsen, Jesper; Kronborg, Gitte; Benfield, Thomas

    2016-10-15

    CD163, a monocyte- and macrophage-specific scavenger receptor, is shed as soluble CD163 (sCD163) during the proinflammatory response. Here, we assessed the association between plasma sCD163 levels and progression to AIDS and all-cause mortality among individuals infected with human immunodeficiency virus type 1 (HIV). Plasma sCD163 levels were measured in 933 HIV-infected individuals. Hazard ratios (HRs) with 95% confidence intervals (CIs) associated with mortality were computed by Cox proportional hazards regression. At baseline, 86% were receiving antiretroviral treatment, 73% had plasma a HIV RNA level of Plasma sCD163 levels were higher in nonsurvivors than in survivors (4.92 mg/L [interquartile range {IQR}, 3.29-8.65 mg/L] vs 3.16 mg/L [IQR, 2.16-4.64 mg/L]; P = .0001). The cumulative incidence of death increased with increasing plasma sCD163 levels, corresponding to a 6% or 35% increased risk of death for each milligram per liter or quartile increase, respectively, in baseline plasma sCD163 level (adjusted HR, 1.06 [95% CI, 1.03-1.09] and 1.35 [95% CI, 1.13-1.63], respectively). Plasma sCD163 was an independent marker of all-cause mortality in a cohort of HIV-infected individuals, suggesting that monocyte/macrophage activation may play a role in HIV pathogenesis and be a target of intervention. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  15. Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal.

    Science.gov (United States)

    Cao, Song; Chen, Shi-Jie

    2011-12-01

    We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ~3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.

  16. Plasma Soluble CD163 Level Independently Predicts All-Cause Mortality in HIV-1-Infected Individuals

    DEFF Research Database (Denmark)

    Knudsen, Troels Bygum; Ertner, Gideon; Petersen, Janne

    2016-01-01

    Background: CD163, a monocyte- and macrophage-specific scavenger receptor, is shed as soluble CD163 (sCD163) during the proinflammatory response. Here, we assessed the association between plasma sCD163 levels and progression to AIDS and all-cause mortality among individuals infected with human...... immunodeficiency virus type 1 (HIV). Methods: Plasma sCD163 levels were measured in 933 HIV–infected individuals. Hazard ratios (HRs) with 95% confidence intervals (CIs) associated with mortality were computed by Cox proportional hazards regression. Results: At baseline, 86% were receiving antiretroviral treatment......, 73% had plasma a HIV RNA level of CD163 levels were higher in nonsurvivors than in survivors (4.92 mg/L [interquartile range {IQR}, 3.29–8.65 mg/L] vs 3.16 mg/L [IQR, 2...

  17. RNA structures, genomic organization and selection of recombinant HIV.

    Science.gov (United States)

    Simon-Loriere, Etienne; Rossolillo, Paola; Negroni, Matteo

    2011-01-01

    Recombination is an evolutionary mechanism intrinsic to the evolution of many RNA viruses. In retroviruses and notably in the case of HIV, recombination is so frequent that it can be considered as part of its mode of replication. This process not only plays a central role in shaping HIV genetic diversity worldwide, but has also been involved in immune escape and development of resistance to antiviral treatments. Recombination does not create new mutations in the existing genetic repertoire of the virus, but creates new combinations of pre-existing polymorphisms. The simultaneous insertion of multiple substitutions in a single replication cycle leaves little room for the progressive coevolution of regions of proteins, RNA or, more in general, genomes, to accommodate these drastic sequence changes. Therefore, recombination, while allowing the virus to rapidly explore larger sequence space than the slow accumulation of point mutations, also runs the risk of generating non functional viruses. Recombination is the consequence of a switch in the template used during reverse transcription and is promoted by the presence of structured regions in the genomic RNA template. In this review, we discuss new observations suggesting that the distribution of RNA structures along the HIV genome may enhance recombination rates in regions where the resultant progeny is less likely to be impaired, and could therefore maximize the evolutionary value of this source of genetic diversity.

  18. Dynamic motions of the HIV-1 frameshift site RNA.

    Science.gov (United States)

    Mouzakis, Kathryn D; Dethoff, Elizabeth A; Tonelli, Marco; Al-Hashimi, Hashim; Butcher, Samuel E

    2015-02-03

    The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (β = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions.

  19. HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1.

    Science.gov (United States)

    Qian, Shuiming; Zhong, Xuehua; Yu, Lianbo; Ding, Biao; de Haan, Peter; Boris-Lawrie, Kathleen

    2009-01-13

    The RNA silencing pathway is an intracellular innate response to virus infections and retro-transposons. Many plant viruses counter this host restriction by RNA silencing suppressor (RSS) activity of a double-stranded RNA-binding protein, e.g., tomato bushy stunt virus P19. Here, we demonstrate P19 and HIV-1 Tat function across the plant and animal kingdoms and suppress a common step in RNA silencing that is downstream of small RNA maturation. Our experiments reveal that RNA silencing in HIV-1 infected human cells severely attenuates the translational output of the unspliced HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenuation in gag mRNA translation is exacerbated by K51A substitution in the Tat double-stranded RNA-binding domain. Tat, plant virus RSS, or Dicer downregulation rescues robust gag translation and bolsters HIV-1 virion production. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by translational inhibition. Our results identify common features between RNA silencing suppression of plant and animal viruses. We suggest that RNA silencing-mediated translation repression plays a strategic role in determining the viral set-point in a newly HIV-1-infected patient.

  20. Diversity of HIV-1 RNA and DNA in breast milk from HIV-1-infected mothers.

    Science.gov (United States)

    Becquart, Pierre; Courgnaud, Valerie; Willumsen, Juana; Van de Perre, Philippe

    2007-07-05

    We compared human immunodeficiency virus type 1 (HIV-1) RNA and DNA populations in the different fractions of breast milk (lactoserum, lipid layer, cell pellet) and between right and left breasts in four HIV-1-infected mothers by analyzing the hypervariable env C2-V5 region. Phylogenetic analyses of the viral quasispecies revealed that RNA populations and DNA populations were clearly distinct and that viral RNA sequences were similar in lipid layer and lactoserum in the milk of 3 out of 4 mothers. Comparison of viral DNA between milk from right and left breast showed a differential distribution of variants in three mothers. In contrast, RNA variants detected from milk of the two breasts were mixed in 3 out of 4 mothers. This study suggests that each mammary gland is subjected to microenvironmental pressure that may differ from the contralateral breast.

  1. Levels and patterns of HIV RNA viral load in untreated pregnant women

    DEFF Research Database (Denmark)

    NN, NN; Patel, Deven; Thorne, Claire;

    2008-01-01

    OBJECTIVE: To assess pregnancy levels and patterns of HIV RNA in the absence of antiretroviral therapy, while appropriately adjusting for potential confounders, including maternal immune status and race. METHODS: Data on > or = 1 antenatal HIV RNA measurements were available for 333 untreated HIV......-infected pregnant women enrolled in the European Collaborative Study. CD4 counts and HIV RNA measurements were routinely collected from 1992 and 1998, respectively. Linear mixed effects models based on 246 women for whom complete data were available examined changes in HIV RNA levels over pregnancy, with a nested...... random effects term accounting for measurement variability within women and period of sample collection. RESULTS: The change in HIV RNA over pregnancy varied significantly by race (p=0.005): from the second trimester until delivery, HIV RNA decreased significantly by an estimated 0.019 log(10) copies...

  2. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

    Directory of Open Access Journals (Sweden)

    Kimberly Bonner

    Full Text Available BACKGROUND: HIV viral load (VL testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures. METHODS AND FINDINGS: Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence, with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL environments. CONCLUSIONS: Whole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in

  3. Changes in HIV RNA and CD4 cell count after acute HCV infection in chronically HIV-infected individuals

    NARCIS (Netherlands)

    Gras, L.; Wolf, F. de; Smit, C.; Prins, M.; Meer, J.T. van der; Vanhommerig, J.W.; Zwinderman, A.H.; Schinkel, J.; Geskus, R.B.; Warris, A.

    2015-01-01

    OBJECTIVE: Little is known about the impact of acute hepatitis C virus (HCV) co-infection on HIV-1 disease progression. We investigated CD4 cell count and HIV RNA concentration changes after HCV infection in individuals chronically infected with HIV-1. METHODS: We selected individuals that had the l

  4. Cell-associated HIV DNA measured early during infection has prognostic value independent of serum HIV RNA measured concomitantly

    DEFF Research Database (Denmark)

    Katzenstein, Terese L; Oliveri, Roberto S; Benfield, Thomas

    2002-01-01

    Using data from the Danish AIDS Cohort of HIV-infected homosexual men established in the 1980s, the prognostic value of early HIV DNA loads was evaluated. In addition to DNA measurements, concomitant serum HIV RNA levels, CD4 cell counts and CCR5 genotypes were determined. The patients were divided...

  5. In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Judith Dalmau

    Full Text Available BACKGROUND: The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. METHODOLOGY/PRINCIPAL FINDINGS: We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. CONCLUSION: This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define

  6. Factors Associated With Plasma IL-6 Levels During HIV Infection

    DEFF Research Database (Denmark)

    Borges, Álvaro H; O'Connor, Jemma L; Phillips, Andrew N

    2015-01-01

    BACKGROUND: Elevated interleukin 6 (IL-6) levels have been linked to cardiovascular disease, cancer and death. Persons with human immunodeficiency virus (HIV) infection receiving treatment have higher IL-6 levels, but few data are available on factors associated with circulating IL-6. METHODS......: Participants in 3 trials with IL-6 measured at baseline were included (N = 9864). Factors associated with IL-6 were identified by linear regression. Demographic and HIV variables (nadir/entry CD4(+) cell count, HIV RNA level, antiretroviral therapy regimen) were investigated in all 3 trials. In the SMART...... education, whereas black race was associated with lower IL-6. Higher HIV RNA levels were associated with higher IL-6 levels, and higher nadir CD4(+) cell counts with lower IL-6 levels. Compared with efavirenz, protease inhibitors were associated with higher and nevirapine with lower IL-6 levels. Smoking...

  7. Novel Biochemical Tools for Probing HIV RNA Structure.

    Science.gov (United States)

    Rausch, Jason W; Sztuba-Solinska, Joanna; Lusvarghi, Sabrina; Le Grice, Stuart F J

    2016-01-01

    Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.

  8. Undetectable plasma viral load predicts normal survival in HIV-2-infected people in a West African village

    Directory of Open Access Journals (Sweden)

    Ricard Dominique

    2010-05-01

    Full Text Available Abstract Background There have been no previous studies of the long-term survival and temporal changes in plasma viral load among HIV-2 infected subjects. Methods 133 HIV-2 infected and 158 HIV-uninfected subjects from a rural area in North-west Guinea-Bissau, West Africa were enrolled into a prospective cohort study in 1991 and followed-up to mid-2009. Data were collected on four occasions during that period on HIV antibodies, CD4% and HIV-2 plasma viral load. Results Median age (interquartile range [IQR] of HIV-2 infected subjects at time of enrollment was 47 (36, 60 years, similar to that of HIV-uninfected control subjects, 49 (38, 62 (p = 0.4. Median (IQR plasma viral load and CD4 percentage were 347 (50, 4,300 copies/ml and 29 (22, 35 respectively. Overall loss to follow-up to assess vital status was small, at 6.7% and 6.3% for HIV-2 infected and uninfected subjects respectively. An additional 17 (12.8% and 16 (10.1% of HIV-2 infected and uninfected subjects respectively were censored during follow-up due to infection with HIV-1. The mortality rate per 100 person-years (95% CI was 4.5 (3.6, 5.8 among HIV-2 infected subjects compared to 2.1 (1.6, 2.9 among HIV-uninfected (age-sex adjusted rate ratio 1.9 (1.3, 2.8, p Viral load measurements were available for 98%, 78%, 77% and 61% HIV-2 infected subjects who were alive and had not become super-infected with HIV-1, in 1991, 1996, 2003 and 2006 respectively. Median plasma viral load (RNA copies per ml (IQR did not change significantly over time, being 150 (50, 1,554; n = 77 in 1996, 203 (50, 2,837; n = 47 in 2003 and 171 (50, 497; n = 31 in 2006. Thirty seven percent of HIV-2 subjects had undetectable viraemia ( Conclusions A substantial proportion of HIV-2 infected subjects in this cohort have stable plasma viral load, and those with an undetectable viral load (37% at study entry had a normal survival rate. However, the sequential laboratory findings need to be interpreted with caution given

  9. Antiretroviral-treated HIV-1 patients can harbour resistant viruses in CSF despite an undetectable viral load in plasma.

    Science.gov (United States)

    Soulie, Cathia; Grudé, Maxime; Descamps, Diane; Amiel, Corinne; Morand-Joubert, Laurence; Raymond, Stéphanie; Pallier, Coralie; Bellecave, Pantxika; Reigadas, Sandrine; Trabaud, Mary-Anne; Delaugerre, Constance; Montes, Brigitte; Barin, Francis; Ferré, Virginie; Jeulin, Hélène; Alloui, Chakib; Yerly, Sabine; Signori-Schmuck, Anne; Guigon, Aurélie; Fafi-Kremer, Samira; Haïm-Boukobza, Stéphanie; Mirand, Audrey; Maillard, Anne; Vallet, Sophie; Roussel, Catherine; Assoumou, Lambert; Calvez, Vincent; Flandre, Philippe; Marcelin, Anne-Geneviève

    2017-08-01

    HIV therapy reduces the CSF HIV RNA viral load (VL) and prevents disorders related to HIV encephalitis. However, these brain disorders may persist in some cases. A large population of antiretroviral-treated patients who had a VL > 1.7 log 10 copies/mL in CSF with detectable or undetectable VL in plasma associated with cognitive impairment was studied, in order to characterize discriminatory factors of these two patient populations. Blood and CSF samples were collected at the time of neurological disorders for 227 patients in 22 centres in France and 1 centre in Switzerland. Genotypic HIV resistance tests were performed on CSF. The genotypic susceptibility score was calculated according to the last Agence Nationale de Recherche sur le Sida et les hépatites virales Action Coordonnée 11 (ANRS AC11) genotype interpretation algorithm. Among the 227 studied patients with VL > 1.7 log 10 copies/mL in CSF, 195 had VL detectable in plasma [median (IQR) HIV RNA was 3.7 (2.7-4.7) log 10 copies/mL] and 32 had discordant VL in plasma (VL  1.7 log 10 copies/mL. Resistance to antiretrovirals was observed in CSF for the two groups of patients. Fourteen percent of this population of patients with cognitive impairment and detectable VL in CSF had well controlled VL in plasma. Thus, it is important to explore CSF HIV (VL and genotype) even if the HIV VL is controlled in plasma because HIV resistance may be observed.

  10. On the Selective Packaging of Genomic RNA by HIV-1

    Directory of Open Access Journals (Sweden)

    Mauricio Comas-Garcia

    2016-09-01

    Full Text Available Like other retroviruses, human immunodeficiency virus type 1 (HIV-1 selectively packages genomic RNA (gRNA during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1 region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

  11. Quantitation of HIV-1 RNA in breast milk by real time PCR.

    Science.gov (United States)

    Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe

    2006-04-01

    HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

  12. Plasma sphingolipids in HIV-associated chronic obstructive pulmonary disease

    Science.gov (United States)

    Hodgson, Shane; Griffin, Timothy J; Reilly, Cavan; Harvey, Stephen; Witthuhn, Bruce A; Sandri, Brian J; Wendt, Chris H

    2017-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity in persons living with HIV (PLWH) and HIV appears to uniquely cause COPD, independent of smoking. The mechanisms by which HIV leads to COPD are not clear. The objective of this study was to identify metabolomic biomarkers and potential mechanistic pathways of HIV-associated COPD (HIV-COPD). Methods We performed case–control metabolite profiling via mass spectrometry in plasma from 38 individuals with HIV-COPD (cases), comparing to matched controls with/without HIV and with/without COPD. Untargeted metabolites of interest were identified with liquid chromatography with mass spectrometry (LC-MS/mass spectrometry (MS)), and targeted metabolomics for tryptophan (Trp) and kynurenine (Kyn) were measured by selective reaction monitoring (SRM) with LC-MS/MS. We used mixed-effects models to compare metabolite concentrations in cases compared with controls while controlling for relevant biological variables. Results We identified 1689 analytes associated with HIV-COPD at a false discovery rate (FDR) of 10%. In PLWH, we identified 263 analytes (10% FDR) between those with and without COPD. LC MS/MS identified Trp and 17 lipids, including sphingolipids and diacylglycerol. After adjusting for relevant covariates, the Kyn/Trp ratio measured by SRM was significantly higher in PLWH (p=0.022), but was not associated with COPD status (p=0.95). Conclusions There is a unique metabolite profile in HIV-COPD that includes sphingolipids. Trp metabolism is increased in HIV, but does not appear to independently contribute to HIV-COPD. Trial registration numbers NCT01810289, NCT01797367, NCT00608764.

  13. siRNA, miRNA and HIV: promises and challenges

    Institute of Scientific and Technical Information of China (English)

    Man Lung YEUNG; Yamina BENNASSER; Shu Yun LE; Kuan Teh JEANG

    2005-01-01

    Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing.The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNAtargeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNAspecified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

  14. Impact of tuberculosis treatment on CD4 cell count, HIV RNA, and p24 antigen in patients with HIV and tuberculosis

    DEFF Research Database (Denmark)

    Wejse, Christian; Furtado, A.; Camara, C.

    2013-01-01

    To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment.......To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment....

  15. HCV-RNA水平对HIV/HCV合并感染者HIV疾病进展影响研究%Impact of HCV-RNA levels on HIV-1 disease progression in Chinese HIV/HCV co-infected individuals

    Institute of Scientific and Technical Information of China (English)

    刘静; 代娣; 丁海波; 尚红; 姜拥军; 张晻; 陈昕; 张子宁; 周立平; 范霞; 王亚男; 胡清海

    2008-01-01

    目的 探讨人免疫缺陷病毒(HIV)和丙型肝炎病毒(HCV)混合感染者HCV-RNA水平对HIV感染疾病进展的影响.方法 采用横断面研究对391例不同途径HIV感染者进行抗HCV-IgG、HCV-RNA、HIV-RNA、T淋巴细胞计数及其他免疫活化指标检测,比较HCV-RNA水平高组和低组病毒学及免疫学相关指标差别,分析HCV-RNA与HIV-RNA、CD4+T淋巴细胞计数的相关性.结果 (1)有偿供血组(93%)和静脉吸毒组(97.5%)抗HCV-IgG阳性率显著高于性接触组(20.1%);在抗HCV-IgG阳性的HIV感染者中,静脉吸毒组HCV-RNA阳性率(89.9%)显著高于有偿供血组(48.3%)及性接触组(62.5%),P均<0.01.(2) HCV-RNA水平和HIV-RNA水平正相关(r=0.237,P<0.01),与CD4计数负相关(r=-0.201,P<0.05).(3) HCV-RNA高组免疫活化标志物HLA-DR表达高于HCV-RNA低组(P<0.01).结论 高水平的HCV-RNA可能是HIV感染疾病进展的危险因素之一.%Objective To investigate the impact of hepatitis C virus (HCV)-RNA levels on human immunodeficiency virus (HIV)-1 disease progression in Chinese HIV/HCV co-infected individuals. Methods Cross-sectional analysis was performed among 391 HIV-infected patients for assessment of HCV-IgG, HCV-RNA, HIV-RNA, CD4 cell counts and cell surface markers of immune activation, to compare the difference of viral and immune indexes between HCV-RNA high group and HCV-RNA low group, and to elucidate the association between HCV-RNA, HIV-RNA and CD4 cell counts in HIV/HCV co-infected patients. Results (1) The percentage of anti HCV-IgG positive of former plasma donor group (93%) and drug-injection group (97.5%) were significantly higher than that of sexual transmission group (20.1%). The percentage of HCV-RNA positive of drug-injection group (89.9%) was significantly higher than that of former plasma donor group (48.3%) and sexual transmission group (62.5%), P<0.01, respectively. (2) HCV-RNA levels were positively correlated to HIV-RNA levels (r=0.237,P

  16. Probing interaction of a fluorescent ligand with HIV TAR RNA

    Science.gov (United States)

    Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

    2017-02-01

    Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

  17. Flow virometric sorting and analysis of HIV quasispecies from plasma

    Science.gov (United States)

    Jones, Jennifer C.; Keele, Brandon F.; Jenkins, Lisa M. Miller; Demberg, Thorsten

    2017-01-01

    Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens. PMID:28239654

  18. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Melissa A McAlexander

    2013-05-01

    Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

  19. RNA interference and HIV-1%RNA干扰和HIV-1

    Institute of Scientific and Technical Information of China (English)

    陈彬; 杨磊; 谭晓华

    2010-01-01

    RNA干扰(RNA interference,RNAi)是指由短双链RNA诱导的同源RNA降解过程或者是指诱导DNA甲基化的方式对其同源序列的基因表达进行干涉的过程.传统观念认为,这种现象发生在转录后水平又称为转录后基因沉默(post-transcriptional gene silence,PTGS).然而,最近研究表明,干扰小RNA(small interfering RNA,siRNA)是一些甲基化转移酶活化的起始信号,能在转录水平调控(沉默)基因的表达(transcriptional gene silence,TGS).该机制广泛存在于从酵母到哺乳动物的细胞中,能调节多种生物学的过程.新近的研究表明细胞和病毒miRNA(vmiRNA)都能调节病毒的复制;还有研究表明有些病毒,比如HIV-1,可以直接调控细胞内的RNA干扰的能力.RNA干扰有可能成为一种新的防治HIV-1感染的基因治疗方法,本文就RNA干扰作用机制以及在HIV-1感染方面的应用进行综述.

  20. DDX3 RNA helicase is required for HIV-1 Tat function.

    Science.gov (United States)

    Yasuda-Inoue, Mariko; Kuroki, Misao; Ariumi, Yasuo

    2013-11-22

    Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function.

  1. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

    Directory of Open Access Journals (Sweden)

    Valentina Vongrad

    Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

  2. Dynamics of breast milk HIV-1 RNA with unilateral mastitis or abscess

    Science.gov (United States)

    Semrau, Katherine; Kuhn, Louise; Brooks, Daniel R.; Cabral, Howard; Sinkala, Moses; Kankasa, Chipepo; Thea, Donald M.; Aldrovandi, Grace M.

    2013-01-01

    Background Mastitis and abscess in HIV-infected women increase risk of breastfeeding transmission of HIV. Guidelines encourage women to stop breastfeeding on the affected breast and feed on the contralateral breast. However, impact of breast pathology on breast milk HIV dynamics is unknown. Methods HIV RNA was quantified in 211 breast milk samples collected before, during and after a clinical mastitis or abscess diagnosis from 38 HIV-infected women participating in a Zambian breastfeeding study. HIV RNA quantity was compared between affected and unaffected breasts over time using generalized estimating equation models. A sample of 115 women without breast pathology was selected as a control group. Results In the affected breast, breast milk HIV RNA quantity increased from the pre- to during-pathology period by log10 0.45 copies/mL (95% CI: 0.16, 0.74) and after symptom resolution, HIV RNA levels were no different from pre-pathology levels (log10 -0.04 copies/mL 95%CI: -0.33, 0.25). In the contralateral unaffected breast, HIV RNA quantity did not significantly increase (log10 0.15 copies/mL, 95% CI: -0.41, 0.10). Increase was more marked in women with abscess or with a greater number of mastitis symptoms. HIV RNA was not significantly different between affected and unaffected women, except at the time of diagnosis. Conclusions Breast milk HIV RNA increased modestly in the affected breast with unilateral mastitis or abscess and returned to pre-pathology levels with symptom resolution. Contralateral HIV RNA was not affected. Results support guidelines encouraging feeding from the contralateral breast to minimize risk of HIV transmission associated with unilateral breast pathology. PMID:23202812

  3. HIV-1 Modulates the tRNA Pool to Improve Translation Efficiency

    Science.gov (United States)

    van Weringh, Anna; Ragonnet-Cronin, Manon; Pranckeviciene, Erinija; Pavon-Eternod, Mariana; Kleiman, Lawrence; Xia, Xuhua

    2011-01-01

    Despite its poorly adapted codon usage, HIV-1 replicates and is expressed extremely well in human host cells. HIV-1 has recently been shown to package non-lysyl transfer RNAs (tRNAs) in addition to the tRNALys needed for priming reverse transcription and integration of the HIV-1 genome. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding codons that are highly used by HIV-1 but avoided by its host are overrepresented in HIV-1 virions. In particular, tRNAs decoding A-ending codons, required for the expression of HIV's A-rich genome, are highly enriched. Because the affinity of Gag-Pol for all tRNAs is nonspecific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Codon usage of HIV-1 early genes is similar to that of highly expressed host genes, but codon usage of HIV-1 late genes was better adapted to the selectively enriched tRNA pool, suggesting that alterations in the tRNA pool are induced late in viral infection. If HIV-1 genes are adapting to an altered tRNA pool, codon adaptation of HIV-1 may be better than previously thought. PMID:21216840

  4. Structure and dynamics of the HIV-1 frameshift element RNA.

    Science.gov (United States)

    Low, Justin T; Garcia-Miranda, Pablo; Mouzakis, Kathryn D; Gorelick, Robert J; Butcher, Samuel E; Weeks, Kevin M

    2014-07-08

    The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.

  5. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

    OpenAIRE

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-01-01

    Experiments are presented which suggest that the binding of the primer tRNA to the primer binding site of the HIV-1 5′ UTR is involved in the dimerization of the genome, as part of the packaging process.

  6. Plasma HIV-1 tropism and risk of short-term clinical progression to AIDS or death

    Directory of Open Access Journals (Sweden)

    Maria Casadellà Fontdevila

    2014-11-01

    Full Text Available Introduction: It is uncertain if plasma HIV-1 tropism is an independent predictor of short-term risk of clinical progression / death, in addition to the CD4 count and HIV RNA level. We conducted a nested case-control study within EuroSIDA to assess this question amongst people with current HIV RNA level >1000 copies/mL, including both people on ART and those ART naïve. Methods: People with an AIDS diagnosis or who died from any causes for whom there was a stored plasma sample with HIV-1 RNA (VL≥1,000 copies/mL available in the time window of 3–12 months prior to the event were identified. At least one control was selected for each case matched for age, VL and HCV status at the time of sampling. Controls were event-free after a matched duration of time from the date of sampling. Plasma HIV tropism was estimated using 454 and population sequencing (PS. Non-R5 HIV was defined as: (a ≥2% of sequences with a Geno2Pheno (G2P FPR≤3.75% by 454, and (b a G2P FPR≤10% by PS. We also compared CD4 slopes over the 12 months following the date of sampling using a linear mixed model with random intercept according to HIV tropism and ART status. Results: The study included 266 subjects, 100 cases and 166 controls, with sample taken on average in 2006; 23% and 24% had non-R5 HIV by 454 and PS respectively. There were 19% women, 25% MSM, 92% Caucasians, 22% HCV+. At the time of sampling, 26% were ART-naïve, 25% had started but were off ART and 49% were receiving ART. The median age, CD4 and viral load was 41 years, 350 cells/mm3 and 4.81 log c/mL, respectively. Baseline characteristics were well balanced by tropism. Factors independently associated with clinical progression or death were female gender (OR=2.12; 95% CI=1.04, 4.36; p=0.038, CD4+ count (OR=0.90 per 100 cells/mm3 higher; 95% CI 0.80, 1.00; p=0.058, being on ART (OR=2.72; 95% CI 1.15, 6.41; p=0.022 and calendar year of sample (OR=0.84 per more recent year; 95% CI=0.77, 0.91; p<0

  7. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array

    Science.gov (United States)

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-beom; Choi, Heon-Jin

    2016-07-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  8. Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

    DEFF Research Database (Denmark)

    Chen, Jianbo; Grunwald, David; Sardo, Luca

    2014-01-01

    Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA...

  9. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    Science.gov (United States)

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  10. Comparing the MicroRNA spectrum between serum and plasma.

    Directory of Open Access Journals (Sweden)

    Kai Wang

    Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that regulate various biological processes, primarily through interaction with messenger RNAs. The levels of specific, circulating miRNAs in blood have been shown to associate with various pathological conditions including cancers. These miRNAs have great potential as biomarkers for various pathophysiological conditions. In this study we focused on different sample types' effects on the spectrum of circulating miRNA in blood. Using serum and corresponding plasma samples from the same individuals, we observed higher miRNA concentrations in serum samples compared to the corresponding plasma samples. The difference between serum and plasma miRNA concentration showed some associations with miRNA from platelets, which may indicate that the coagulation process may affect the spectrum of extracellular miRNA in blood. Several miRNAs also showed platform dependent variations in measurements. Our results suggest that there are a number of factors that might affect the measurement of circulating miRNA concentration. Caution must be taken when comparing miRNA data generated from different sample types or measurement platforms.

  11. Frequent intratype neutralization by plasma immunoglobulin a identified in HIV type 2 infection.

    Science.gov (United States)

    Özkaya Şahin, Gülşen; Månsson, Fredrik; Palm, Angelica A; Vincic, Elzbieta; da Silva, Zacarias; Medstrand, Patrik; Norrgren, Hans; Fenyö, Eva Maria; Jansson, Marianne

    2013-03-01

    Human immunodeficiency virus type 2 (HIV-2) is less transmissible and less pathogenic compared to HIV-1 and, when matched for CD4(+) T cell count, the plasma viral load in HIV-2-infected individuals is approximately one log lower than in HIV-1-infected individuals. The explanation for these observations is elusive, but differences in virus controlling immunity generated in the two infections may be contributing factors. In the present study, we investigated neutralization by immunoglobulin A (IgA), in parallel with IgG, purified from plasma of HIV-1, HIV-2, and HIV-1/HIV-2 dually (HIV-D) infected individuals. Neutralization was analyzed against HIV-1 and HIV-2 isolates using a plaque reduction assay. In HIV-2 infection, intratype-specific neutralization by IgA was frequently detected, although at a lesser magnitude then the corresponding IgG neutralizing titers. In contrast, neutralization by IgA could rarely be demonstrated in HIV-1 infection despite similar plasma IgA levels in both infections. In addition, IgA and IgG of HIV-D plasma neutralized the HIV-2 isolate more potently than the HIV-1 isolate, suggesting that the difference between neutralizing activity of plasma IgA and IgG depends on the virus itself. Taken together, these findings suggest that both IgA and IgG add to the potent intratype neutralizing activity detected in HIV-2 plasma, which may contribute to virus control in HIV-2 infection.

  12. Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

    Directory of Open Access Journals (Sweden)

    Devidas N Chaturbhuj

    Full Text Available OBJECTIVES: Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples. DESIGN: The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity. METHODS: The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88 with known viral loads ranges from 1000-1.8 million RNA copies/ml. RESULTS: Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$, thus making it cost effective. CONCLUSIONS: The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.

  13. Assessing an improved protocol for plasma microRNA extraction.

    Directory of Open Access Journals (Sweden)

    Inés Moret

    Full Text Available The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  14. Assessing an improved protocol for plasma microRNA extraction.

    Science.gov (United States)

    Moret, Inés; Sánchez-Izquierdo, Dolors; Iborra, Marisa; Tortosa, Luis; Navarro-Puche, Ana; Nos, Pilar; Cervera, José; Beltrán, Belén

    2013-01-01

    The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  15. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

    Science.gov (United States)

    Bonner, Kimberly; Siemieniuk, Reed A; Boozary, Andrew; Roberts, Teri; Fajardo, Emmanuel; Cohn, Jennifer

    2014-01-01

    HIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures. Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (PPT in field conditions are needed.

  16. Validation of the Gen-Probe Aptima qualitative HIV-1 RNA assay for diagnosis of human immunodeficiency virus infection in infants.

    Science.gov (United States)

    Fiscus, Susan A; McMillion, Takesha; Nelson, Julie A E; Miller, William C

    2013-12-01

    The qualitative Roche HIV-1 DNA Amplicor assay has been used for the past 20 years to diagnose HIV infection in infants and young children but is being phased out; hence, alternative assays must be found. The Gen-Probe Aptima qualitative HIV-1 RNA assay is currently the only FDA-cleared HIV-1 nucleic acid assay approved for diagnosis, but data on the use of this assay with infant plasma are limited. We assessed Aptima's performance using control material for reproducibility and limit of detection and 394 plasma samples (0.2 to 0.5 ml) from HIV-exposed infected and uninfected infants and children for analytical sensitivity and specificity. Assays to assess within-run repeatability and between-run reproducibility indicated that the controls with 10,000 (5 of 5), 200 (5 of 5), 100 (16 of 16), 50 (12 of 12), and 25 (20 of 20) HIV-1 RNA copies/ml (cp/ml) were always positive, and negatives were always negative (20 of 20). The limit of detection was 14 cp/ml, as determined by probit analysis. The analytic sensitivity of the assay was 99.5% (189/190 samples; 95% confidence interval [CI], 97.1 to 99.9%) and specificity was 99.5% (199/200 samples; 95% CI, 97.2 to 99.9%). These results suggest that the assay is suitable for early infant diagnosis of HIV-1.

  17. Strong epistatic selection on the RNA secondary structure of HIV.

    Directory of Open Access Journals (Sweden)

    Raquel Assis

    2014-09-01

    Full Text Available A key question in evolutionary genomics is how populations navigate the adaptive landscape in the presence of epistasis, or interactions among loci. This problem can be directly addressed by studying the evolution of RNA secondary structures, for which there is constraint to maintain pairing between Watson-Crick (WC sites. Replacement of a nucleotide at one site of a WC pair reduces fitness by disrupting binding, which can be restored via a compensatory replacement at the interacting site. Here, I present the first genome-scale analysis of epistasis on the RNA secondary structure of human immunodeficiency virus type 1 (HIV-1. Comparison of polymorphism frequencies at ancestrally conserved sites reveals that selection against replacements is ∼ 2.7 times stronger at WC than at non-WC sites, such that nearly 50% of constraint can be attributed to epistasis. However, almost all epistatic constraint is due to selection against conversions of WC pairs to unpaired (UP nucleotides, whereas conversions to GU wobbles are only slightly deleterious. This disparity is also evident in pairs with second-site compensatory replacements; conversions from UP nucleotides to WC pairs increase median fitness by ∼ 4.2%, whereas conversions from GU wobbles to WC pairs only increase median fitness by ∼ 0.3%. Moreover, second-site replacements that convert UP nucleotides to GU wobbles also increase median fitness by ∼ 4%, indicating that such replacements are nearly as compensatory as those that restore WC pairing. Thus, WC peaks of the HIV-1 epistatic adaptive landscape are connected by high GU ridges, enabling the viral population to rapidly explore distant peaks without traversing deep UP valleys.

  18. Effects of medical male circumcision (MC on plasma HIV viral load in HIV+ HAART naive men; Rakai, Uganda.

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    Godfrey Kigozi

    Full Text Available BACKGROUND: Medical male circumcision (MC of HIV-infected men may increase plasma HIV viral load and place female partners at risk of infection. We assessed the effect of MC on plasma HIV viral load in HIV-infected men in Rakai, Uganda. METHODS: 195 consenting HIV-positive, HAART naïve men aged 12 and above provided blood for plasma HIV viral load testing before surgery and weekly for six weeks and at 2 and 3 months post surgery. Data were also collected on baseline social demographic characteristics and CD4 counts. Change in log10 plasma viral load between baseline and follow-up visits was estimated using paired t tests and multivariate generalized estimating equation (GEE. RESULTS: Of the 195 men, 129 had a CD4 count ≧ 350 and 66 had CD4 <350 cells/mm3. Men with CD4 counts <350 had higher baseline mean log10 plasma viral load than those with CD4 counts ≧ 350 cells/mm3 (4.715 vs 4.217 cps/mL, respectively, p = 0.0005. Compared to baseline, there was no statistically significant increase in post-MC HIV plasma viral loads irrespective of CD4. Multivariate analysis showed that higher baseline log10 plasma viral load was significantly associated with reduction in mean log10 plasma viral load following MC (coef.  = -0.134, p<0.001. CONCLUSION: We observed no increase in plasma HIV viral load following MC in HIV-infected, HAART naïve men.

  19. Changes in HIV-1 Subtypes B and C Genital Tract RNA in Women and Men After Initiation of Antiretroviral Therapy

    Science.gov (United States)

    Fiscus, Susan A.; Cu-Uvin, Susan; Eshete, Abel Tilahun; Hughes, Michael D.; Bao, Yajing; Hosseinipour, Mina; Grinsztejn, Beatriz; Badal-Faesen, Sharlaa; Dragavon, Joan; Coombs, Robert W.; Braun, Ken; Moran, Laura; Hakim, James; Flanigan, Timothy; Kumarasamy, N.; Campbell, Thomas B.; Klingman, Karin L.; Nair, Apsara; Walawander, Ann; Smeaton, Laura M.; De Gruttola, Victor; Martinez, Ana I.; Swann, Edith; Barnett, Ronald L.; Brizz, Barbara; Delph, Yvette; Gettinger, Nikki; Mitsuyasu, Ronald T.; Eshleman, Susan; Safren, Steven; Andrade, Adriana; Haas, David W.; Amod, Farida; Berthaud, Vladimir; Bollinger, Robert C.; Bryson, Yvonne; Celentano, David; Chilongozi, David; Cohen, Myron; Collier, Ann C.; Currier, Judith Silverstein; Eron, Joseph; Firnhaber, Cynthia; Flexner, Charles; Gallant, Joel E.; Gulick, Roy M.; Hammer, Scott M.; Hoffman, Irving; Kazembe, Peter; Kumwenda, Johnstone; Kumwenda, Newton; Lama, Javier R.; Lawrence, Jody; Maponga, Chiedza; Martinson, Francis; Mayer, Kenneth; Nielsen, Karin; Pendame, Richard B.; Ramratnam, Bharat; Rooney, James F.; Sanchez, Jorge; Sanne, Ian; Schooley, Robert T.; Snowden, Wendy; Solomon, Suniti; Tabet, Steve; Taha, Taha; Uy, Jonathan; van der Horst, Charles; Wanke, Christine; Gormley, Joan; Marcus, Cheryl J.; Putnam, Beverly; Ntshele, Smanga; Loeliger, Edde; Pappa, Keith A.; Webb, Nancy; Shugarts, David L.; Winters, Mark A.; Descallar, Renard S.; Sharma, Jabin; Poongulali, S.; Cardoso, Sandra Wagner; Faria, Deise Lucia; Berendes, Sima; Burke, Kelly; Kanyama, Cecelia; Kayoyo, Virginia; Samaneka, Wadzanai P.; Chisada, Anthony; Santos, Breno; La Rosa, Alberto; Infante, Rosa; Balfour, Henry H.; Mullan, Beth; Kim, Ge-Youl; Klebert, Michael K.; Mildvan, Donna; Revuelta, Manuel; Jan Geiseler, P.; Santos, Bartolo; Daar, Eric S.; Lopez, Ruben; Frarey, Laurie; Currin, David; Haas, David H.; Bailey, Vicki L.; Tebas, Pablo; Zifchak, Larisa; Sha, Beverly E.; Fritsche, Janice M.

    2013-01-01

    Background. Combination antiretroviral therapy (cART) reduces genital tract human immunodeficiency virus type 1 (HIV-1) load and reduces the risk of sexual transmission, but little is known about the efficacy of cART for decreasing genital tract viral load (GTVL) and differences in sex or HIV-1 subtype. Methods. HIV-1 RNA from blood plasma, seminal plasma, or cervical wicks was quantified at baseline and at weeks 48 and 96 after entry in a randomized clinical trial of 3 cART regimens. Results. One hundred fifty-eight men and 170 women from 7 countries were studied (men: 55% subtype B and 45% subtype C; women: 24% subtype B and 76% subtype C). Despite similar baseline CD4+ cell counts and blood plasma viral loads, women with subtype C had the highest GTVL (median, 5.1 log10 copies/mL) compared to women with subtype B and men with subtype C or B (4.0, 4.0, and 3.8 log10 copies/mL, respectively; P < .001). The proportion of participants with a GTVL below the lower limit of quantification (LLQ) at week 48 (90%) and week 96 (90%) was increased compared to baseline (16%; P < .001 at both times). Women were significantly less likely to have GTVL below the LLQ compared to men (84% vs 94% at week 48, P = .006; 84% vs 97% at week 96, P = .002), despite a more sensitive assay for seminal plasma than for cervical wicks. No difference in GTVL response across the 3 cART regimens was detected. Conclusions. The female genital tract may serve as a reservoir of persistent HIV-1 replication during cART and affect the use of cART to prevent sexual and perinatal transmission of HIV-1. PMID:23532477

  20. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Directory of Open Access Journals (Sweden)

    Gregory Upert

    2012-01-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  1. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

    Science.gov (United States)

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S; Morris, Kevin V; Burnett, John C; Rossi, John J

    2015-03-19

    The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here, we combine the live-cell-based SELEX with high-throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as small interfering RNA [siRNA] delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5-expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4(+) T cells with a nanomolar inhibitory concentration 50%. G-3 was also capable of transferring functional siRNAs to CCR5-expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties.

  2. Viro-immunological characterization of naïve patients with high cerebrospinal fluid (CSF HIV RNA

    Directory of Open Access Journals (Sweden)

    Francesca Iannuzzi

    2014-11-01

    Full Text Available Background: HIV can spread into the central nervous system (CNS early in the course of infection and this turns into intrathecal inflammation and neuronal damage. We aimed to investigate clinical and immunological parameters associated with elevated CSF VL in HIV-infected ART-naïve patients. Materials and Methods: HIV+ ART-naïve patients underwent a comprehensive battery of neurocognitive (NC tests and lumbar puncture (LP for CSF HIV-RNA detection. Plasma HIV-RNA and peripheral T-cell immune-phenotypes (CD38/CD45RA/CD45R0/CD127 on CD4/CD8 were also assessed (flow cytometry. High-CSF HIV RNA was defined as≥10000cp/mL (H-CSF, while CSF HIV RNA10000 cp/mL.Table 1 shows the features of H- versus L-CSF patients. Compared to L-CSF patients, H-CSF patients displayed lower current CD4+%, lower CD4/CD8 ratio and higher CD8%. No differences in NC tests performance were observed between groups (p=0.6. Regarding T-cell immuno-phenotypes, H-CSF patients displayed a higher proportion of CD45R0+CD38+CD8+ (11 vs 7%, p=0.02 and lower expression of CD45RA+CD8+ % (16 vs 20%, p=0.007, in comparison to L-CSF patients. In multivariate analysis CD45RA+CD8+ T-cells % (OR 0.917, CI 95% 0.852–0.987, p=0.002 was associated with H-CSF, even after adjustment for plasma VL, CD8 and CD4 count. Globally, in univariate CSF VL inversely correlated with CD45RA+CD8+ % (r=−0.223, p=0.0217 and CD127+CD4+ % (r= −0.204, p= 0.0225, while a positive association was found between CSF and plasma VL (r=0.303, p=0.0004 and CD8 % (r=0.211, p=0.016. In multivariate linear regression, in addition to positive association between plasma and CSF VL (β: 0.212, 95% CI 0.02–0.41, p=0.032, also CD45RA+CD8+ % were confirmed inversely associated to CSF VL (β: 0.21, 95% CI −0.5 to −0.002, p=0.036, adjusting for CD4/CD8 and CD4CD127 %. Conclusions: We hereby describe a 32% prevalence of H-CSF in a cohort of HIV+ ART-naïve patients. Subjects with high-CSF viral replication are mostly

  3. Multiplexing seven miRNA-Based shRNAs to suppress HIV replication.

    Science.gov (United States)

    Choi, Jang-Gi; Bharaj, Preeti; Abraham, Sojan; Ma, Hongming; Yi, Guohua; Ye, Chunting; Dang, Ying; Manjunath, N; Wu, Haoquan; Shankar, Premlata

    2015-02-01

    Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

  4. The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

    Directory of Open Access Journals (Sweden)

    Kyle A. Nilson

    2011-01-01

    Full Text Available HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II, leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

  5. Selection and characterization of small molecules that bind the HIV-1 frameshift site RNA.

    Science.gov (United States)

    Marcheschi, Ryan J; Mouzakis, Kathryn D; Butcher, Samuel E

    2009-10-16

    HIV-1 requires a -1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of approximately 5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (K(d) = 2.8 microM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 degrees C and decreased translational frameshifting by 28% (+/-14%), as measured in vitro.

  6. Longitudinal serum HIV RNA quantification: correlation to viral phenotype at seroconversion and clinical outcome

    DEFF Research Database (Denmark)

    Katzenstein, T L; Pedersen, C; Nielsen, C

    1996-01-01

    OBJECTIVE: To investigate the longitudinal changes in serum HIV RNA, and to clarify whether the viral load early in infection has a predictive value for the clinical outcome; also, to correlate viral phenotype at seroconversion and changes in CD4 cell counts with viral burden. DESIGN: Twenty...... seroconverters with HIV isolates available at seroconversion had HIV RNA quantified by polymerase chain reaction (PCR) at seroconversion and thereafter every 6 months. Mean follow-up time was 65 months. Patients were classified according to viral phenotype at seroconversion, time to AIDS progression, serum viral....... Harbouring syncytium-inducing (SI) virus at seroconversion was associated with faster progression to AIDS than non-SI (NSI; P RNA. CONCLUSION: Serum HIV RNA is high around the time...

  7. Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm.

    Science.gov (United States)

    Chang, Ming; Wong, Audrey J S; Raugi, Dana N; Smith, Robert A; Seilie, Annette M; Ortega, Jose P; Bogusz, Kyle M; Sall, Fatima; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S; Coombs, Robert W

    2017-01-01

    The 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients' blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. To validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2-infected Senegalese study participants. We evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. All PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. Our TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time PCR for quantification of HIV-1 RNA on dried blood spots.

    Science.gov (United States)

    Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance

    2014-01-01

    Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

  9. Plasma lipidomic profiling of treated HIV-positive individuals and the implications for cardiovascular risk prediction.

    Science.gov (United States)

    Wong, Gerard; Trevillyan, Janine M; Fatou, Benoit; Cinel, Michelle; Weir, Jacquelyn M; Hoy, Jennifer F; Meikle, Peter J

    2014-01-01

    The increased risk of coronary artery disease in human immunodeficiency virus (HIV) positive patients is collectively contributed to by the human immunodeficiency virus and antiretroviral-associated dyslipidaemia. In this study, we investigate the characterisation of the plasma lipid profiles of treated HIV patients and the relationship of 316 plasma lipid species across multiple lipid classes with the risk of future cardiovascular events in HIV-positive patients. In a retrospective case-control study, we analysed plasma lipid profiles of 113 subjects. Cases (n = 23) were HIV-positive individuals with a stored blood sample available 12 months prior to their diagnosis of coronary artery disease (CAD). They were age and sex matched to HIV-positive individuals without a diagnosis of CAD (n = 45) and with healthy HIV-negative volunteers (n = 45). Association of plasma lipid species and classes with HIV infection and cardiovascular risk in HIV were determined. In multiple logistic regression, we identified 83 lipids species and 7 lipid classes significantly associated with HIV infection and a further identified 74 lipid species and 8 lipid classes significantly associated with future cardiovascular events in HIV-positive subjects. Risk prediction models incorporating lipid species attained an area under the receiver operator characteristic curve (AUC) of 0.78 (0.775, 0.785)) and outperformed all other tested markers and risk scores in the identification of HIV-positive subjects with increased risk of cardiovascular events. Our results demonstrate that HIV-positive patients have significant differences in their plasma lipid profiles compared with healthy HIV-negative controls and that numerous lipid species were significantly associated with elevated cardiovascular risk. This suggests a potential novel application for plasma lipids in cardiovascular risk screening of HIV-positive patients.

  10. Frequent Intratype Neutralization by Plasma Immunoglobulin A Identified in HIV Type 2 Infection

    OpenAIRE

    Özkaya Şahin, Gülşen; Månsson, Fredrik; Palm, Angelica A.; Vincic, Elzbieta; da Silva, Zacarias; Medstrand, Patrik; Norrgren, Hans; Fenyö, Eva Maria; Jansson, Marianne

    2013-01-01

    Human immunodeficiency virus type 2 (HIV-2) is less transmissible and less pathogenic compared to HIV-1 and, when matched for CD4+ T cell count, the plasma viral load in HIV-2-infected individuals is approximately one log lower than in HIV-1-infected individuals. The explanation for these observations is elusive, but differences in virus controlling immunity generated in the two infections may be contributing factors. In the present study, we investigated neutralization by immunoglobulin A (I...

  11. Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H.

    Directory of Open Access Journals (Sweden)

    Vanessa B Soros

    2007-02-01

    Full Text Available APOBEC3G (A3G is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older "mature" A3G already residing in high-molecular-mass RNA-protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.

  12. Crystal methamphetamine injection predicts slower HIV RNA suppression among injection drug users.

    Science.gov (United States)

    Fairbairn, Nadia; Kerr, Thomas; Milloy, M-J; Zhang, Ruth; Montaner, Julio; Wood, Evan

    2011-07-01

    We examined the impact of crystal methamphetamine injection on HIV RNA suppression among a prospective cohort of HIV-positive injection drug users initiating antiretroviral therapy. A multivariate Cox regression analysis found crystal methamphetamine injection to be negatively associated with viral load suppression (RH=0.63 [95% CI: 0.40-0.98]; p=0.039). This study is the first to our knowledge to demonstrate an association between crystal methamphetamine use and HIV RNA suppression. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Efavirenz Plasma Concentrations and HIV Viral Load in HIV/AIDS-tuberculosis Infection Patients Treated with Rifampicin.

    Science.gov (United States)

    Mariana, Nina; Purwantyastuti; Instiaty; Rusli, Adria

    2016-01-01

    to determine the effect of a rifampicin-containing tuberculosis regimen on efavirenz plasma concentrations and viral load in HIV/AIDS-Tuberculosis infection patients who received efavirenz-based antiretroviral therapy. plasma efavirenz concentrations and HIV viral load were measured in HIV/AIDS patients treated with 600 mg efavirenz-based antiretroviral for 3 to 6 months and in HIV/AIDS-Tuberculosis infection patients treated with similar antiretroviral regimen plus rifampicin-containing antituberculosis in Sulianti Saroso Infectious disease Hospital, Jakarta. Plasma efavirenz concentration in both groups were compared using Mann-Whitney test, while proportion of patients with viral load >40 copy/mL were analyzed with chi-square test. forty five patients (27 with HIV/AIDS and 18 with HIV/AIDS-Tuberculosis infections) were recruited during the period of February to May 2015. The median efavirenz plasma concentration obtained from HIV/AIDS group was 0,680 mg/L(range 0,24 to 5,67 mg/L and that obtained from HIV/AIDS-Tuberculosis group was 0.685 mg/L (0.12 -2.23 mg/L) which was not significantly different statistically. The proportion of patients with viral load 40 copies/mL after 3-6 months of ARV treatment in the HIV/AIDS group was 51.9%, and in the HIV/AIDS-Tuberculosis group was 72.2%, which was not significantly different statistically (Chi Square test, p=0.291). plasma efavirenz concentration in HIV/AIDS-tuberculosis patients receiving antiretroviral and rifampicin is not significantly different from that on HIV/AIDS patients without tuberculosis. Proportion of patients with viral load of >40 copy/mL is higher in HIV/AIDS-tuberculosis patients receiving rifampicin compared to HIV/AIDS patients that not receive rifampicin. However, this difference did not reach statistical significance. Confirmatory studies with bigger sample size are needed to clarify the influence of rifampicin on plasma level of efavirenzand and on viral load.

  14. Plasma levels of soluble CD27: a simple marker to monitor immune activation during potent antiretroviral therapy in HIV-1-infected subjects

    Science.gov (United States)

    DE MILITO, A; ALEMAN, S; MARENZI, R; SÖNNERBORG, A; FUCHS, D; ZAZZI, M; CHIODI, F

    2002-01-01

    Plasma levels of soluble CD27 (sCD27) are elevated in diseases characterized by T cell activation and are used as a marker of immune activation. We assessed the usefulness of determining plasma sCD27 as a marker for monitoring immune activation in HIV-1-infected patients treated with highly active antiretroviral therapy (HAART). A first cross-sectional examination of 68 HIV-1-infected and 18 normal subjects showed high levels of sCD27 in HIV-1 infection; plasma sCD27 was correlated to HIV-1 viraemia and inversely correlated to CD4+ T cell count. Twenty-six HIV-1-infected patients undergoing HAART were studied at baseline and after 6, 12, 18 and 24 months of therapy. Seven additional patients under HAART were analysed at baseline, during and after interruption of therapy. In the total population, HAART induced a significant and progressive reduction, but not a normalization, of plasma levels of sCD27 after 24 months. A full normalization of plasma sCD27 was observed in the virological responders (undetectable HIV-1 RNA at months 18 and 24) and also in patients with moderate immunodeficiency at baseline (CD4+ T cell count >200 cells/mm3). Changes in plasma neopterin paralleled the changes in sCD27 but only baseline sCD27 levels were predictive of a greater increase in CD4+ T cell count during the follow-up. Discontinuation of therapy resulted in a rapid increase of sCD27 plasma levels associated with viraemia rebound and drop in CD4+ T cell count. Our findings suggest that plasma sCD27 may represent an alternative and simple marker to monitor immune activation during potent antiretroviral therapy. HIV-1-induced immune activation can be normalized by HAART in successfully treated patients where the disease is not advanced. PMID:11966765

  15. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging.

    Science.gov (United States)

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-10-01

    The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

  16. Adenosine deaminase acting on RNA-1 (ADAR1 inhibits HIV-1 replication in human alveolar macrophages.

    Directory of Open Access Journals (Sweden)

    Michael D Weiden

    Full Text Available While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1 in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

  17. Modulation of innate and adaptive cellular immunity relevant to HIV-1 vaccine design by seminal plasma.

    Science.gov (United States)

    Selva, Kevin J; Kent, Stephen J; Parsons, Matthew S

    2017-01-28

    Mucosal exposure to HIV-1 infection generally occurs in the presence of semen. Immunomodulation by seminal plasma is well described in the reproductive biology literature. Little is known, however, about the impact of seminal plasma on innate and adaptive anti-HIV-1 cellular immunity. The study investigated the effects of seminal plasma on immune responses considered important for prophylactic HIV-1 vaccine development, namely innate and adaptive cellular immunity mediated by natural killer (NK) cells and T cells, respectively. The ability of seminal plasma to modulate direct, antibody-dependent and cytokine-stimulated NK cell activation was assessed utilizing intracellular cytokine staining. Direct and antibody-dependent cellular cytotoxicity was assessed using lactate dehydrogenase release assays. The effects of seminal plasma on T-cell activation upon stimulation with staphylococcus enterotoxin B or HIV-1 Gag peptides were assessed by intracellular cytokine staining. The impact of seminal plasma on redirected cytolysis mediated by T cells was measured using lactate dehydrogenase release assays. Both direct and antibody-dependent NK cell activation were dramatically impaired by the presence of either HIV-1-uninfected or HIV-1-infected seminal plasma in a dose-dependent manner. Additionally, seminal plasma suppressed both direct and antibody-dependent NK cell-mediated cytolysis, including anti-HIV-1 antibody-dependent cytolysis of gp120-pulsed CEM.NKr-CCR5 cells. Finally, seminal plasma attenuated both HIV-1 Gag-specific and staphylococcus enterotoxin B-induced CTL activation. Semen contains potent immunosuppressors of both NK cell and CD8 T-cell-mediated anti-HIV-1 immune responses. This could impede attempts to provide vaccine-induced immunity to HIV-1.

  18. The silent defense: Micro-RNA directed defense against HIV-1 replication

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    Kumar Ajit

    2007-04-01

    Full Text Available Abstract MicroRNAs play critical role in regulating gene expression. MicroRNA profile of particular cell type bears the signature of cell type specific gene expression. Given that viral pathogens replicate by evading host defenses, research is now focused on the miRNA-regulated genes that critically regulate HIV-1 propagation in human host cells.

  19. HIV-1 Continues To Replicate and Evolve in Patients with Natural Control of HIV Infection

    DEFF Research Database (Denmark)

    Mens, Helene; Kearney, Mary; Wiegand, Ann

    2010-01-01

    Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical...

  20. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    Energy Technology Data Exchange (ETDEWEB)

    López, Claudia S., E-mail: lopezcl@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Sloan, Rachel; Cylinder, Isabel [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Kozak, Susan L.; Kabat, David [Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Barklis, Eric, E-mail: barklis@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States)

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  1. Plasma Selenium Concentrations Are Sufficient and Associated with Protease Inhibitor Use in Treated HIV-Infected Adults123

    Science.gov (United States)

    Hileman, Corrilynn O; Dirajlal-Fargo, Sahera; Lam, Suet Kam; Kumar, Jessica; Lacher, Craig; Combs, Gerald F; McComsey, Grace A

    2015-01-01

    Background: Selenium is an essential constituent of selenoproteins, which play a substantial role in antioxidant defense and inflammatory cascades. Selenium deficiency is associated with disease states characterized by inflammation, including cardiovascular disease (CVD). Although HIV infection has been associated with low selenium, the role of selenium status in HIV-related CVD is unclear. Objectives: We sought to assess associations between plasma selenium and markers of inflammation, immune activation, and subclinical vascular disease in HIV-infected adults on contemporary antiretroviral therapy (ART) and to determine if statin therapy modifies selenium status. Methods: In the Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN trial, HIV-infected adults on stable ART were randomly assigned 1:1 to rosuvastatin or placebo. Plasma selenium concentrations were determined at entry, week 24, and week 48. Spearman correlation and linear regression analyses were used to assess relations between baseline selenium, HIV-related factors and markers of inflammation, immune activation, and subclinical vascular disease. Changes in selenium over 24 and 48 wk were compared between groups. Results: One hundred forty-seven HIV-infected adults were included. All participants were on ART. Median current CD4+ count was 613, and 76% had HIV-1 RNA ≤48 copies/mL (range: contemporary ART. The association between current PI use and higher selenium may have implications for ART allocation, especially in resource-limited countries. Also, it appears that statin therapy may increase selenium concentrations; however, larger studies are necessary to confirm this finding. This trial was registered at clinicaltrials.gov as NCT01218802. PMID:26269240

  2. Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

    Science.gov (United States)

    Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

    2010-04-01

    Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

  3. Kinetics of HIV-1 in cerebrospinal fluid and plasma in cryptococcal meningitis

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    Jorge A. Benetucci

    2012-04-01

    Full Text Available In order to determine HIV-1 kinetics in cerebrospinal fluid (CSF and plasma in patients with cryptococcal meningitis (CM, we undertook a prospective collection of paired CSF/plasma samples from antiretroviral therapy- free HIV-infected patients with CM. Samples were obtained at baseline (S1 and at the second (S2 and third (S3 weeks of antifungal therapy. HIV-1 CSF concentrations were significantly lower in both S2 and S3 with respect to S1. Plasma concentrations remained stable. HIV-1 concentrations were higher in plasma than CSF in all cases. Patients who survived the episode of CM (but not those who died showed a decrease in CSF viral load, what suggests different viral kinetics of HIV-1 in the CSF according to the clinical course of this opportunistic disease.

  4. Kinetics of HIV-1 in cerebrospinal fluid and plasma in cryptococcal meningitis

    Science.gov (United States)

    Cecchini, Diego M.; Cañizal, Ana M.; Rojas, Haroldo; Arechavala, Alicia; Negroni, Ricardo; Bouzas, María B.; Benetucci, Jorge A.

    2012-01-01

    In order to determine HIV-1 kinetics in cerebrospinal fluid (CSF) and plasma in patients with cryptococcal meningitis (CM), we undertook a prospective collection of paired CSF/plasma samples from antiretroviral therapy-free HIV-infected patients with CM. Samples were obtained at baseline (S1) and at the second (S2) and third (S3) weeks of antifungal therapy. HIV-1 CSF concentrations were significantly lower in both S2 and S3 with respect to S1. Plasma concentrations remained stable. HIV-1 concentrations were higher in plasma than CSF in all cases. Patients who survived the episode of CM (but not those who died) showed a decrease in CSF viral load, what suggests different viral kinetics of HIV-1 in the CSF according to the clinical course of this opportunistic disease. PMID:24470944

  5. Kinetics of HIV-1 in cerebrospinal fluid and plasma in cryptococcal meningitis.

    Science.gov (United States)

    Cecchini, Diego M; Cañizal, Ana M; Rojas, Haroldo; Arechavala, Alicia; Negroni, Ricardo; Bouzas, María B; Benetucci, Jorge A

    2012-04-27

    In order to determine HIV-1 kinetics in cerebrospinal fluid (CSF) and plasma in patients with cryptococcal meningitis (CM), we undertook a prospective collection of paired CSF/plasma samples from antiretroviral therapy-free HIV-infected patients with CM. Samples were obtained at baseline (S1) and at the second (S2) and third (S3) weeks of antifungal therapy. HIV-1 CSF concentrations were significantly lower in both S2 and S3 with respect to S1. Plasma concentrations remained stable. HIV-1 concentrations were higher in plasma than CSF in all cases. Patients who survived the episode of CM (but not those who died) showed a decrease in CSF viral load, what suggests different viral kinetics of HIV-1 in the CSF according to the clinical course of this opportunistic disease.

  6. In vivo SELEX of single-stranded domains in the HIV-1 leader RNA.

    Science.gov (United States)

    van Bel, Nikki; Das, Atze T; Berkhout, Ben

    2014-02-01

    The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two

  7. Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct

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    Zhang Tao

    2012-06-01

    Full Text Available Abstract Background RNA interference (RNAi has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1 replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study. Results An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed. Conclusions The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.

  8. Predictive factors of plasma HIV suppression during pregnancy: a prospective cohort study in Benin.

    Directory of Open Access Journals (Sweden)

    Lise Denoeud-Ndam

    Full Text Available OBJECTIVE: To investigate the factors associated with HIV1 RNA plasma viral load (pVL below 40 copies/mL at the third trimester of pregnancy, as part of prevention of mother-to-child transmission (PMTCT in Benin. DESIGN: Sub study of the PACOME clinical trial of malaria prophylaxis in HIV-infected pregnant women, conducted before and after the implementation of the WHO 2009 revised guidelines for PMTCT. METHODS: HIV-infected women were enrolled in the second trimester of pregnancy. Socio-economic characteristics, HIV history, clinical and biological characteristics were recorded. Malaria prevention and PMTCT involving antiretroviral therapy (ART for mothers and infants were provided. Logistic regression helped identifying factors associated with virologic suppression at the end of pregnancy. RESULTS: Overall 217 third trimester pVLs were available, and 71% showed undetectability. Virologic suppression was more frequent in women enrolled after the change in PMTCT recommendations, advising to start ART at 14 weeks instead of 28 weeks of pregnancy. In multivariate analysis, Fon ethnic group (the predominant ethnic group in the study area, regular job, first and second pregnancy, higher baseline pVL and impaired adherence to ART were negative factors whereas higher weight, higher antenatal care attendance and longer ART duration were favorable factors to achieve virologic suppression. CONCLUSIONS: This study provides more evidence that ART has to be initiated before the last trimester of pregnancy to achieve an undetectable pVL before delivery. In Benin, new recommendations supporting early initiation were well implemented and, together with a high antenatal care attendance, led to high rate of virologic control.

  9. PEGylated poly(ethylene imine) copolymer-delivered siRNA inhibits HIV replication in vitro.

    Science.gov (United States)

    Weber, Nick D; Merkel, Olivia M; Kissel, Thomas; Muñoz-Fernández, María Ángeles

    2012-01-10

    RNA interference is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including targeting viral infections such as HIV. T lymphocytes, the primary target for HIV, are very difficult to treat with gene therapy applications such as RNA interference because of issues with drug delivery. To circumvent these problems, we investigated poly(ethylene imine) (PEI) as a method of improving transfection efficiency of siRNA to T lymphocytes. Additionally, polyethylene glycol (PEG) moieties were engrafted to the PEI polymers with the goals of improving stability and reducing cytotoxicity. Initial studies on PEG-PEI/siRNA polyplex formation, size and their interaction with cell membranes demonstrated their feasibility as drug delivery agents. Assays with lymphocytes revealed low cytotoxicity profiles of the polyplexes at pharmacologically relevant concentrations with PEGylated copolymers obtaining the best results. Successful transfection of a T cell line or primary T cells with siRNA was observed via flow cytometry and confocal microscopy. Finally, the biological effect of copolymer-delivered siRNA was measured. Of particular significance, siRNA targeted to the HIV gene nef and delivered by one of the PEG-PEI copolymers in repetitive treatments every 2-3 days was observed to inhibit HIV replication to the same extent as azidothymidine over the course of 15 days. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. RNA structures facilitate recombination-mediated gene swapping in HIV-1.

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P; Weeks, Kevin M; Negroni, Matteo

    2010-12-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.

  11. RNA Structures Facilitate Recombination-Mediated Gene Swapping in HIV-1 ▿

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P.; Weeks, Kevin M.; Negroni, Matteo

    2010-01-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny. PMID:20881047

  12. A Comprehensive Analysis of the Impact of HIV on HCV Immune Responses and Its Association with Liver Disease Progression in a Unique Plasma Donor Cohort

    Science.gov (United States)

    Rajapaksa, Ushani S.; Lawrence, Tessa M.; Peng, Yan-Chun; Liu, Jinghua; Xu, Keyi; Hu, Ke; Qin, Ling; Liu, Ning; Sun, Huanqin; Yan, Hui-Ping; Repapi, Emmanouela; Rowland-Jones, Sarah; Thimme, Robert; McKeating, Jane A.; Dong, Tao

    2016-01-01

    Objective Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV) co-infection is recognized as a major cause of morbidity and mortality among HIV-1 infected patients. Our understanding of the impact of HIV infection on HCV specific immune responses and liver disease outcome is limited by the heterogeneous study populations with genetically diverse infecting viruses, varying duration of infection and anti-viral treatment. Methods Viral-specific immune responses in a cohort of 151 HCV mono- and HIV co-infected former plasma donors infected with a narrow source of virus were studied. HCV and HIV specific T cell responses were correlated with clinical data. Results HIV-1 accelerated liver disease progression and decreased HCV specific T cell immunity. The magnitude of HCV specific T cell responses inversely correlated with lower HCV RNA load and reduced liver injury as assessed by non-invasive markers of liver fibrosis. HIV co-infection reduced the frequency of HCV specific CD4+ T cells with no detectable effect on CD8+ T cells or neutralizing antibody levels. Conclusion Our study highlights the impact of HIV co-infection on HCV specific CD4+ T cell responses in a unique cohort of patients for both HCV and HIV and suggests a crucial role for these cells in controlling chronic HCV replication and liver disease progression. PMID:27455208

  13. The control of HIV transcription: keeping RNA polymerase II on track.

    Science.gov (United States)

    Ott, Melanie; Geyer, Matthias; Zhou, Qiang

    2011-11-17

    Thirteen years ago, human cyclin T1 was identified as part of the positive transcription elongation factor b (P-TEFb) and the long-sought host cofactor for the HIV-1 transactivator Tat. Recent years have brought new insights into the intricate regulation of P-TEFb function and its relationship with Tat, revealing novel mechanisms for controlling HIV transcription and fueling new efforts to overcome the barrier of transcriptional latency in eradicating HIV. Moreover, the improved understanding of HIV and Tat forms a basis for studying transcription elongation control in general. Here, we review advances in HIV transcription research with a focus on the growing family of cellular P-TEFb complexes, structural insights into the interactions between Tat, P-TEFb, and TAR RNA, and the multifaceted regulation of these interactions by posttranscriptional modifications of Tat. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging.

    Science.gov (United States)

    Olson, Erik D; Cantara, William A; Musier-Forsyth, Karin

    2015-08-24

    Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

  15. New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging

    Directory of Open Access Journals (Sweden)

    Erik D. Olson

    2015-08-01

    Full Text Available Two copies of unspliced human immunodeficiency virus (HIV-1 genomic RNA (gRNA are preferentially selected for packaging by the group-specific antigen (Gag polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

  16. HIV/HCV混合感染对HIV RNA及T淋巴细胞亚群的影响%Impact of HIV/HCV coinfection on HIV RNA and T-lymphocyte subsets

    Institute of Scientific and Technical Information of China (English)

    王海斌; 游晶; 刘娜

    2013-01-01

    目的:探讨HIV/HCV混合感染对HIV RNA及T淋巴细胞亚群的影响.方法:采用荧光定量PCR技术和流式细胞技术分别检测HIV单纯感染与HIV/HCV混合感染者的HIV、HCV病毒载量及T淋巴细胞亚群,分析HIV RNA、T淋巴细胞亚群与HCV RNA的相关性.结果:(1)HIV单纯感染组与HIV/HCV混合感染组间的HIV RNA (log10 copies/mL)、CD4+、CD8+差异有统计学意义(P<0.05).(2)HIV/HCV混合感染组中HIV RNA(log10 copies/mL)与HCV RNA (log10 copies/mL)之间存在正相关关系(r=0.365,P< 0.05).(3)HIV单纯感染组及HIV/HCV混合感染组中HIV RNA (log10copies/mL)与CD4+、CD4+/CD8+之间均存在明显的负相关关系(r=-0.421、-0.533、-0.534、-0.408,P<0.01).结论:HIV/HCV混合感染很可能加速了HW RNA的复制,进而因HIV RNA复制的增长加速了对T淋巴细胞亚群的损害.

  17. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression

    Directory of Open Access Journals (Sweden)

    Meiri Eti

    2009-02-01

    Full Text Available Abstract Background RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression. Results Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well. Conclusion The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

  18. Comparative expression profile of miRNA and mRNA in primary peripheral blood mononuclear cells infected with human immunodeficiency virus (HIV-1.

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    Ankit Gupta

    Full Text Available Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.

  19. Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

    Science.gov (United States)

    Kutky, Meherzad; Hudak, Katalin A

    2017-09-01

    HIV-1 transcription is primarily controlled by the virally encoded Tat protein and its interaction with the viral TAR RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In this study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant ( Phytolacca americana ), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1, NF-κB and SP-1, which modulate HIV-1 transcription by binding to the viral LTR, were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein. ©2017 The Author(s).

  20. Interaction of drugs of abuse and microRNA with HIV: a brief review

    Directory of Open Access Journals (Sweden)

    Sudheesh ePilakka-Kanthikeel

    2015-09-01

    Full Text Available MicroRNAs (miRNAs, the post-transcriptional regulators of gene expression, play key roles in modulating many cellular processes. The changes in the expression profiles of several specific miRNAs affect the interactions between miRNA and their targets in various illnesses, including addiction, HIV, cancer etc. The presence of anti-HIV-1 microRNAs (which regulate the level of infectivity of HIV-1 have been validated in the cells which are the primary targets of HIV infection. Drugs of abuse impair the intracellular innate anti-HIV mechanism(s in monocytes, contributing to cell susceptibility to HIV infection. Emerging evidence has implicated miRNAs are differentially expressed in response to chronic morphine treatment. Activation of mu opioid receptors (MOR by morphine is shown to down regulate the expression of anti-HIV miRNAs. In this review, we summarize the results which demonstrate that several drugs of abuse related miRNAs have roles in the mechanisms that define addiction, and how they interact with HIV.

  1. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

    Directory of Open Access Journals (Sweden)

    Das Atze T

    2012-07-01

    Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

  2. Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function.

    Science.gov (United States)

    Yedavalli, Venkat S R K; Neuveut, Christine; Chi, Ya-Hui; Kleiman, Lawrence; Jeang, Kuan-Teh

    2004-10-29

    A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins. Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts. Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA. Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3. We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores. Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs. Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.

  3. Stress and coping in HIV-positive former plasma/blood donors in China: a test of cognitive appraisal theory.

    Science.gov (United States)

    Meade, Christina S; Wang, Jianping; Lin, Xiuyun; Wu, Hao; Poppen, Paul J

    2010-04-01

    Throughout the 1990s, many villagers in rural China were infected with HIV through commercial plasma/blood donation. These former plasma/blood donors (FPDs) experienced many HIV-related stressors. This study tested a cognitive appraisal model of stress and coping in a sample of HIV-positive adult FPDs. Participants (N = 207) from multiple villages completed a battery of questionnaires assessing HIV-related stress, HIV symptoms, cognitive appraisal, coping behaviors, and psychological distress. Participants reported high levels of HIV-related stress, depression, and anxiety. In a structural equation model, greater HIV-related stress, HIV symptoms, and threat appraisal were directly associated with psychological distress. HIV-related stress was also indirectly associated with psychological distress through threat appraisal. In a second model, coping was found to mediate the relationship between challenge appraisal and psychological distress. Results support the utility of cognitive appraisal theory. Stress management interventions targeting HIV-positive FPDs in China are indicated.

  4. Lipidomic dataset of plasma from patients infected with wild type and nef-deficient HIV-1 strain

    Directory of Open Access Journals (Sweden)

    Peter Meikle

    2016-03-01

    Full Text Available Previous in vitro and in vivo studies demonstrated that HIV protein nef plays a key role in impairing cellular and systemic cholesterol metabolism in HIV disease, but clinical support for these findings is lacking. Here we present the data of comparative lipidomic analysis (330 lipid species of plasma samples from HIV-negative subjects, patients infected with WT HIV-1 strain and patients infected with nef-deficient strain of HIV-1. We determine which effects of HIV on plasma lipidome are explained by the presence of nef. The data can be used to evaluate cardiovascular risk in HIV disease and to assess the role of nef in HIV-induced disturbances in systemic lipid metabolism. The full impact of nef deficiency on lipid and lipoprotein metabolism in HIV-infected patients is presented in the accompanying study “Lipid Metabolism in Patients Infected with Nef-deficient HIV-1 Strain” [1].

  5. A lateral flow assay for quantitative detection of amplified HIV-1 RNA.

    Directory of Open Access Journals (Sweden)

    Brittany A Rohrman

    Full Text Available Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA, this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings.

  6. Multiple shRNA combinations for near-complete coverage of all HIV-1 strains

    Directory of Open Access Journals (Sweden)

    Tran Anna

    2011-01-01

    Full Text Available Abstract Background Combinatorial RNA interference (co-RNAi approaches are needed to account for viral variability in treating HIV-1 with RNAi, as single short hairpin RNAs (shRNA are rapidly rendered ineffective by resistant strains. Current work suggests that 4 simultaneously expressed shRNAs may prevent the emergence of resistant strains. Results In this study we assembled combinations of highly-conserved shRNAs to target as many HIV-1 strains as possible. We analyzed intersecting conservations of 10 shRNAs to find combinations with 4+ matching the maximum number of strains using 1220+ HIV-1 sequences from the Los Alamos National Laboratory (LANL. We built 26 combinations of 2 to 7 shRNAs with up to 87% coverage for all known strains and 100% coverage of clade B subtypes, and characterized their intrinsic suppressive activities in transient expression assays. We found that all combinations had high combined suppressive activities, though there were also large changes in the individual activities of the component shRNAs in our multiple expression cassette configurations. Conclusion By considering the intersecting conservations of shRNA combinations we have shown that it is possible to assemble combinations of 6 and 7 highly active, highly conserved shRNAs such that there is always at least 4 shRNAs within each combination covering all currently known variants of entire HIV-1 subtypes. By extension, it may be possible to combine several combinations for complete global coverage of HIV-1 variants.

  7. Acquisition of HIV-1 resistance in T lymphocytes using an ACA-specific E. coli mRNA interferase.

    Science.gov (United States)

    Chono, Hideto; Matsumoto, Kazuya; Tsuda, Hiroshi; Saito, Naoki; Lee, Karim; Kim, Sujeong; Shibata, Hiroaki; Ageyama, Naohide; Terao, Keiji; Yasutomi, Yasuhiro; Mineno, Junichi; Kim, Sunyoung; Inouye, Masayori; Kato, Ikunoshin

    2011-01-01

    Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.

  8. RNA interactions in the 5' region of the HIV-1 genome

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Andersen, Ebbe Sloth; Knudsen, Bjarne

    2004-01-01

    a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we...

  9. Ebola Virus RNA in Semen from an HIV-Positive Survivor of Ebola

    Science.gov (United States)

    Rogers, Emerson; Baller, April; White, Stephen; Soka, Moses; Choi, Mary J.; Mahmoud, Nuha; Wasunna, Christine; Massaquoi, Moses; Kollie, Jomah; Dweh, Straker; Bemah, Philip; Ladele, Victor; Kpaka, Jonathan; Jawara, Mary; Mugisha, Margaret; Subah, Onyekachi; Faikai, Mylene; Bailey, Jeff A.; Rollin, Pierre; Marston, Barbara; Nyenswah, Tolbert; Gasasira, Alex; Knust, Barbara; Nichol, Stuart; Williams, Desmond

    2017-01-01

    Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD. PMID:28287374

  10. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    DEFF Research Database (Denmark)

    Sükösd, Zsuzsanna; Andersen, Ebbe Sloth; Seemann, Ernst Stefan;

    2015-01-01

    of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping...

  11. A new approach for sequencing virion genome of Chinese HIV-1 strains subtype B and BC from plasma

    Institute of Scientific and Technical Information of China (English)

    MENG Zhe-feng; ZHANG Xiao-yan; XIN Ruo-lei; XING Hui; HE Xiang; XU Jian-qing; SHAO Yi-ming

    2011-01-01

    Background Although it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate,to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide.Up to date,no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments.In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.Methods The methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs.Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains.Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains,a three-amplicon strategy (including 4.3-kb gag-pol,2.9-kb pol-env and 2.7-kb env-ne) was developed.In addition,one amplicon (9 kb near full-length genome) was also used in 32 samples with varied viral loads.All amplicons were directly sequenced by DNA automated sequencer.Results Twenty-five percent(8/32) amplification efficiency was achieved by the one-amplicon strategy and 65.6%(21/32) by three-amplicon strategy.For one amplicon strategy,none of complete near full-length genome sequences was obtained by DNA sequencing.For three-amplicon strategy,75% sequences were achieved in DNA sequencing.Amplification efficiency but not sequencing efficiency was closely associated with viral loads.Conclusion Three-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.

  12. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA.

    Science.gov (United States)

    Elbeik, Tarek; Nassos, Patricia; Kipnis, Patricia; Haller, Barbara; Ng, Valerie L

    2005-08-01

    Separation and storage of plasma within 2 h of phlebotomy is required for the VACUTAINER PPT Plasma Preparation Tube (PPT) versus 4 h for the predecessor VACUTAINER EDTA tube for human immunodeficiency virus type 1 (HIV-1) viral load (HIVL) testing by the VERSANT HIV-1 RNA 3.0 assay (branched DNA). The 2-h limit for PPT imposes time constraints for handling and transporting to the testing laboratory. This study compares HIVL reproducibility from matched blood in EDTA tubes and PPTs and between PPT pairs following processing within 4 h of phlebotomy, stability of plasma HIV-1 RNA at 24- and 72-h room temperature storage in the tube, and comparative labor and supply requirements. Blood from 159 patients was collected in paired tubes (EDTA/PPT or PPT/PPT): 86 paired EDTA tubes and PPTs were processed 4 h following phlebotomy and their HIVLs were compared, 42 paired PPT/PPT pairs were analyzed for intertube HIVL reproducibility, and 31 PPT/PPT pairs were analyzed for HIV-1 RNA stability by HIVL. Labor and supply requirements were compared between PPT and EDTA tubes. PPTs produce results equivalent to standard EDTA tube results when processed 4 h after phlebotomy. PPT intertube analyte results are reproducible. An average decrease of 13% and 37% in HIVL was observed in PPT plasma after 24 and 72 h of room temperature storage, respectively; thus, plasma can be stored at room temperature up to 24 h in the original tube. PPTs offer labor and supply savings over EDTA tubes.

  13. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  14. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    LIU YingChun; ZHANG Yan; YE GuoZhu; YANG ZhenJun; ZHANG LiangRen; ZHANG LiHe

    2008-01-01

    Aptamers that interact with various HIV-1 proteins, such as reverse transcriptase, Rev, Tat protein, and nuclear capsule protein, have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However, there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation, we selected alternative RNA aptamers specific for the HIV-1 Aptamer IN1, IN2, IN3 had similar and the highest Kd values from 145 to 239 nmol. L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition, structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure, it might directly interact with the DDE motif of integrase, which is the virus DNA-binding site, because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together, this study generated a novel RNA aptamer IN1, which could be useful in basic research and anti-HIV drug screening.

  15. Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

    Science.gov (United States)

    Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

    2008-05-01

    The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

  16. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers that interact with various HIV-1 proteins,such as reverse transcriptase,Rev,Tat protein,and nuclear capsule protein,have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However,there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation,we selected alternative RNA aptamers specific for the HIV-1 integrase by using a different binding buffer containing 10 mmol·L-1 MgCl2 and 100 mmol·L-1 KCl. Aptamer IN1,IN2,IN3 had similar and the highest Kd values from 145 to 239 nmol·L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition,structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure,it might directly interact with the DDE motif of integrase,which is the virus DNA-binding site,because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together,this study generated a novel RNA aptamer IN1,which could be useful in basic research and anti-HIV drug screening.

  17. The miRNA Plasma Signature in Response to Acute Aerobic Exercise and Endurance Training

    DEFF Research Database (Denmark)

    Nielsen, Søren; Åkerström, Thorbjörn; Rinnov, Anders

    2014-01-01

    the miRNA signature in human plasma in response to acute exercise and chronic endurance training by utilizing a novel methodological approach. RNA was isolated from human plasma collected from young healthy men before and after an acute endurance exercise bout and following 12 weeks of endurance training...

  18. 对五种国产核酸筛查试剂检测HIV-1RNA效果的初步评价%Primary Evaluation on the Capacity of Five Domestic NAT Donor Screening Assays in Detecting HIV-1 RNA

    Institute of Scientific and Technical Information of China (English)

    许四宏; 宋爱京; 聂建辉; 李秀华; 王佑春

    2011-01-01

    目的 对5种国产HBV/HCV/HIV-1核酸筛查试剂(A、B1、B2、C和D)检测HIV-1 RNA的能力进行初步评价.方法 从我国不同地区收集60份HIV-1感染者样品(包含1份HIV-1感染窗口期样品)及540份HIV阴性样品,将60份HIV-1感染者样品随机分布于540份HIV阴性样品中,按照合并检测模式(pool模式)对该600份样品进行检测,将每种试剂检测结果为阳性的pool分别按说明书进一步拆分和/或鉴别试验.结果 A、B1、B2和C四种试剂检测HIV-1RNA的效果较强,其阴性和阳性符合率均为100%;D试剂则较差,其中2份HIV-1感染者样品(基因型分别为BC和B/B′,HIV-1 RNA含量分别为9.70×102 copies/ml和5.20×103 copies/ml)分别处于2个pool中,该2个pool经D试剂检测,均为HIV-1 RNA阴性.对检测结果为阳性的35个pool进行拆分检测时,D试剂检测1份HIV-1感染者样品(B/B′亚型、HIV-1 RNA含量为1.09×103 copies/ml)为HIV-1 RNA阴性,3份HIV-1 RNA阴性样品为HIV-1 RNA阳性.对于1份HIV-1感染窗口期样品,5种试剂均检测为HIV-1 RNA阳性.结论 A、B1、B2和C四种试剂均有较高的一致性,但D试剂具有一定的假阳性和漏检,应进一步提高质量.%Objective To evaluate the capacity of five domestic NAT donor screening assays of HBV DNA, HCV RNA and HIV-1 RNA (A, B1, B2, C and D assays) in detecting HIV-1 RNA. Methods 60 HIV-1 positive plasma were collected from HIV-1-infected individuals and 540 HIV-1 negative plasma were collected from donors with negative for anti-HIV in different regions in China. The 60 samples positive for HIV-1 RNA were randomly distributed into 540 negative samples for HIV-1 RNA. Mini-pool test and further discrimination test were completed according to the manufactures' instruction of the five assays. Results For A, B1, B2 and C assays,the coincidence rates of HIV-1 infected and negative samples for HIV were 100%. However, for D assay, 2 pools containing one HIV-1 genotype BC sample (viral load: 9

  19. HIV-1 pre-mRNA commitment to Rev mediated export through PSF and Matrin 3

    Energy Technology Data Exchange (ETDEWEB)

    Kula, Anna; Gharu, Lavina [The Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste (Italy); Marcello, Alessandro, E-mail: marcello@icgeb.org [The Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste (Italy)

    2013-01-20

    Human immunodeficiency virus gene expression and replication are regulated at several levels. Incompletely spliced viral RNAs and full-length genomic RNA contain the RRE element and are bound by the viral trans-acting protein Rev to be transported out of the nucleus. Previously we found that the nuclear matrix protein MATR3 was a cofactor of Rev-mediated RNA export. Here we show that the pleiotropic protein PSF binds viral RNA and is associated with MATR3. PSF is involved in the maintenance of a pool of RNA available for Rev activity. However, while Rev and PSF bind the viral pre-mRNA at the site of viral transcription, MATR3 interacts at a subsequent step. We propose that PSF and MATR3 define a novel pathway for RRE-containing HIV-1 RNAs that is hijacked by the viral Rev protein.

  20. HIV-1 buds predominantly at the plasma membrane of primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Sonja Welsch

    2007-03-01

    Full Text Available HIV-1 assembly and release are believed to occur at the plasma membrane in most host cells with the exception of primary macrophages, for which exclusive budding at late endosomes has been reported. Here, we applied a novel ultrastructural approach to assess HIV-1 budding in primary macrophages in an immunomarker-independent manner. Infected macrophages were fed with BSA-gold and stained with the membrane-impermeant dye ruthenium red to identify endosomes and the plasma membrane, respectively. Virus-filled vacuolar structures with a seemingly intracellular localization displayed intense staining with ruthenium red, but lacked endocytosed BSA-gold, defining them as plasma membrane. Moreover, HIV budding profiles were virtually excluded from gold-filled endosomes while frequently being detected on ruthenium red-positive membranes. The composition of cellular marker proteins incorporated into HIV-1 supported a plasma membrane-derived origin of the viral envelope. Thus, contrary to current opinion, the plasma membrane is the primary site of HIV-1 budding also in infected macrophages.

  1. HIV-Infected Ugandan Women on Antiretroviral Therapy Maintain HIV-1 RNA Suppression Across Periconception, Pregnancy, and Postpartum Periods.

    Science.gov (United States)

    Matthews, Lynn T; Ribaudo, Heather B; Kaida, Angela; Bennett, Kara; Musinguzi, Nicholas; Siedner, Mark J; Kabakyenga, Jerome; Hunt, Peter W; Martin, Jeffrey N; Boum, Yap; Haberer, Jessica E; Bangsberg, David R

    2016-04-01

    HIV-infected women risk sexual and perinatal HIV transmission during conception, pregnancy, childbirth, and breastfeeding. We compared HIV-1 RNA suppression and medication adherence across periconception, pregnancy, and postpartum periods, among women on antiretroviral therapy (ART) in Uganda. We analyzed data from women in a prospective cohort study, aged 18-49 years, enrolled at ART initiation and with ≥1 pregnancy between 2005 and 2011. Participants were seen quarterly. The primary exposure of interest was pregnancy period, including periconception (3 quarters before pregnancy), pregnancy, postpartum (6 months after pregnancy outcome), or nonpregnancy related. Regression models using generalized estimating equations compared the likelihood of HIV-1 RNA ≤400 copies per milliliter, <80% average adherence based on electronic pill caps (medication event monitoring system), and likelihood of 72-hour medication gaps across each period. One hundred eleven women contributed 486 person-years of follow-up. Viral suppression was present at 89% of nonpregnancy, 97% of periconception, 93% of pregnancy, and 89% of postpartum visits, and was more likely during periconception (adjusted odds ratio, 2.15) compared with nonpregnant periods. Average ART adherence was 90% [interquartile range (IQR), 70%-98%], 93% (IQR, 82%-98%), 92% (IQR, 72%-98%), and 88% (IQR, 63%-97%) during nonpregnant, periconception, pregnant, and postpartum periods, respectively. Average adherence <80% was less likely during periconception (adjusted odds ratio, 0.68), and 72-hour gaps per 90 days were less frequent during periconception (adjusted relative risk, 0.72) and more frequent during postpartum (adjusted relative risk, 1.40). Women with pregnancy were virologically suppressed at most visits, with an increased likelihood of suppression and high adherence during periconception follow-up. Increased frequency of 72-hour gaps suggests a need for increased adherence support during postpartum periods.

  2. Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

    Science.gov (United States)

    Freisz, Séverine; Mezher, Joelle; Hafirassou, Lamine; Wolff, Philippe; Nominé, Yves; Romier, Christophe; Dumas, Philippe; Ennifar, Eric

    2012-07-01

    The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

  3. Intrinsic network connectivity abnormalities in HIV-infected individuals over age 60.

    Science.gov (United States)

    Guha, Anika; Wang, Liang; Tanenbaum, Aaron; Esmaeili-Firidouni, Pardis; Wendelken, Lauren A; Busovaca, Edgar; Clifford, Katherine; Desai, Akash; Ances, Beau M; Valcour, Victor

    2016-02-01

    Individuals infected with HIV are living longer due to effective treatment with combination antiretroviral therapy (cART). Despite these advances, HIV-associated neurocognitive disorders (HAND) remain prevalent. In this study, we analyzed resting state functional connectivity (rs-fc) data from HIV-infected and matched HIV-uninfected adults aged 60 years and older to determine associations between HIV status, neuropsychological performance, and clinical variables. HIV-infected participants with detectable plasma HIV RNA exhibited decreased rs-fc within the salience (SAL) network compared to HIV-infected participants with suppressed plasma HIV RNA. We did not identify differences in rs-fc within HIV-infected individuals by HAND status. Our analysis identifies focal deficits in the SAL network that may be mitigated with suppression of plasma virus. However, these findings suggest that rs-fc may not be sensitive as a marker of HAND among individuals with suppressed plasma viral loads.

  4. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  5. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    Science.gov (United States)

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-07-21

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.

  6. p21(WAF1/CIP1 RNA expression in highly HIV-1 exposed, uninfected individuals.

    Directory of Open Access Journals (Sweden)

    Joshua Herbeck

    Full Text Available Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN. However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior, p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80. Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year and lowest 40 LESC (median = 2 partners/year showed no difference between the groups (P = 0.84. There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12, but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.

  7. Platelets confound the measurement of extracellular miRNA in archived plasma

    OpenAIRE

    Mitchell, Adam J.; Gray, Warren D; Hayek, Salim S.; Yi-An Ko; Sheena Thomas; Kim Rooney; Mosaab Awad; John D. Roback; Arshed Quyyumi; Searles, Charles D.

    2016-01-01

    Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a c...

  8. Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls

    DEFF Research Database (Denmark)

    Carlsen, Anting Liu; Joergensen, Maiken Thyregod; Knudsen, Steen;

    2013-01-01

    There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles...

  9. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals

    DEFF Research Database (Denmark)

    Grint, D; Tedaldi, Ellen; Peters, L

    2017-01-01

    OBJECTIVES: Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among tho...

  10. Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies.

    Science.gov (United States)

    Marin, Mariana; Golem, Sheetal; Kozak, Susan L; Kabat, David

    2016-02-02

    APOBEC3 cytidine deaminases and viral genomic RNA (gRNA) occur in virions, polysomes, and cytoplasmic granules, but have not been tracked together. Moreover, gRNA traffic is important, but the factors that move it into granules are unknown. Using in situ hybridization of transfected cells and protein synthesis inhibitors that drive mRNAs between locales, we observed APOBEC3F cotrafficking with gRNA without altering its movements. Whereas cells with little cytoplasmic gRNA were translationally active and accumulated Gag, suprathreshold amounts induced autophosphorylation of the cytoplasmic double-stranded RNA (dsRNA)-dependent protein kinase (PKR), causing eIF2α phosphorylation, protein synthesis suppression, and gRNA sequestration in stress granules. Additionally, we confirmed recent evidence that PKR is activated by chromosome-associated cellular dsRNAs after nuclear membranes disperse in prophase. By arresting cells in G2, HIV-1 blocks this mechanism for PKR activation and eIF2α phosphorylation. However, cytopathic membrane damage in CD4- and coreceptor-positive cultures infected with laboratory-adapted fusogenic HIV-1LAI eventually enabled PKR entry and activation in interphase nuclei. These results reveal multiple stages in the PKR-HIV-1 battleground that culminate in cell death. We discuss evidence suggesting that HIV-1s evolve in vivo to prevent or delay PKR activation by all these mechanisms. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

    Science.gov (United States)

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  12. HIV-1 subtype C primary isolates exhibit high sensitivity to an anti-gp120 RNA aptamer

    CSIR Research Space (South Africa)

    Mufhandu, Hazel T

    2012-09-01

    Full Text Available Globally, HIV-1 subtype C is the most prevalent subtype, yet most antiretroviral drugs are developed against subtype B. UCLA1 RNA aptamer, which we previously showed neutralizes HIV-1 subtype C Env-pseudotyped viruses was examined for neutralization...

  13. Multicenter comparison of Roche COBAS AMPLICOR MONITOR version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex version 3.0 for quantification of human immunodeficiency virus type 1 RNA in plasma.

    Science.gov (United States)

    Murphy, D G; Côté, L; Fauvel, M; René, P; Vincelette, J

    2000-11-01

    The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1. 5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log(10) RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log(10) lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.

  14. Using plasma viral load to guide antiretroviral therapy initiation to prevent HIV-1 transmission.

    Directory of Open Access Journals (Sweden)

    Pamela M Murnane

    Full Text Available BACKGROUND: Current WHO guidelines recommend antiretroviral therapy (ART initiation at CD4 counts ≤350 cells/µL. Increasing this threshold has been proposed, with a primary goal of reducing HIV-1 infectiousness. Because the quantity of HIV-1 in plasma is the primary predictor of HIV-1 transmission, consideration of plasma viral load in ART initiation guidelines is warranted. METHODS: Using per-sex-act infectivity estimates and cross-sectional sexual behavior data from 2,484 HIV-1 infected persons with CD4 counts >350 enrolled in a study of African heterosexual HIV-1 serodiscordant couples, we calculated the number of transmissions expected and the number potentially averted under selected scenarios for ART initiation: i CD4 count 350 while averting 40.5% of expected transmissions (ratio 2.0; treating at viral load ≥10,0000 copies/mL had a ratio of 1.5. In contrast, initiation at CD4 count <500 would require treating 41.8%, while averting 48.4% (ratio 1.1. CONCLUSION: Inclusion of viral load in ART initiation guidelines could permit targeting ART resources to HIV-1 infected persons who have a higher risk of transmitting HIV-1. Further work is needed to estimate costs and feasibility.

  15. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    Science.gov (United States)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

  16. Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals.

    Science.gov (United States)

    Konadu, Kateena Addae; Huang, Ming Bo; Roth, William; Armstrong, Wendy; Powell, Michael; Villinger, Francois; Bond, Vincent

    2016-01-05

    Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

  17. Bone marrow plasma cells are a primary source of serum HIV-1-specific antibodies in chronically infected individuals.

    Science.gov (United States)

    Montezuma-Rusca, Jairo M; Moir, Susan; Kardava, Lela; Buckner, Clarisa M; Louie, Aaron; Kim, Leo J Y; Santich, Brian H; Wang, Wei; Fankuchen, Olivia R; Diaz, Gabriella; Daub, Janine R; Rosenzweig, Sergio D; Chun, Tae-Wook; Li, Yuxing; Braylan, Raul C; Calvo, Katherine R; Fauci, Anthony S

    2015-03-15

    Several potent and broadly neutralizing Abs to HIV-1 have been isolated recently from peripheral blood B cells of infected individuals, based on prescreening of Ab activity in the serum. However, little is known regarding the cells that make the Abs that circulate in the blood. Accordingly, we investigated the most likely source, the bone marrow, of chronically HIV-1-infected individuals who were not receiving antiretroviral therapy. Increased frequencies of plasma cells, as well as B cell precursors, namely preB-I and preB-II, and decreased frequencies of mature B cells were observed in bone marrow aspirates of these individuals compared with HIV-negative counterparts. Increased frequencies of bone marrow plasma cells are consistent with known hallmarks of HIV-1 infection, namely hypergammaglobulinemia and increased frequencies of peripheral blood plasmablasts. Levels of HIV-1 envelope (Env)-binding and HIV-1-neutralizing Abs were measured in serum, and corresponding frequencies of Ab-secreting or Env-binding cells were measured in the blood (plasmablasts and memory B cells) and in the bone marrow (plasma cells). A strong correlation was observed between serum HIV-1-specific Abs and Env-specific bone marrow-derived plasma cells, but not circulating plasmablasts or memory B cells. These findings demonstrate that, despite HIV-1-induced phenotypic and functional B cell dysregulation in the peripheral blood and secondary lymphoid tissues, bone marrow plasma cells remain a primary source for circulating HIV-1-specific Abs in HIV-1-infected individuals.

  18. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.

  19. Associations between plasma tenofovir concentration and renal function markers in HIV-infected women

    Directory of Open Access Journals (Sweden)

    Mwila Mulubwa

    2016-02-01

    Full Text Available Background: Tenofovir disoproxil fumarate (TDF has been associated with kidney tubulardys function and reduced renal function. Limited studies were performed in Europe and Asia that related plasma tenofovir (TFV concentration with renal function; no such studies to date have been performed on Africans.Objective: To investigate the correlation between plasma tenofovir (TFV concentration and certain renal function markers in HIV-infected women on TDF antiretroviral therapy (ART.These markers were also compared to a HIV-uninfected control group.Methods: HIV-infected women (n = 30 on TDF-based ART were matched with 30 controls forage and body mass index. Renal markers analysed were estimated glomerular filtration rate (eGFR, creatinine clearance (CrCl, serum creatinine, albuminuria, glucosuria, serum urea, serum uric acid, urine sodium and maximum tubular reabsorption of phosphate. Baseline eGFR and CrCl data were obtained retrospectively for the HIV-infected women. Plasma TFV was assayed using a validated HPLC-MS/MS method. Step wise regression, Mann–Whitney test, unpaired and paired t-tests were applied in the statistical analyses.Results: TFV concentration was independently associated with albuminuria (adjusted r2 = 0.339; p = 0.001 in HIV-infected women. In the adjusted (weight analysis, eGFR (p = 0.038,CrCl (p = 0.032 and albuminuria (p = 0.048 were significantly higher in HIV-infected compared to the uninfected women, but eGFR was abnormally high in HIV-infected women. Both eGFR (p < 0.001 and CrCl (p = 0.008 increased from baseline to follow-up in HIV-infected women.Conclusion: Plasma TFV concentration was associated with increased albuminuria in HIV infected women in this sub-study. Both eGFR and CrCl were increased in HIV-infected women from baseline. These findings should be confirmed in larger studies, and hyperfiltration in HIV-infected women warrants further investigation.

  20. Plasma Biomarkers for Detecting Hodgkin's Lymphoma in HIV Patients

    OpenAIRE

    VARNUM, SUSAN M.; Webb-Robertson, Bobbie-Jo M.; Hessol, Nancy A.; Smith, Richard D.; Zangar, Richard C.

    2011-01-01

    The lifespan of people with human immunodeficiency virus (HIV) infection has increased as a result of effective antiretroviral therapy, and the incidences of the AIDS-defining cancers, non-Hodgkin's lymphoma and Kaposi sarcoma, have declined. Even so, HIV-infected individuals are now at greater risk of other cancers, including Hodgkin's lymphoma (HL). To identify candidate biomarkers for the early detection of HL, we undertook an accurate mass and elution time tag proteomics analysis of indiv...

  1. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  2. An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1.

    Science.gov (United States)

    Méndez-Ortega, María C; Restrepo, Silvia; Rodríguez-R, Luis M; Pérez, Iván; Mendoza, Juan C; Martínez, Andrés P; Sierra, Roberto; Rey-Benito, Gloria J

    2010-12-20

    HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases. A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings. Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing. HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted under specific antiretroviral selective

  3. An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1

    Directory of Open Access Journals (Sweden)

    Mendoza Juan C

    2010-12-01

    Full Text Available Abstract Background HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases. Methods A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings. Results Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing. Conclusions HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant

  4. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  5. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Science.gov (United States)

    Lai, Ming-Chih; Wang, Shainn-Wei; Cheng, Lie; Tarn, Woan-Yuh; Tsai, Shaw-Jenq; Sun, H Sunny

    2013-01-01

    Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  6. [Synthesis of messenger-like RNA in plasma cels producing different clases of immunoglobulins].

    Science.gov (United States)

    Nikitin, A V; Babichev, V A

    1976-01-01

    Electrophoretic analysis of the nuclear poly A RNA cells of the MOPC 21, MOPC 406 and MOPC 41 plasmocytomas demonstrated a similarity in their distribution by the mol wt. Kinetics of the label accumulation in the nuclear poly A RNA of different plasma cells was unitypical. Individual peculiarities of the distribution of the cytoplasmic poly A RNA were expressed for each individual line of the myeloma cells. The majority of the molecules of the heterogenous RNA in the plasma cells were subjected to posttranscription polyadenylation.

  7. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    DEFF Research Database (Denmark)

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe

    2007-01-01

    OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin......: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p insulin resistance. While acute exercise did not affect IL-18 mRNA expression...... at the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes. DISCUSSION: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial effects...

  8. Platelets confound the measurement of extracellular miRNA in archived plasma.

    Science.gov (United States)

    Mitchell, Adam J; Gray, Warren D; Hayek, Salim S; Ko, Yi-An; Thomas, Sheena; Rooney, Kim; Awad, Mosaab; Roback, John D; Quyyumi, Arshed; Searles, Charles D

    2016-09-13

    Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a case-control study of archived plasma, we noted a significant correlation between miRNA levels and platelet counts despite post-thaw processing. We thus examined the effects of a single freeze/thaw cycle on microparticles (MPs) and miRNA levels, and show that a single freeze/thaw cycle of plasma dramatically increases the number of platelet-derived MPs, contaminates the extracellular miRNA pool, and profoundly affects the levels of miRNAs detected. The measurement of extracellular miRNAs in archived samples is critically dependent on the removal of residual platelets prior to freezing plasma samples. Many previous clinical studies of extracellular miRNA in archived plasma should be interpreted with caution and future studies should avoid the effects of platelet contamination.

  9. Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing Detecção do RNA do HIV e HCV em sêmen de homens brasileiros, usando PCR multiplex antes e depois do "semen washing"

    Directory of Open Access Journals (Sweden)

    Cynthia Liliane Motta do Canto

    2006-08-01

    Full Text Available INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.O aumento da sobrevida dos pacientes que utilizam terapêutica antiretroviral altamente eficaz (HAART- Highly Active Antiretroviral Therapy trouxe uma nova demanda de casais sorodiscordantes que desejam filhos. Como esses casais não podem abandonar o uso de preservativos, torna-se indispensável tratar o sêmen infectado com técnicas laboratoriais eficazes que além de isolar os melhores espermatozóides, reduzam a carga viral do HIV e HCV a níveis indetectáveis. Para isso, são utilizadas técnicas de semen washing, associadas a testes ultra sensíveis de biologia molecular. Após análise seminal, sêmen de 20 pacientes co-infectados HIV-HCV foram submetidos a fracionamento celular e

  10. Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV- 2LTR y ARN de HIV Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

    Directory of Open Access Journals (Sweden)

    Rosana Gariglio

    2004-10-01

    study, we analyzed the presence of total HIV DNA (T-HIV DNA, non-integrated DNA with 2LTR (2LTR-HIV DNA and HIV RNA in a group of 55 HIV-positive subjects from Rosario City, with different clinical stages, with and without HAART. All markers were evaluated by PCR assays optimized in our laboratory that included colorimetric detection in microplate. HIV RNA clinical sensitivity was compared with a reference test, bDNA, resulting in 74% and 64% respectively, with an 85% of agreement. Thus, our HIV RNA assay could be used to monitor patients under HAART and at risk of infection. The 2LTR-HIV DNA was 54% positive although it was absent in patients with high VL. This marker was considered a labile product therefore its presence was associated with recent infection. However, current evidences question its stability. Thus, its clinical significance should be reconsidered. The absence of 2LTR-HIV DNA in patients with detectable VL may relate to the heterogeneity of the sequence used for its detection. T-HIV DNA was present in 100% of the samples and could be a relevant remission marker when therapies that effectively eradicate the infection became available.

  11. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    Science.gov (United States)

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  12. A plasma biomarker signature of immune activation in HIV patients on antiretroviral therapy.

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    Anupa Kamat

    Full Text Available BACKGROUND: Immune activation is a strong predictor of disease progression in HIV infection. Combinatorial plasma biomarker signatures that represent surrogate markers of immune activation in both viremic and aviremic HIV patients on combination antiretroviral therapy (cART have not been defined. Here, we identify a plasma inflammatory biomarker signature that distinguishes between both viremic and aviremic HIV patients on cART and healthy controls and examine relationships of this signature to markers of disease progression. METHODS: Multiplex profiling and ELISA were used to detect 15 cytokines/chemokines, soluble IL-2R (sIL-2R, and soluble CD14 (sCD14 in plasma from 57 HIV patients with CD4 nadir <300 cells/µl and 29 healthy controls. Supervised and unsupervised analyses were used to identify biomarkers explaining variance between groups defined by HIV status or drug abuse. Relationships between biomarkers and disease markers were examined by Spearman correlation. RESULTS: The majority (91% of HIV subjects were on cART, with 38% having undetectable viral loads (VL. Hierarchical clustering identified a biomarker cluster in plasma consisting of two interferon-stimulated gene products (CXCL9 and CXCL10, T cell activation marker (sIL-2R, and monocyte activation marker (sCD14 that distinguished both viremic and aviremic HIV patients on cART from controls (p<0.0001 and were top-ranked in variables important in projection plots. IL-12 and CCL4 were also elevated in viremic and aviremic patients compared to controls (p<0.05. IL-12 correlated with IFNα, IFNγ, CXCL9, and sIL-2R (p<0.05. CXCL10 correlated positively with plasma VL and percentage of CD16+ monocytes, and inversely with CD4 count (p = 0.001, <0.0001, and 0.04, respectively. CONCLUSION: A plasma inflammatory biomarker signature consisting of CXCL9, CXCL10, sIL-2R, and sCD14 may be useful as a surrogate marker to monitor immune activation in both viremic and aviremic HIV patients on c

  13. Plasma membrane is the site of productive HIV-1 particle assembly.

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    Nolwenn Jouvenet

    2006-12-01

    Full Text Available Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

  14. HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The human immunodeficiency virus (HIV type-1 viral protein U (Vpu protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.

  15. Plasma concentrations of generic lopinavir/ritonavir in HIV type-1-infected individuals.

    NARCIS (Netherlands)

    Lugt, J. van der; Lange, J.; Avihingsanon, A.; Ananworanich, J.; Sealoo, S.; Burger, D.M.; Gorowara, M.; Phanuphak, P.; Ruxrungtham, K.

    2009-01-01

    BACKGROUND: Generic drugs can contribute to access to treatment for HIV-infected patients. However quality and safety remains an issue of concern. Therefore, we evaluated minimal plasma concentrations and short-term safety of a generic lopinavir/ritonavir 200/50 mg tablet formulation. METHODS: In a

  16. Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

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    Soros Vanessa

    2008-10-01

    Full Text Available Abstract HIV-1 structural proteins are translated from incompletely spliced 9 kb and 4 kb mRNAs, which are transported to the cytoplasm by Crm1. It has been assumed that once in the cytoplasm, translation of incompletely spliced HIV-1 mRNAs occurs in the same manner as host mRNAs. Previous analyses have demonstrated that Sam68 and a mutant thereof, Sam68ΔC, have dramatic effects on HIV gene expression, strongly enhancing and inhibiting viral structural protein synthesis, respectively. While investigating the inhibition of incompletely spliced HIV-1 mRNAs by Sam68ΔC, we determined that the effect was independent of the perinuclear bundling of the viral RNA. Inhibition was dependent upon the nuclear export pathway used, as translation of viral RNA exported via the Tap/CTE export pathway was not blocked by Sam68ΔC. We demonstrate that inhibition of HIV expression by Sam68ΔC is correlated with a loss of PABP1 binding with no attendant change in polyadenosine tail length of the affected RNAs. The capacity of Sam68ΔC to selectively inhibit translation of HIV-1 RNAs exported by Crm1 suggests that it is able to recognize unique characteristics of these viral RNPs, a property that could lead to new therapeutic approaches to controlling HIV-1 replication.

  17. CCR5 genotype and plasma ß-chemokine concentration of Brazilian HIV-infected individuals

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    Mikawa A.Y.

    2002-01-01

    Full Text Available The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1ß and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26 and among HIV-1-infected individuals (N = 129. The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (delta32ccr5 in this population was 0.032; however, no delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating ß-chemokines (MIP-1alpha, RANTES and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5 (28.23 ng/ml were higher than in the control samples (16.07 ng/ml; P<0.05; however, this HIV+ patient group (mean 26.23 pg/ml had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05. Both HIV-1-infected and uninfected individuals heterozygous for the delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05. These findings suggest that the CCR5 allele and ß-chemokine production may affect the immunopathogenesis of HIV-1.

  18. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    Science.gov (United States)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  19. Role of uncontrolled HIV RNA level and immunodeficiency in the occurrence of malignancy in HIV-infected patients during the combination antiretroviral therapy era: Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort.

    Science.gov (United States)

    Bruyand, Mathias; Thiébaut, Rodolphe; Lawson-Ayayi, Sylvie; Joly, Pierre; Sasco, Annie Jeanne; Mercié, Patrick; Pellegrin, Jean Luc; Neau, Didier; Dabis, François; Morlat, Philippe; Chêne, Geneviève; Bonnet, Fabrice

    2009-10-01

    Human immunodeficiency virus (HIV)-infected patients are at higher risk of malignancies. In addition to traditional determinants, a specific deleterious effect of HIV and immunodeficiency is speculated. We aimed at studying the association between immunological and virological characteristics of HIV-infected patients in care and the risk of acquired immunodeficiency syndrome (AIDS)-defining and non-AIDS-defining malignancies. Patients consecutively enrolled in the hospital-based Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort were included if the duration of follow-up was >3 months during the period 1998-2006. Multivariate modeling used an extended Cox proportional hazards model for time-dependent covariates and delayed entry. The 4194 patients included in the study developed 251 first malignancies during 22,389 person-years. A higher incidence of AIDS-defining malignancies (107 cases) was independently associated with (1) both longer and current exposures to a plasma HIV RNA level >500 copies/mL (hazard ratio [HR], 1.27 per year [P500 cells/mm(3) to prevent the occurrence of malignancy, this should be integrated to malignancy-prevention policies.

  20. Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses.

    Directory of Open Access Journals (Sweden)

    Michael D Moore

    2009-10-01

    Full Text Available Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

  1. HIV diversity and drug resistance from plasma and non-plasma analytes in a large treatment programme in western Kenya

    Directory of Open Access Journals (Sweden)

    Rami Kantor

    2014-11-01

    Full Text Available Introduction: Antiretroviral resistance leads to treatment failure and resistance transmission. Resistance data in western Kenya are limited. Collection of non-plasma analytes may provide additional resistance information. Methods: We assessed HIV diversity using the REGA tool, transmitted resistance by the WHO mutation list and acquired resistance upon first-line failure by the IAS–USA mutation list, at the Academic Model Providing Access to Healthcare (AMPATH, a major treatment programme in western Kenya. Plasma and four non-plasma analytes, dried blood-spots (DBS, dried plasma-spots (DPS, ViveSTTM-plasma (STP and ViveST-blood (STB, were compared to identify diversity and evaluate sequence concordance. Results: Among 122 patients, 62 were treatment-naïve and 60 treatment-experienced; 61% were female, median age 35 years, median CD4 182 cells/µL, median viral-load 4.6 log10 copies/mL. One hundred and ninety-six sequences were available for 107/122 (88% patients, 58/62 (94% treatment-naïve and 49/60 (82% treated; 100/122 (82% plasma, 37/78 (47% attempted DBS, 16/45 (36% attempted DPS, 14/44 (32% attempted STP from fresh plasma and 23/34 (68% from frozen plasma, and 5/42 (12% attempted STB. Plasma and DBS genotyping success increased at higher VL and shorter shipment-to-genotyping time. Main subtypes were A (62%, D (15% and C (6%. Transmitted resistance was found in 1.8% of plasma sequences, and 7% combining analytes. Plasma resistance mutations were identified in 91% of treated patients, 76% NRTI, 91% NNRTI; 76% dual-class; 60% with intermediate-high predicted resistance to future treatment options; with novel mutation co-occurrence patterns. Nearly 88% of plasma mutations were identified in DBS, 89% in DPS and 94% in STP. Of 23 discordant mutations, 92% in plasma and 60% in non-plasma analytes were mixtures. Mean whole-sequence discordance from frozen plasma reference was 1.1% for plasma-DBS, 1.2% plasma-DPS, 2.0% plasma-STP and 2

  2. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

    Directory of Open Access Journals (Sweden)

    Kevin A Wilkinson

    2008-04-01

    Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

  3. The role of RNA pooling technique in the diagnosis of acute HIV infection and the estimation on HIV incidence among low-grade-venues female sex workers%集合核酸检测用于低档场所女性性工作者HIV急性感染诊断及发病率估计

    Institute of Scientific and Technical Information of China (English)

    李剑军; 张鸿满; 沈智勇; 周月姣; 方宁烨; 王斌; 王江伟; 唐振柱

    2014-01-01

    Objective To examine the feasibility of RNA pooling technique in the diagnosis on acute HIV infection among female sex workers (FSWs) working at the low-grade venues.Methods Plasma samples from the low-grade-venue FSWs in Guangxi,in 2011 were tested for HIV antibody using the rapid testing method.All samples which were HIV antibody negative in the rapid testing were tested for HIV RNA with RNA pooling technique.FSWs who showed HIV RNA positive were tested for HIV antibody by Western blot method in 3 months.The HIV incidence in the low-grade venue FSWs was counted under the estimation formula.Results There were 6 469 cases of FSWs who were recruited in this study.Through rapid testing,results showed that HIV antibody was positive in 139 cases,with the positive rate as 2.15%.6 330 FSWs with HIV antibody negative were tested by HIV RNA pooling method,with 7 of them showing HIV RNA positive,in which 6 cases showed HIV-1 antibody seroconversion,thus were diagnosed as acute HIV infection.HIV incidence in low-grade FSWs appeared to be 1.45 per 100 person years (95%CI:1.17-1.76 per 100 person years) in Guangxi.Conclusion Other than regular routine HIV antibody testing,it seemed necessary to adopt the HIV RNA pooling strategy in high-risk groups such as FSWs,so as to early detect the HIV infection and to timely perform the intervention or treatment programs to prevent sexual transmission of HIV.%目的 评价集合核酸检测方法在低档场所女性性工作者(FSWs)HIV急性感染诊断中的应用价值.方法 对广西壮族自治区2011年低档场所FSWs进行调查,并采集血液,采用胶体硒法对HIV抗体初筛检测;对初筛阴性者采用集合核酸检测方法检测HIVRNA,若为阳性,3个月内随访并采集血液进行确证;应用估算公式计算HIV年发病率.结果 共收集6 469例FSWs的血液样本,HIV抗体初筛阳性139例,阳性率为2.15%.刘6 330例初筛阴性者进行集合核酸检测,7例为HIVRNA阳性.3

  4. Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

    Science.gov (United States)

    Sampey, Gavin C; Saifuddin, Mohammed; Schwab, Angela; Barclay, Robert; Punya, Shreya; Chung, Myung-Chul; Hakami, Ramin M; Zadeh, Mohammad Asad; Lepene, Benjamin; Klase, Zachary A; El-Hage, Nazira; Young, Mary; Iordanskiy, Sergey; Kashanchi, Fatah

    2016-01-15

    HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

  5. HIV Type 1 Nef Is Released from Infected Cells in CD45+ Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

    Science.gov (United States)

    Raymond, A.D.; Campbell-Sims, T.C.; Khan, M.; Lang, M.; Huang, M.B.; Bond, V.C.

    2011-01-01

    Abstract HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIVMN. Interestingly, plasma mvNef levels in HIV+ patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS. PMID:20964480

  6. Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

    Institute of Scientific and Technical Information of China (English)

    LI Shuli; ZENG Linglan; LUO Duande; LIU Wei; GUO Jingsong; YANG Xiaoming

    2001-01-01

    The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus(HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %, 15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22. 2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

  7. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA.

    Science.gov (United States)

    Parkin, Neil T

    2014-01-01

    World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can

  8. Systematic identification of spontaneous preterm birth-associated RNA transcripts in maternal plasma.

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    Stephen S C Chim

    Full Text Available BACKGROUND: Spontaneous preterm birth (SPB, before 37 gestational weeks is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls. PRINCIPAL FINDINGS: Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the IL1RL1 mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60% plasma samples collected during the presentation of preterm labor (≤32.9 weeks in women eventually giving SPB, but was detected in only 1 of 27 (4% samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056. CONCLUSION: We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the IL1RL1 mRNA was more frequently detected in predelivery maternal plasma samples collected from women

  9. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects

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    Takahashi, Kei; Yokota, Shin-ichi; Tatsumi, Naoyuki; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki, E-mail: nmiki@p.kanazawa-u.ac.jp

    2013-10-01

    Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two-thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases. - Highlights: • Plasma miRNA profiles were unambiguously different between smokers and non-smokers. • Smoking status might interfere with the diagnosis of disease using plasma miRNAs. • Changes of plasma miRNA profiles may be a signal of smoking-related diseases.

  10. RNA silencing and HIV: A hypothesis for the etiology of the severe combined immunodeficiency induced by the virus

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    Ludwig Linda B

    2008-09-01

    Full Text Available Abstract A novel intrinsic HIV-1 antisense gene was previously described with RNA initiating from the region of an HIV-1 antisense initiator promoter element (HIVaINR. The antisense RNA is exactly complementary to HIV-1 sense RNA and capable of forming ~400 base-pair (bp duplex RNA in the region of the long terminal repeat (LTR spanning the beginning portion of TAR in the repeat (R region and extending through the U3 region. Duplex or double-stranded RNA of several hundred nucleotides in length is a key initiating element of RNA interference (RNAi in several species. This HIVaINR antisense RNA is also capable of forming multiple stem-loop or hairpin-like secondary structures by M-fold analysis, with at least one that perfectly fits the criteria for a microRNA (miRNA precursor. MicroRNAs (miRNAs interact in a sequence-specific manner with target messenger RNAs (mRNAs to induce either cleavage of the message or impede translation. Human mRNA targets of the predicted HIVaINR antisense RNA (HAA microRNAs include mRNA for the human interleukin-2 receptor gamma chain (IL-2RG, also called the common gamma (γc receptor chain, because it is an integral part of 6 receptors mediating interleukin signalling (IL-2R, IL-4R, IL-7R, IL-9R, IL-15R and IL-21R. Other potential human mRNA targets include interleukin-15 (IL-15 mRNA, the fragile × mental retardation protein (FMRP mRNA, and the IL-1 receptor-associated kinase 1 (IRAK1 mRNA, amongst others. Thus the proposed intrinsic HIVaINR antisense RNA microRNAs (HAAmiRNAs of the human immunodeficiency virus form complementary targets with mRNAs of a key human gene in adaptive immunity, the IL-2Rγc, in which genetic defects are known to cause an X-linked severe combined immunodeficiency syndrome (X-SCID, as well as mRNAs of genes important in innate immunity. A new model of intrinsic RNA silencing induced by the HIVaINR antisense RNA in the absence of Tat is proposed, with elements suggestive of both small

  11. Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides

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    Li Yun-Yao

    2006-10-01

    Full Text Available Abstract Background Evaluation of microbicides for prevention of HIV-1 infection in macaque models for vaginal infection has indicated that the concentrations of active compounds needed for protection by far exceed levels sufficient for complete inhibition of infection in vitro. These experiments were done in the absence of seminal plasma (SP, a vehicle for sexual transmission of the virus. To gain insight into the possible effect of SP on the performance of selected microbicides, their anti-HIV-1 activity in the presence, and absence of SP, was determined. Methods The inhibitory activity of compounds against the X4 virus, HIV-1 IIIB, and the R5 virus, HIV-1 BaL was determined using TZM-bl indicator cells and quantitated by measuring β-galactosidase induced by infection. The virucidal properties of cellulose acetate 1,2-benzene-dicarboxylate (CAP, the only microbicide provided in water insoluble, micronized form, in the presence of SP was measured. Results The HIV-1 inhibitory activity of the polymeric microbicides, poly(naphthalene sulfonate, cellulose sulfate, carrageenan, CAP (in soluble form and polystyrene sulfonate, respectively, was considerably (range ≈ 4 to ≈ 73-fold diminished in the presence of SP (33.3%. Formulations of micronized CAP, providing an acidic buffering system even in the presence of an SP volume excess, effectively inactivated HIV-1 infectivity. Conclusion The data presented here suggest that the in vivo efficacy of polymeric microbicides, acting as HIV-1 entry inhibitors, might become at least partly compromised by the inevitable presence of SP. These possible disadvantages could be overcome by combining the respective polymers with acidic pH buffering systems (built-in for formulations of micronized CAP or with other anti-HIV-1 compounds, the activity of which is not affected by SP, e.g. reverse transcriptase and zinc finger inhibitors.

  12. Role of microRNA modulation in the interferon-α/ribavirin suppression of HIV-1 in vivo.

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    Mohamed Abdel-Mohsen

    Full Text Available Interferon-α (IFN-α treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV. We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037. Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

  13. Efficacy of raltegravir switching strategies in HIV-infected patients with suppressed viraemia according to the genotypic sensitivity score.

    Science.gov (United States)

    Caby, F; Schneider, L; Blanc, C; Soulié, C; Tindel, M; Peytavin, G; Agher, R; Valantin, M A; Tubiana, R; Wirden, M; Calvez, V; Marcelin, A G; Katlama, C

    2014-04-01

    The lack of antiretroviral (ARV) backbone activity associated with raltegravir has been proposed as the main explanation for virological relapse observed in patients with undetectable viraemia who are switched from a ritonavir-boosted protease inhibitor (PI) to raltegravir. However ARV activity remains difficult to assess in this context. The aim of our study was to precisely assess the ARV backbone activity in patients with undetectable viraemia who underwent raltegravir switching strategies and to evaluate the efficacy of such switching strategies based on the genotypic sensitivity score (GSS). Patients with a plasma human immunodeficiency virus type 1 (HIV-1) RNA level of HIV-1 RNA of the last plasma measurement with a HIV-1 RNA level of >50 copies/mL before the switch and on the results of all previous genotyping tests. The primary endpoint was the proportion of patients with a plasma HIV-1 RNA level of HIV-1 RNA level of HIV-RNA genotyping test results.

  14. Dynamic of Mixed HCV Infection in Plasma and PBMC of HIV/HCV Patients Under Treatment With Peg-IFN/Ribavirin.

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    Bagaglio, Sabrina; Uberti-Foppa, Caterina; Di Serio, Clelia; Trentini, Filippo; Andolina, Andrea; Hasson, Hamid; Messina, Emanuela; Merli, Marco; Porrino, Lucy; Lazzarin, Adriano; Morsica, Giulia

    2015-10-01

    The extent of mixed hepatitis C virus (HCV) genotype in different compartments (plasma and peripheral blood mononuclear cell, PBMC) and possible association with treatment efficacy in HIV/HCV coinfected patients remains to be unknown.The objective of this study was to elucidate the frequency of mixed genotype infection (MG), its profile in different compartments during anti-HCV treatment, and the possible influence of different genotypes on the response rate.The compartmentalization of HCV population was investigated by next-generation sequencing in 19 HIV/HCV coinfected patients under anti-HCV treatment with peginterferon/ribavirin (P-R). Ten individuals were nonresponder (NR) or relapser (RE) to P-R treatment and 9 had a sustained virological response (SVR).Eleven/nineteen (58%) patients had MG in plasma compartment. Ten or 12 patients infected by a difficult to treat genotype (DTG) 1 or 4 as dominant strain, had an MG, whereas only 1/7 individuals infected by easy to treat genotype (ETG) harbored a mixed genotype, P = 0.006. HCV-RNA was more frequently detected in PBMC of NR (10/10) than in those of SVR (5/9), P = 0.032. Mixed genotype infection was detected in 6/15 (40%) PBMC-positive cases and was not associated with P-R treatment response. By multivariate analysis, MG in plasma samples was the most important viral factor affecting the treatment response (P = 0.0237).Detection of MG in plasma of HIV/HCV coinfected patients seems to represent the major determinant of response to P-R treatment. This finding may have important clinical implication in light of the new therapeutic approach in HIV/HCV coinfected individuals suggesting that combination treatment with direct acting antivirals could be less effective in MG.

  15. The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

    Science.gov (United States)

    Booth, David S; Cheng, Yifan; Frankel, Alan D

    2014-12-08

    The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

  16. Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

    Science.gov (United States)

    Rivero, Matias; Cohen, Éric A.; Lopez-Lastra, Marcelo; Mouland, Andrew J.

    2013-01-01

    The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production. PMID:23861855

  17. Dual mechanisms of translation initiation of the full-length HIV-1 mRNA contribute to gag synthesis.

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    Anne Monette

    Full Text Available The precursor group-specific antigen (pr55(Gag is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES-mediated mechanisms. In support of this notion, pr55(Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55(Gag expression, the synthesis of other HIV-1 proteins, including that of pr160(Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55(Gag for virus assembly and production.

  18. Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

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    Vanesa Garcia

    Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

  19. Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment Comparação longitudinal entre cargas virais seminais e plasmáticas do HIV-1 durante terapia anti-retroviral

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    Lauro Ferreira da Silva Pinto Neto

    2003-12-01

    Full Text Available This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21. Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma and 3.2 log10 copies/ml (semen. At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.Este estudo foi desenhado para investigar o impacto do tratamento com anti-retrovirais na evolução das cargas virais plasmáticas e seminais do HIV-1. A carga viral do HIV-1 e a contagem de linfócitos T CD4+ e CD8+ foi determinada em amostras pareadas de sangue e sêmen de 36 pacientes virgem de tratamento nos dias 0, 45, 90 e 180 após o início da terapia. As curvas de declínio das cargas virais plasmática e seminal foram semelhantes (p= 0.21. As medianas da carga viral plasmática e seminal no pré-tratamento (dia 0 foram 4.95 e 4.48 log10 cópias/ml, respectivamente. Seis meses após o início da terapia, a mediana da carga viral plasmática era 3.15 log10 cópias/ml e a seminal 3.2 log10 cópias/ml. Neste mesmo periodo, 22 pacientes apresentavam carga viral abaixo de 400 cópias/ml no plasma e/ou sêmen, enquanto apenas 9 pacientes apresentavam carga viral abaixo do limite de detecção nos dois compartimentos.

  20. Assessment of Population-Based HIV RNA Levels in a Rural East African Setting Using a Fingerprick-Based Blood Collection Method

    OpenAIRE

    Jain, Vivek; Liegler, Teri; Kabami, Jane; Chamie, Gabriel; Clark, Tamara D; Black, Douglas; Geng, Elvin H.; Kwarisiima, Dalsone; Wong, Joseph K.; Abdel-Mohsen, Mohamed; Sonawane, Nitin; Aweeka, Francesca T.; THIRUMURTHY, Harsha; Petersen, Maya L; Charlebois, Edwin D.

    2012-01-01

    Population-based human immunodeficiency virus (HIV) RNA metrics can help estimate antiretroviral therapy effectiveness within a community. We developed a fingerprick-based viral load technique and measured population HIV RNA levels in a rural Ugandan community, providing the first report from a resource limited setting.

  1. Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects

    Science.gov (United States)

    Yeri, Ashish; Courtright, Amanda; Reiman, Rebecca; Carlson, Elizabeth; Beecroft, Taylor; Janss, Alex; Siniard, Ashley; Richholt, Ryan; Balak, Chris; Rozowsky, Joel; Kitchen, Robert; Hutchins, Elizabeth; Winarta, Joseph; McCoy, Roger; Anastasi, Matthew; Kim, Seungchan; Huentelman, Matthew; Van Keuren-Jensen, Kendall

    2017-01-01

    Interest in circulating RNAs for monitoring and diagnosing human health has grown significantly. There are few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. In this study, total extracellular RNA (exRNA) was isolated and sequenced from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 male college athletes ages 18–25 years. Many participants provided more than one sample, allowing us to investigate variability in an individual’s exRNA expression levels over time. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. The small RNA profile of each biofluid is distinct. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid, and profiles of miRNA are different for each biofluid. Not unexpectedly, saliva samples have high levels of exogenous sequence that can be traced to bacteria. These data significantly contribute to the current number of sequenced exRNA samples from normal healthy individuals. PMID:28303895

  2. Implications of HIV RNA structure for recombination, speciation, and the neutralism-selectionism controversy.

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    Forsdyke, Donald R

    2014-02-01

    The conflict between the needs to encode both a protein (impaired by non-synonymous mutation), and nucleic acid structure (impaired by synonymous or non-synonymous mutation), can sometimes be resolved in favour of the nucleic acid because its structure is critical for a selectively advantageous genome-wide activity--recombination. However, above a sequence difference threshold, recombination is impaired. It may then be advantageous for new species to arise. Building on the work of Grantham and others critical of the neutralist viewpoint, heuristic support for this hypothesis emerged from studies of the base composition and structure of retroviral genomes. The extreme enrichment in the purine A of the RNA of human immunodeficiency virus (HIV-1), parallels the mild purine-loading of the RNAs of most organisms, for which there is an adaptive explanation--immune evasion. However, human T cell leukaemia virus (HTLV-1), with the potential to invade the same host cell, shows extreme enrichment in the pyrimidine C. Assuming the low GC% HIV and the high GC% HTLV-1 to share a common ancestor, it was postulated that differences in GC% had arisen to prevent homologous recombination between these emerging lentiviral species. Sympatrically isolated by this intracellular reproductive barrier, prototypic HIV-1 seized the AU-rich (low GC%) high ground (thus committing to purine A rather than purine G). Prototypic HTLV-1 forwent this advantage and evolved an independent evolutionary strategy--similar to that of the GC%-rich Epstein-Barr virus--profound latency maintained by transcription of one purine-rich mRNA. The evidence supporting these interpretations is reviewed. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. An example of genetically distinct HIV type 1 variants in cerebrospinal fluid and plasma during suppressive therapy

    OpenAIRE

    Dahl, V.; Gisslen, M.; Hagberg, L.; Peterson, J.; Shao, W.; Spudich, S.; Price, RW; Palmer, S.

    2014-01-01

    We sequenced the genome of human immunodeficiency virus type 1 (HIV-1) recovered from 70 cerebrospinal fluid (CSF) specimens and 29 plasma samples and corresponding samples obtained before treatment initiation from 17 subjects receiving suppressive therapy. More CSF sequences than plasma sequences were hypermutants. We determined CSF sequences and plasma sequences in specimens obtained from 2 subjects after treatment initiation. In one subject, we found genetically distinct CSF and plasma seq...

  4. Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

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    Myers Michael P

    2011-07-01

    Full Text Available Abstract Background Central to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1 is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev posttranscriptional activator and the Rev response element (RRE. Results Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3 and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA. Conclusions In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.

  5. Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

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    Frange, Pierre; Meyer, Laurence; Jung, Matthieu; Goujard, Cecile; Zucman, David; Abel, Sylvie; Hochedez, Patrick; Gousset, Marine; Gascuel, Olivier; Rouzioux, Christine; Chaix, Marie-Laure

    2013-01-01

    Objective Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”). Methods Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively. Results Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors. Conclusions These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and

  6. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  7. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle.

    Directory of Open Access Journals (Sweden)

    Seyed Hanif Mahboobi

    Full Text Available Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC, which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV's reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA, computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  8. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle.

    Science.gov (United States)

    Mahboobi, Seyed Hanif; Javanpour, Alex A; Mofrad, Mohammad R K

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV's reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  9. Coordinated modulation of circulating miR-21 in HIV, HIV-associated pulmonary arterial hypertension, and HIV/HCV co-infection

    Science.gov (United States)

    Parikh, Victoria N.; Park, Joseph; Nikolic, Ivana; Channick, Richard; Yu, Paul B.; De Marco, Teresa; Hsue, Priscilla; Chan, Stephen Y.

    2015-01-01

    Dysregulation of microRNA-21 (miR-21) is independently associated with human immunodeficiency virus (HIV) infection, pulmonary arterial hypertension (PAH), and hepatitis C virus (HCV) infection. To assess expression of miR-21 in these overlapping comorbidities, we measured plasma miR-21 in HIV with and without PAH and then stratified by concomitant HCV infection. miR-21 was increased in HIV and HIV-PAH versus uninfected subjects, but did not differ between these groups. HIV/HCV co-infection correlated with even higher miR-21 levels within the HIV-infected population. These data reveal specific regulation of plasma miR-21 in HIV, HIV/HCV co-infection, and PAH and suggest that miR-21 may integrate complex disease-specific signaling in the setting of HIV infection. PMID:26473639

  10. Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient

    Science.gov (United States)

    Bordi, Licia; Castilletti, Concetta; Colavita, Francesca; Quartu, Serena; Nicastri, Emanuele; Lauria, Francesco Nicola; Petrosillo, Nicola; Lanini, Simone; Kobinger, Gary; Zumla, Alimuddin; Di Caro, Antonino; Ippolito, Giuseppe; Capobianchi, Maria Rosaria; Lalle, Eleonora

    2017-01-01

    An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract. PMID:28056096

  11. PD-1 predicts CD4 loss rate in chronic HIV-1 infection better than HIV RNA and CD38 but not in cryopreserved samples.

    Science.gov (United States)

    Holm, Malin; Pettersen, Frank Olav; Kvale, Dag

    2008-01-01

    The immunopathogenic factor programmed cell death 1 (PD-1) was compared to CD38 and HIV RNA in predicting actual CD4+ T cell loss rate indicative for clinical progression. This cross sectional exploratory study included 50 consecutive, healthy HIV-infected patients off antiretroviral therapy (ART); 43 had the required observation times > 12 months. PD-1 and CD38 were determined on various T cell subsets by FACS analyses in fresh and later in parallel cryopreserved samples. Here more rapid progressors were relatively defined by having CD4 loss rates < median at -45.7/microl/year. PD-1 and CD38 densities in fresh blood were lower (p<0.001) in patients on ART (n=14) and seronegative controls (n=8). CD4 loss rates correlated significantly to current HIV RNA (R=-0.30), CD38 (R=-0.33) and PD-1 densities (R=-0.38) on CD8+ T cells, and best to DeltaCD38, i.e. the difference in CD38 between the PD-1+CD8+ and CD8+ subsets (R=-0.51). PD-1 was highest on the CD27+CD28-CD8+ subset with best correlation to progression (R=-0.54) in rapid progressors. Logistic regression models from HIV RNA, CD38 and PD-1 predicting rapid progression included PD-1 as best independent variable in combination with DeltaCD38 or CD38, supported by similar results from multiple regression analyses. PD-1 did not correlate with any of the other candidate variables. Cryopreservation reduced the CD38+ and PD-1+ fractions but corresponding densities became more suppressed through a non-linear loss most pronounced in CD38hi/PD-1hi cells with loss of predictive power. In conclusion, PD-1 was the best independent predictor for CD4 loss rates in fresh blood compared with CD38 and HIV RNA.

  12. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

    Science.gov (United States)

    Tanaka, Motoko; Robinson, Bridget A; Chutiraka, Kasana; Geary, Clair D; Reed, Jonathan C; Lingappa, Jaisri R

    2015-12-09

    The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues

  13. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  14. Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis.

    Science.gov (United States)

    Ishaq, Musarat; Hu, Jiajie; Wu, Xiaoyun; Fu, Qiong; Yang, Yalin; Liu, Qingzhen; Guo, Deyin

    2008-07-01

    The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

  15. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  16. Effect of Raltegravir-containing Intensification on HIV Burden and T Cell Activation in Multiple Gut Sites of HIV+ Adults on Suppressive Antiretroviral Therapy

    Science.gov (United States)

    Yukl, Steven A.; Shergill, Amandeep; McQuaid, Kenneth; Gianella, Sara; Lampiris, Harry; Hare, C. Bradley; Pandori, Mark; Sinclair, Elizabeth; Günthard, Huldrych F.; Fischer, Marek; Wong, Joseph K.; Havlir, Diane V.

    2010-01-01

    Objective To determine whether raltegravir-containing antiretroviral therapy (ART) intensification reduces HIV levels in the gut. Design Open-label study in HIV+ adults on ART with plasma HIV RNA<40 copies/ml. Methods Seven HIV+ adults received 12 weeks of ART intensification with raltegravir alone or in combination with efavirenz or darunavir. Gut cells were obtained by upper and lower endoscopy with biopsies from duodenum, ileum, colon, and rectum at baseline and 12 weeks. Study outcomes included plasma HIV RNA, HIV DNA and RNA from PBMC and 4 gut sites, T cell subsets, and activation markers. Results Intensification produced no consistent decrease in HIV RNA in the plasma, PBMC, duodenum, colon, or rectum. However, 5 of 7 participants had a decrease in unspliced HIV RNA per 106 CD4+ T cells in the ileum. There was a trend towards decreased T cell activation in all sites, which was greatest for CD8+ T cells in the ileum and PBMC, and a trend towards increased CD4+ T cells in the ileum. Conclusion Most HIV RNA and DNA in the blood and gut is not the result of ongoing replication that can be impacted by short-term intensification with raltegravir. However, the ileum may support ongoing productive infection in some patients on ART, even if the contribution to plasma RNA is not discernible. PMID:20827162

  17. High plasma levels of intact and cleaved soluble urokinase receptor reflect immune activation and are independent predictors of mortality in HIV-1-infected patients

    DEFF Research Database (Denmark)

    Ostrowski, Sisse Rye; Piironen, Timo; Høyer-Hansen, Gunilla

    2005-01-01

    ). In multivariate Cox analysis adjusting for CD4+ count, HIV RNA, beta2-microglobulin, hemoglobin and clinical stage, higher levels of suPAR(I-III) and suPAR(II-III) were independent predictors of increased mortality risk (P ...BACKGROUND: High blood levels of soluble urokinase receptor (suPAR) measured by enzyme-linked immunoassay (ELISA) (bulk measurement of 3-domain and 2-domain suPAR [suPAR(I-III), suPAR(II-III)], and suPAR(I-III) ligand complexes) strongly predict mortality in HIV-1-infected patients. This study...... investigated plasma levels of suPAR(I-III), suPAR(II-III), and 1-domain suPAR [suPAR(I)] and their predictive value for survival in HIV patients. METHODS: Plasma suPAR was measured by ELISA and 3 different time-resolved fluorescence immunoassays detecting suPAR(I-III), suPAR(I-III) plus suPAR(II-III), and su...

  18. Thromboelastography on plasma reveals delayed clot formation and accelerated clot lyses in HIV-1 infected persons compared with healthy controls

    DEFF Research Database (Denmark)

    Rönsholt, Frederikke Falkencrone; Gerstoft, Jan; Ullum, Henrik;

    2015-01-01

    in the hemostatic system in a pro-coagulant direction based on measurements of isolated components of the coagulation pahways. In disease conditions, the flowing blood may change from "normal" to hyper- or hypocoagulant or to hyper- or hypofibrinolytic. A balance may exist in the flowing blood, i.e. between blood......BACKGROUND: Thromboembolic events among HIV infected persons are a recognized clinical problem but the underlying mechanisms are poorly understood. To assess whether coagulation and fibrinolysis differ between long-term treated HIV infected individuals (HIV+) and healthy controls (CON), we...... =-0.651, p = 0.012). DISCUSSION: No previous studies have examined plasma coagulation by TEG in HIV, however, we have previously demonstrated that HIV+ display hypocoagulability in whole blood by TEG in accordance with the results of this study. Others have reported of HIV associated changes...

  19. Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual.

    Science.gov (United States)

    Khan, Lubina; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Nair, Ambili; Andrabi, Raiees; Clark, Brenda E; Auyeung, Kate; Bhattacharya, Jayanta; Vajpayee, Madhu; Wig, Naveet; Pantophlet, Ralph; Luthra, Kalpana

    2015-01-01

    Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

  20. Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual.

    Directory of Open Access Journals (Sweden)

    Lubina Khan

    Full Text Available Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9% studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO revealed that five (AIIMS 617, 619, 627, 642, 660 contained RSC3-reactive plasma antibodies and only one (AIIMS 660 contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

  1. Isolation and characterization of 20'-F-RNA aptamers against whole HIV-1 subtype C envelope pseudovirus

    CSIR Research Space (South Africa)

    London, GM

    2015-01-01

    Full Text Available Aptamers, which are artificial nucleic acid ligands akin to antibodies in function, represent a new class of molecules that can prevent HIV infection. In this study, we isolated RNA aptamers against whole HV-1CAP45 enveloped pseudotyped virus...

  2. A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability

    NARCIS (Netherlands)

    M. Lutzelberger; L.S. Reinert; A.T. Das; B. Berkhout; J. Kjems

    2006-01-01

    Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-c

  3. Maternal characteristics during pregnancy and risk factors for positive HIV RNA at delivery: a single-cohort observational study (Brescia, Northern Italy

    Directory of Open Access Journals (Sweden)

    Magoni Michele

    2011-02-01

    Full Text Available Abstract Background Detectable HIV RNA in mothers at delivery is an important risk factor for HIV transmission to newborns. Our hypothesis was that, in migrant women, the risk of detectable HIV RNA at delivery is greater owing to late HIV diagnosis. Therefore, we examined pregnant women by regional provenance and measured variables that could be associated with detectable HIV RNA at delivery. Methods A observational retrospective study was conducted from January 1999 to May 2008. Univariate and multivariable regression analyses (generalized linear models were used, with detectable HIV RNA at delivery as dependent variable. Results The overall population comprised 154 women (46.8% migrants. Presentation was later in migrant women than Italians, as assessed by CD4-T-cell count at first contact (mean 417/mm3 versus 545/mm3, respectively; p = 0.003. Likewise, HIV diagnosis was made before pregnancy and HAART was already prescribed at the time of pregnancy in more Italians (91% and 75%, respectively than migrants (61% and 42.8%, respectively. A subgroup of women with available HIV RNA close to term (i.e., ≤30 days before labour was studied for risk factors of detectable HIV RNA (≥50 copies/ml at delivery. Among 93 women, 25 (26.9% had detectable HIV RNA. A trend toward an association between non-Italian nationality and detectable HIV RNA at delivery was demonstrated by univariate analysis (relative risk, RR = 1.86; p = 0.099. However, by multivariable regression analysis, the following factors appeared to be more important: lack of stable (i.e., ≥14 days antiretroviral therapy at the time of HIV RNA testing (RR = 4.3; p 3, RR = 0.94; p = 0.038. Conclusions These results reinforce the importance of extensive screening for HIV infection, earlier initiation of antiretroviral therapy and stricter monitoring of pregnant women to reduce the risk of detectable HIV RNA at delivery. Public health interventions should be particularly targeted to migrant

  4. Cytokine expression during syphilis infection in HIV-1-infected individuals

    DEFF Research Database (Denmark)

    Knudsen, Andreas; Benfield, Thomas; Kofoed, Kristian

    2009-01-01

    syphilis.IL-10 and TNF-alpha levels correlated positively with plasma HIV RNA values at the time of diagnosis (r = 0.38, P = 0.023, and r = 0.64, P ... stage syphilis coinfection were associated with an increase in IL-10. IL-10 and TNF-alpha both decreased after treatment of syphilis. TNF-alpha and IL-10 correlated with low CD4 T cell counts and high plasma HIV RNA values....

  5. Altered relationship of plasma triglycerides to HDL cholesterol in patients with HIV/HAART-associated dyslipidemia: further evidence for a unique form of metabolic syndrome in HIV patients.

    Science.gov (United States)

    Vu, Catherine N; Ruiz-Esponda, Raul; Yang, Eric; Chang, Evelyn; Gillard, Baiba; Pownall, Henry J; Hoogeveen, Ron C; Coraza, Ivonne; Balasubramanyam, Ashok

    2013-07-01

    Plasma triglycerides (TG) and HDL-C are inversely related in Metabolic Syndrome (MetS), due to exchange of VLDL-TG for HDL-cholesteryl esters catalyzed by cholesteryl ester transfer protein (CETP). We investigated the relationship of TG to HDL-C in highly-active antiretroviral drug (HAART)-treated HIV patients. Fasting plasma TG and HDL-C levels were compared in 179 hypertriglyceridemic HIV/HAART patients and 71 HIV-negative persons (31 normotriglyceridemic (NL) and 40 hypertriglyceridemic due to type IV hyperlipidemia (HTG)). CETP mass and activity were compared in 19 NL and 87 HIV/HAART subjects. Among the three groups, a plot of HDL-C vs. TG gave similar slopes but significantly different y-intercepts (9.24±0.45, 8.16±0.54, 6.70±0.65, sqrt(HDL-C) for NL, HIV and HTG respectively; P<0.001); this difference persisted after adjusting HDL-C for TG, age, BMI, gender, glucose, CD4 count, viral load and HAART strata (7.18±0.20, 6.20±0.05 and 4.55±0.15 sqrt(HDL-C) for NL, HIV and HTG, respectively, P<0.001). CETP activity was not different between NL and HIV, but CETP mass was significantly higher in HIV (1.47±0.53 compared to 0.93±0.27μg/mL, P<0.0001), hence CETP specific activity was lower in HIV (22.67±13.46 compared to 28.46±8.24nmol/μg/h, P=0.001). Dyslipidemic HIV/HAART patients have a distinctive HDL-C plasma concentration adjusted for TG. The weak inverse relationship between HDL-C and TG is not explained by altered total CETP activity; it could result from a non-CETP-dependent mechanism or a decrease in CETP function due to inhibitors of CETP activity in HIV patients' plasma. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Differences in HIV Burden and Immune Activation within the Gut of HIV+ Patients on Suppressive Antiretroviral Therapy

    Science.gov (United States)

    Yukl, Steven; Gianella, Sara; Sinclair, Elizabeth; Epling, Lorrie; Li, Qingsheng; Duan, Lijie; Choi, Alex L. M.; Girling, Valerie; Ho, Terence; Li, Peilin; Fujimoto, Katsuya; Lampiris, Harry; Hare, C. Bradley; Pandori, Mark; Haase, Ashley T.; Günthard, Huldrych F.; Fischer, Marek; Shergill, Amandeep; McQuaid, Kenneth; Havlir, Diane V.; Wong, Joseph K.

    2010-01-01

    Background The gut is a major reservoir for HIV in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV. Methods In 8 HIV-1+ adults on ART with CD4>200 and plasma VL<40, levels of HIV and T-cell activation were measured in blood and endoscopic biopsies from the duodenum, ileum, right colon, and rectum. Results HIV DNA and RNA per CD4+T-cell were higher in all four gut sites compared to blood. HIV DNA increased from the duodenum to the rectum, while the median HIV RNA peaked in the ileum. HIV DNA correlated positively with T-cell activation in the PBMC but negatively with T-cell activation in the gut. Multiply-spliced RNA was infrequently detected in gut, and unspliced RNA/DNA ratios were lower in the colon and rectum relative to PBMC, reflecting paradoxically low HIV transcription given the higher T-cell activation in the gut. Conclusions HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA and T-cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut. PMID:20939732

  7. Modest attenuation of HIV-1 Vpu alleles derived from elite controller plasma.

    Directory of Open Access Journals (Sweden)

    Jingyan Chen

    Full Text Available In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1 can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.

  8. Soluble urokinase receptor levels in plasma during 5 years of highly active antiretroviral therapy in HIV-1-infected patients

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Katzenstein, Terese L; Piironen, Timo;

    2004-01-01

    High blood levels of the soluble urokinase receptor (suPAR) strongly predict increased mortality in human immunodeficiency virus-1 (HIV-1)-infected patients. This study investigated the plasma concentration of suPAR in 29 treatment-naive HIV-1-infected patients during 5 years treatment with highly...... active antiretroviral therapy (HAART). Plasma suPAR decreased after introducing HAART, most pronounced during the first treatment year. The change in plasma suPAR was independent of changes in viral replication and CD4+ cells but it was strongly correlated with plasma levels of the soluble TNF receptor...... is linked to inflammation in untreated as well as HAART-treated HIV-1-infected patients....

  9. Associations between HIV-RNA-based indicators and virological and clinical outcomes

    DEFF Research Database (Denmark)

    Laut, Kamilla G.; Shepherd, Leah C.; Pedersen, Court

    2016-01-01

    Objectives: To evaluate and compare the performance of six HIV-RNA-based quality of care indicators for predicting short-term and long-term outcomes. Design: Multinational cohort study. Methods: We included EuroSIDA patients on antiretroviral therapy (ART) with at least three viral load...... rate ratios for all outcomes tended to increase with increasing viraemia copy years, number of consecutive months with viral load ≥50 copies/ml, current viral load and with lower %FS, but the gradient of increased risk was weak across strata. None of the indicators reliably identified those at risk...... of long-term outcomes (AUC 0.54-0.58), but performed consistently better with short-term outcomes [triple class failure (AUC 0.67-0.76) and resistance (AUC 0.64-0.79)]. Goodness of fit varied with the outcome evaluated, but differences between indicators were small. Conclusion: Differences between quality...

  10. Plasma microRNA profiles distinguish lethal injury in acetaminophen toxicity: A research study

    Institute of Scientific and Technical Information of China (English)

    Jeanine Ward; Shashi Bala; Jan Petrasek; Gyongyi Szabo

    2012-01-01

    AIM:To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice.METHODS:Using plasma from APAP poisoned mice,either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed,we screened commercially available murine microRNA libraries (SABiosciences,Qiagen Sciences,MD) to evaluate for unique miRNA profiles between these two dosing parameters.RESULTS:We distinguished numerous,unique plasma miRNAs both up- and downregulated in lethally compared to sublethally dosed mice.Of note,many of the greatest up- and downregulated miRNAs,namely 574-5p,466g,466f-3p,375,29c,and 148a,have been shown to be associated with asthma in prior studies.Interestingly,a relationship between APAP and asthma has been previously well described in the literature,with an as yet unknown mechanism of pathology.There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point (P <0.001).There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point (P =0.011).CONCLUSION:We identified unique plasma miRNAs both up- and downregulated in APAP poisoning which are correlated to asthma development.

  11. Association between the miRNA Signatures in Plasma and Bronchoalveolar Fluid in Respiratory Pathologies

    Directory of Open Access Journals (Sweden)

    Sonia Molina-Pinelo

    2012-01-01

    Full Text Available The identification of new less invasive biomarkers is necessary to improve the detection and prognostic outcome of respiratory pathological processes. The measurement of miRNA expression through less invasive techniques such as plasma and serum have been suggested to analysis of several lung malignancies including lung cancer. These studies are assuming a common deregulated miRNA expression both in blood and lung tissue. The present study aimed to obtain miRNA representative signatures both in plasma and bronchoalveolar cell fraction that could serve as biomarker in respiratory diseases. Ten patients were evaluated to assess the expression levels of 381 miRNAs. We found that around 50% miRNAs were no detected in both plasma and bronchoalveolar cell fraction and only 20% of miRNAs showed similar expression in both samples. These results show a lack of association of miRNA signatures between plasma and bronchoalveolar cytology in the same patient. The profiles are not comparable; however, there is a similarity in the relative expression in a very small subset of miRNAs (miR-17, miR-19b, miR-195 and miR-20b between both biological samples in all patients. This finding supports that the miRNAs profiles obtained from different biological samples have to be carefully validated to link with respiratory diseases.

  12. HIV-1 genomic RNA diversification following sexual and parenteral virus transmission.

    Science.gov (United States)

    Wolfs, T F; Zwart, G; Bakker, M; Goudsmit, J

    1992-07-01

    Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by epidemiological history and taken within a time bracket of three months of the recipient seroconversion, were analyzed by polymerase chain reaction amplification followed by sequencing of eight cDNA clones of 276 bp, including the V3 coding region. The sequence populations of the recipients were without exception homogeneous, while the sequence populations of the transmitters showed varying degrees of heterogeneity. Nucleotide distance between consensus sequences of unrelated individuals from the Amsterdam population (interpatient variation) averaged 11% (range 7-15%). The largest distance between two clonal sequences of one individual (intrapatient variation) was also 11%. Consensus sequences of five recipients differed by only 0-1% from the consensus sequence of the presumed transmitter, including two pairs of which the transmission was either proven or highly probable. This contrasted with a difference of 10-12% in two pairs, casting doubt on the epidemiological relatedness. Antibody reactivity to a panel of V3 peptides with varying degrees of similarity to the V3 sequences obtained did not augment the discriminatory power of sequence analysis. Results of the sequential sequencing of samples of one transmitter suggest that this was due to an anamnestic antibody response of the transmitter to early variants. From the loss of sequence heterogeneity following transmission and the consensus sequence similarities observed within five transmitter-recipient pairs, we conclude that HIV-1 transmission results in the selection of a limited number of genomes carrying on the infection in the new host, but does not generally lead to a shift in the sequence population as defined by

  13. Cyclic PNA-based compound directed against HIV-1 TAR RNA: modelling, liquid-phase synthesis and TAR binding.

    Science.gov (United States)

    Depecker, Geoffrey; Patino, Nadia; Di Giorgio, Christophe; Terreux, Raphael; Cabrol-Bass, Daniel; Bailly, Christian; Aubertin, Anne-Marie; Condom, Roger

    2004-01-07

    A cyclic molecule including a hexameric PNA sequence has been designed and synthesized in order to target the TAR RNA loop of HIV-1 through the formation of a "kissing complex". For comparison, its linear analogue has also been investigated. The synthesis of the cyclic and linear PNA has been accomplished following a liquid-phase strategy using mixed PNA and fully N-protected (aminoethylglycinamide) fragments. The interactions of this cyclic PNA and its linear analogue with TAR RNA have been studied and the results indicate clearly that no interaction occurs between the cyclic antisense PNA and TAR RNA, whereas a tenuous interaction has been detected with its linear PNA analogue.

  14. Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease

    Directory of Open Access Journals (Sweden)

    Chi-Sheng Wu

    2016-01-01

    Full Text Available Background. Various microRNAs (miRNAs are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25 U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25 U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis.

  15. Maternal And Neonatal Plasma MicroRNA Biomarkers For Fetal Alcohol Exposure In An Ovine Model

    Science.gov (United States)

    Balaraman, Sridevi; Lunde, E. Raine; Sawant, Onkar; Cudd, Timothy A.; Washburn, Shannon E.; Miranda, Rajesh C.

    2014-01-01

    Background Plasma or circulating miRNAs (cirmiRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. Methods Pregnant sheep were exposed to a binge model of ethanol consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a three-trimester equivalent period from gestational day (GD) 4 to GD 132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte and leukocytes obtained from non-pregnant ewes, and plasma from pregnant ewes 24 hours following the last binge ethanol episode, and from newborn lambs, at birth on ~GD 147. Results Pregnant ewe and newborn lamb cirmiRNA profiles were similar to each other and different from non-pregnant female plasma, erythrocyte or leukocyte miRNAs. Significant changes in cirmiRNA profiles were observed in the ethanol-exposed ewe, and at birth, in the in utero, ethanol-exposed lamb. CirmiRNAs including miR-9, -15b, -19b and -20a were sensitive and specific measures of ethanol exposure in both pregnant ewe and newborn lamb. Additionally, ethanol exposure altered guide to passenger strand cirmiRNA ratios in the pregnant ewe, but not in the lamb. Conclusion Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. CirmiRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment. PMID:24588274

  16. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    Science.gov (United States)

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

  17. Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA.

    Science.gov (United States)

    Boltz, Valerie F; Rausch, Jason; Shao, Wei; Hattori, Junko; Luke, Brian; Maldarelli, Frank; Mellors, John W; Kearney, Mary F; Coffin, John M

    2016-12-20

    Although next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the "gold-standard" of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth. Primer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations. First, the resultant cDNA was divided and NGS libraries were generated in parallel using two methods: uSGS and a method applying long PCR primers to attach the NGS adaptors (LP-PCR-1). Second, cDNA was divided and NGS libraries were generated in parallel comparing 3 methods: uSGS and 2 methods adapted from more recent reports using variations of the long PCR primers to attach the adaptors (LP-PCR-2 and LP-PCR-3). Consistently, the uSGS method amplified a greater proportion of cDNAs, averaging 30% compared to 13% for LP-PCR-1, 21% for LP-PCR-2 and 14% for LP-PCR-3. Most importantly, when the uSGS sequences were binned according to their primer IDs, 94% of the bins did not contain PCR recombinant sequences versus only 55, 75 and 65% for LP-PCR-1, 2 and 3, respectively. Finally, when uSGS was applied to plasma samples from HIV-1 infected donors, both frequent and rare variants were detected in each sample and neighbor-joining trees revealed clusters of genomes driven by the linkage of these mutations, showing the lack of PCR recombinants in the datasets. The uSGS assay can be used for accurate detection of rare variants and for identifying linkage of rare alleles associated with HIV-1 drug resistance. In addition

  18. On-chip purification and detection of hepatitis C virus RNA from human plasma.

    Science.gov (United States)

    Vaghi, V; Potrich, C; Pasquardini, L; Lunelli, L; Vanzetti, L; Ebranati, E; Lai, A; Zehender, G; Mombello, D; Cocuzza, M; Pirri, C F; Pederzolli, C

    2016-01-01

    Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Correlation between lamivudine plasma concentrations and patient self-reported adherence to antiretroviral treatment in experienced HIV patients

    Directory of Open Access Journals (Sweden)

    Minzi OM

    2011-11-01

    Full Text Available OM Minzi1, V Mugoyela2, LL Gustafsson31Unit of Pharmacology and Therapeutics, 2Department of Medicinal Chemistry, School of Pharmacy, Muhimbili University of Health and Allied Sciences, Dar Es Salaam, Tanzania; 3Division of Clinical Pharmacology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenBackground: Adherence to antiretroviral treatment (ART is important to achieve treatment success in human immunodeficiency virus (HIV-infected patients. Most HIV clinics apply the patient self-report (PSR method. However, the reliability of this method in experienced HIV patients remains questionable.Purpose: To validate the PSR method for measuring adherence to ART using lamivudine (3TC plasma concentrations in experienced HIV patients.Methods: The study was conducted in Dar Es Salaam and involved 220 patients who were receiving ART services at HIV clinics for more than 12 months. Self-reported adherence information to ART was obtained on the day of HIV clinic visit. The patients were asked to mention the number of doses missed within the past 7 days. In addition, blood samples (2 mL were collected from each patient on the same day. The blood samples were determined for 3TC plasma concentrations. The target 3TC plasma concentration as indicator concentration for adherent patients was determined in 20 patients who took their evening dose of antiretrovirals under supervision. The blood from these patients was drawn 3 hours after drug administration.Results: Complete drug levels of 3TC and self-reported adherence data was obtained in 200 treatment-experienced HIV patients. Lamivudine plasma concentrations obtained in these patients ranged between 0.02–17.36 µg/mL. The mean time from dose administration to blood drawing was 3.1 ± 1.2 hours with coefficient of variation >39%. The mean 3TC plasma concentration obtained in 20 patients who took their antiretroviral dose under supervision was

  20. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Larsen, Carsten Schade;

    2000-01-01

    Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma...... of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  1. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C

    2000-01-01

    chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta......Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma...... of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...

  2. Genomic HIV RNA induces innate immune responses through RIG-I-dependent sensing of secondary-structured RNA

    NARCIS (Netherlands)

    Berg, R.K.; Melchjorsen, J.; Rintahaka, J.; Diget, E.; Søby, S.; Horan, K.A.; Gorelick, R.J.; Matikainen, S.; Larsen, C.S.; Ostergaard, L.; Paludan, S.R.; Mogensen, T.H.

    2012-01-01

    BACKGROUND: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses la

  3. Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

    Science.gov (United States)

    Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

    2017-02-01

    HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.

  4. Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

    Directory of Open Access Journals (Sweden)

    Friew Yeshitila N

    2009-06-01

    Full Text Available Abstract Background Host restriction factor APOBEC3G (A3G blocks human immunodeficiency virus type 1 (HIV-1 replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2 mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22 to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.

  5. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  6. Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy.

    Science.gov (United States)

    Anderson, Joseph S; Javien, John; Nolta, Jan A; Bauer, Gerhard

    2009-12-01

    Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

  7. HIV-infected former plasma donors in rural Central China: from infection to survival outcomes, 1985-2008.

    Directory of Open Access Journals (Sweden)

    Zhihui Dou

    Full Text Available BACKGROUND: The HIV epidemic among former plasma donors (FPDs in rural Central China in the early-mid 1990s is likely the largest known HIV-infected cohort in the world related to commercial plasma donation but has never been fully described. The objectives of this study are to estimate the timing and geographic spread of HIV infection in this cohort and to demonstrate the impact of antiretroviral therapy on survival outcomes. METHODOLOGY/PRINCIPAL FINDINGS: HIV-infected FPDs were identified using the national HIV epidemiology and treatment databases. Locations of subjects were mapped. Dates of infection and survival were estimated using the midpoint date between initial-final plasma donation dates from 1985-2008 among those with plasma donation windows ≤2 years. Among 37,084 FPDs in the two databases, 36,110 were included. 95% were located in focal areas of Henan Province and adjacent areas of surrounding provinces. Midpoint year between initial-final plasma donation dates was 1994 among FPDs with known donation dates. Median survival from infection to AIDS was 11.8 years and, among those not treated, 1.6 years from AIDS to death. Among those on treatment, 71% were still alive after five years. Using Cox proportional hazard modeling, untreated AIDS patients were 4.9 times (95% confidence interval 4.6-5.2 more likely to die than those on treatment. CONCLUSIONS/SIGNIFICANCE: The epidemic of HIV-infected FPD in China was not widespread throughout China but rather was centered in Henan Province and the adjacent areas of surrounding provinces. Even in these areas, infections were concentrated in focal locations. Overall, HIV infections in this cohort peaked in 1994, with median survival of 13.4 years from infection to death among those not treated. Among AIDS patients on treatment, 71% were still alive after five years.

  8. Control of HIV-1 in Elite Suppressors despite Ongoing Replication and Evolution in Plasma Virus▿

    OpenAIRE

    O'Connell, Karen A; Brennan, Timothy P.; Bailey, Justin R.; Ray, Stuart C.; Robert F. Siliciano; Blankson, Joel N.

    2010-01-01

    A subset of HIV-1-infected patients known as elite controllers or suppressors (ES) control the virus naturally. We have previously demonstrated sequence discordance between proviral and plasma gag clones in ES, much of which can be attributed to selective pressure from the host (J. R. Bailey, T. M. Williams, R. F. Siliciano, and J. N. Blankson, J. Exp. Med. 203:1357-1369, 2006). However, it is not clear whether ongoing viral replication continues in ES once the control of viremia has been est...

  9. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

    Directory of Open Access Journals (Sweden)

    Lara Ajamian

    2015-10-01

    Full Text Available Unspliced, genomic HIV-1 RNA (vRNA is a component of several ribonucleoprotein complexes (RNP during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1, a key factor in nonsense-mediated mRNA decay (NMD, colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

  10. Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.

    Directory of Open Access Journals (Sweden)

    Yu Yamaura

    Full Text Available MicroRNAs (miRNAs are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis and chronic liver injury (steatosis, steatohepatitis and fibrosis using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.

  11. MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism

    Directory of Open Access Journals (Sweden)

    Liu Yi

    2011-09-01

    Full Text Available Abstract Background Acute pulmonary embolism (APE remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown. Methods Thirty-two APE patients, 32 healthy controls, and 22 non-APE patients (reported dyspnea, chest pain, or cough were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA identified as the candidate biomarker. Results Plasma miRNA-134 (miR-134 level was significantly higher in the APE patients than in the healthy controls or non-APE patients. The ROC curve showed that plasma miR-134 was a specific diagnostic predictor of APE with an area under the curve of 0.833 (95% confidence interval, 0.737 to 0.929; P Conclusions Our findings indicated that plasma miR-134 could be an important biomarker for the diagnosis of APE. Because of this finding, large-scale investigations are urgently needed to pave the way from basic research to clinical utilization.

  12. Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitrios

    2011-01-01

    Full Text Available Abstract Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0 assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD difference of -0.206 (0.298 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD difference: 0.160 (0.287 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86% differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3

  13. IP-10 predicts the first phase decline of HCV RNA and overall viral response to therapy in patients co-infected with chronic hepatitis C virus infection and HIV

    DEFF Research Database (Denmark)

    Falconer, Karolin; Askarieh, Galia; Weis, Nina Margrethe

    2010-01-01

    The aim of this study was to investigate the utility of baseline plasma interferon-gamma inducible protein-10 (IP-10) levels in human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected patients. Baseline IP-10 was monitored during HCV combination therapy in 21 HIV-HCV co-infected pa......The aim of this study was to investigate the utility of baseline plasma interferon-gamma inducible protein-10 (IP-10) levels in human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected patients. Baseline IP-10 was monitored during HCV combination therapy in 21 HIV-HCV co......-infected patients (HCV genotype 1 (n = 16), 2 (n = 2), and 3 (n = 3)). Lower baseline IP-10 was significantly associated with a rapid decline in HCV RNA, in particular with the first phase reduction, and similar cut-off levels ( 600 pg/ml) as in HCV mono-infected patients apply. In conclusion, baseline IP...

  14. Immunoelectron microscopic evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Jason Hammonds

    2010-02-01

    Full Text Available Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence.

  15. The miRNA plasma signature in response to acute aerobic exercise and endurance training.

    Directory of Open Access Journals (Sweden)

    Søren Nielsen

    Full Text Available MiRNAs are potent intracellular posttranscriptional regulators and are also selectively secreted into the circulation in a cell-specific fashion. Global changes in miRNA expression in skeletal muscle in response to endurance exercise training have been reported. Therefore, our aim was to establish the miRNA signature in human plasma in response to acute exercise and chronic endurance training by utilizing a novel methodological approach. RNA was isolated from human plasma collected from young healthy men before and after an acute endurance exercise bout and following 12 weeks of endurance training. Global miRNA (742 miRNAs measurements were performed as a screening to identify detectable miRNAs in plasma. Using customized qPCR panels we quantified the expression levels of miRNAs detected in the screening procedure (188 miRNAs. We demonstrate a dynamic regulation of circulating miRNA (ci-miRNA levels following 0 hour (miR-106a, miR-221, miR-30b, miR-151-5p, let-7i, miR-146, miR-652 and miR-151-3p, 1 hour (miR-338-3p, miR-330-3p, miR-223, miR-139-5p and miR-143 and 3 hours (miR-1 after an acute exercise bout (P<0.00032. Where ci-miRNAs were all downregulated immediately after an acute exercise bout (0 hour the 1 and 3 hour post exercise timepoints were followed by upregulations. In response to chronic training, we identified seven ci-miRNAs with decreased levels in plasma (miR-342-3p, let-7d, miR-766, miR-25, miR-148a, miR-185 and miR-21 and two miRNAs that were present at higher levels after the training period (miR-103 and miR-107 (P<0.00032. In conclusion, acute exercise and chronic endurance training, likely through specific mechanisms unique to each stimulus, robustly modify the miRNA signature of human plasma.

  16. A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models.

    Directory of Open Access Journals (Sweden)

    Sandhya Boyapalle

    Full Text Available Human immunodeficiency virus (HIV types 1 and 2 (HIV-1 and HIV-2 are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162 viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections.

  17. A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models.

    Science.gov (United States)

    Boyapalle, Sandhya; Xu, Weidong; Raulji, Payal; Mohapatra, Subhra; Mohapatra, Shyam S

    2015-01-01

    Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections.

  18. Application on pooled HIV-1 RNA RT-PCR in detecting acute HIV infection among MSM in Guangzhou%集合PCR在检测MSM HIV急性期感染中的应用

    Institute of Scientific and Technical Information of China (English)

    陈绮珊; 韩志刚; 徐慧芳; 梁彩云; 高凯

    2013-01-01

    目的 应用集合聚合酶链反应(PCR)方法,发现广州市男男性行为人群(MSM)中艾滋病病毒(HIV)急性感染者,并估计该人群HIV感染的发病率.方法 采用10∶1、5∶1、1∶1三级集合HIV-1核糖核酸(RNA)反转录PCR(RT-PCR)方法进行检测.结果 2008年,检测1 250份MSM HIV-1抗体阴性血浆,发现HIV-1 RNA 阳性3例,追踪随访,2例HIV-1抗体阳转,1例失访;初步估计HIV-1年发病率约为8.50%[95%可信区间(CI):1.75%~24.86%].2009年,检测1002份MSM HIV-1抗体阴性血浆,发现HIV-1 RNA阳性4例,追踪随访,4例均HIV-1抗体阳转;估计HIV-1年发病率约为14.17%(95%CI:3.85%~36.21%).结论 集合HIV-1 RNA RT-PCR是发现急性感染者的有效方法,并可估计人群HIV发病率.%Objective To detect acute human immunodeficiency virus (HIV) infection (AHI) and estimate HIV-1 incidence among men who have sex with men (MSM) in Guangzhou using pooled polymerase chain reaction (PCR).Methods Pooled HIV-1 ribonucleic acid (RNA) RT-PCR (3-staged pooling by 10 ∶ 1,5 ∶ 1,1 ∶ 1) was conducted.Results Three positives for HIV-1 RNA were found among 1250 HIV antibody negative samples collected from MSM in 2008,and four RNA positives were found among 1002 HIV antibody negative samples collected from MSM in 2009.Six of above seven RNA positive individuals seroconverted to HIV-1 in following up visits except one lost to follow-up.Estimated HIV incidence among MSM was 8.50% [95% conficence interval (CI),1.75%~24.86%] in2008 and 14.17% (95%CI,3.85%~36.21%) in 2009,respectively.Conclusion Pooled HIV-1 RNA RT-PCR is an available method for detecting acute HIV infection,and can be applied in estimating population' s HIV incidence.

  19. HIV-1 vif基因的RNA干扰体外研究%In vitro study of RNA interference on vif gene of HIV-1

    Institute of Scientific and Technical Information of China (English)

    江雪艳; 卢洪洲; 陈秋丽; 潘卫; 张云智; 沈银忠

    2009-01-01

    目的 利用RNAi技术对HIV-1 vif基因的转录后水平进行下凋,达到抑制vif蛋白表达的目的 ,为新的抗HIV治疗和预防研究提供理论基础.方法 通过体外转录法合成siRNA,与转接有vif基因的表达质粒共同转染HEK 293T细胞,荧光显微镜下观察干扰效果,并利用Real-time PCR和Western blot对HIV-1 vif分别从转录和表达水平进行验证.结果 将pEGFP-N1-vif重组载体转染HEK 293T细胞后,结果显示vif蛋白可以在细胞中高水平表达;针对vif设计的3段特异的siRNA可以有效且特异地降低vif蛋白的表达.结论 RNAi技术可以下调HIV-1蛋白的表达,此技术可以作为一项新的治疗和预防HIV-1的方法用于进一步的研究.

  20. Stability of hepatitis C virus (HCV) RNA levels among interferon-naïve HIV/HCV-coinfected individuals treated with combination antiretroviral therapy

    DEFF Research Database (Denmark)

    Grint, D; Peters, Lars; Reekie, J;

    2013-01-01

    Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals.......Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals....

  1. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    Science.gov (United States)

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  2. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients

    Science.gov (United States)

    Rodriguez-Auad, Juan Pablo; Rojas-Montes, Othon; Maldonado-Rodriguez, Angelica; Alvarez-Muñoz, Ma. Teresa; Muñoz, Onofre; Torres-Ibarra, Rocio; Vazquez-Rosales, Guillermo

    2015-01-01

    Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings. PMID:26779533

  3. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients

    Directory of Open Access Journals (Sweden)

    Juan Pablo Rodriguez-Auad

    2015-01-01

    Full Text Available Monitoring antiretroviral therapy using measurements of viral load (VL and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6% of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

  4. Targeting folded RNA: A branched peptide boronic acid that binds to a large surface area of HIV-1 RRE RNA†

    Science.gov (United States)

    Zhang, Wenyu; Bryson, David I.; Crumpton, Jason B.; Wynn, Jessica; Santos, Webster L.

    2013-01-01

    On-bead high throughput screening of a medium sized (1000–2000 Da) branched peptide boronic acid (BPBA) library consisting of 46,656 unique sequences against HIV-1 RRE RNA generated peptides with binding affinities in the low micromolar range. In particular, BPBA1 had a Kd of 1.4 µM with RRE IIB, preference for RNA over DNA (27 fold), and selectivity of up to >75 fold against a panel of RRE IIB variants. Structure-activity studies suggest that the boronic acid moiety and “branching” in peptides are key structural features for efficient binding and selectivity for the folded RNA target. BPBA1 was efficiently taken up by HeLa and A2780 cells. RNA-footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. PMID:23925474

  5. Plasma viraemia in HIV-positive pregnant women entering antenatal care in South Africa

    Directory of Open Access Journals (Sweden)

    Landon Myer

    2015-07-01

    Full Text Available Introduction: Plasma HIV viral load (VL is the principle determinant of mother-to-child HIV transmission (MTCT, yet there are few data on VL in populations of pregnant women in sub-Saharan Africa. We examined the distribution and determinants of VL in HIV-positive women seeking antenatal care (ANC in Cape Town, South Africa. Methods: Consecutive HIV-positive pregnant women making their first antenatal clinic visit were recruited into a cross-sectional study of viraemia in pregnancy, including a brief questionnaire and specimens for VL testing and CD4 cell enumeration. Results & discussion: Overall 5551 pregnant women sought ANC during the study period, of whom 1839 (33% were HIV positive and 1521 (85% were included. Approximately two-thirds of HIV-positive women in the sample (n=947 were not on antiretrovirals at the time of the first ANC visit, and the remainder (38%, n=574 had initiated antiretroviral therapy (ART prior to conception. For women not on ART, the median VL was 3.98 log10 copies/mL; in this group, the sensitivity of CD4 cell counts ≤350 cells/µL in detecting VL>10,000 copies/mL was 64% and this increased to 78% with a CD4 threshold of ≤500 cells/µL. Among women on ART, 78% had VL1000 copies/mL at the time of their ANC visit. Conclusions: VL >10,000 copies/mL was commonly observed in women not on ART with CD4 cell counts >350 cells/µL, suggesting that CD4 cell counts may not be adequately sensitive in identifying women at greatest risk of MTCT. A large proportion of women entering ANC initiated ART before conception, and in this group more than 10% had VL>1000 copies/mL despite ART use. VL monitoring during pregnancy may help to identify pregnancies that require additional clinical attention to minimize MTCT risk and improve maternal and child health outcomes.

  6. Evaluation of microRNA stability in plasma and serum from healthy dogs

    DEFF Research Database (Denmark)

    Enelund, Lars; Nielsen, Lise Nikolic; Cirera, Susanna

    2017-01-01

    . METHODS: The levels of four microRNAs (cfa-let-7a, cfa-miR-16, cfa-miR-23a and cfa-miR-26a) known to be stably expressed from other canine studies, have been measured by real-time quantitative PCR. RESULTS: MicroRNA levels were found sufficiently stable for gene profiling in serum- and plasma stored...

  7. Antiretroviral treatment effect on immune activation reduces cerebrospinal fluid HIV-1 infection.

    Science.gov (United States)

    Sinclair, Elizabeth; Ronquillo, Rollie; Lollo, Nicole; Deeks, Steven G; Hunt, Peter; Yiannoutsos, Constantin T; Spudich, Serena; Price, Richard W

    2008-04-15

    To define the effect of antiretroviral therapy (ART) on activation of T cells in cerebrospinal fluid (CSF) and blood, and interactions of this activation with CSF HIV-1 RNA concentrations. Cross-sectional analysis of 14 HIV-negative subjects and 123 neuroasymptomatic HIV-1-infected subjects divided into 3 groups: not on ART (termed "offs"), on ART with plasma HIV-1 RNA >500 copies/mL ("failures"), and on ART with plasma HIV-1 RNA plasma HIV-1, it maintained a coincident relation to CSF HIV-1 in both viremic groups. In addition to correlation with CSF HIV-1 concentrations, CD8 activation in blood and CSF correlated with CSF WBCs and CSF neopterin. Multivariate analysis confirmed the association of blood CD8 T-cell activation, along with plasma HIV-1 RNA and CSF neopterin, with CSF HIV-1 RNA levels. The similarity of CD8 T-cell activation in blood and CSF suggests these cells move from blood to CSF with only minor changes in CD38/HLA-DR expression. Differences in the relation of CD8 activation to HIV-1 concentrations in the blood and CSF in the 2 viremic groups suggest that changes in immune activation not only modulate CSF HIV-1 replication but also contribute to CSF treatment effects. The magnitude of systemic HIV-1 infection and intrathecal macrophage activation are also important determinants of CSF HIV-1 RNA levels.

  8. 基于RNA干扰的抗HIV-1研究%Study on Anti-HIV-1 Based on RNA Interference

    Institute of Scientific and Technical Information of China (English)

    王静; 孙奉玉; 周云; 王芳宇

    2012-01-01

    艾滋病是由艾滋病毒感染而引起的传染病,目前在全世界广为流行,但目前还没有彻底的根治方法。该文简要综述了RNA干扰技术在抑制艾滋病毒感染中的研究进展、存在的问题和发展前景。%AIDS is an infectious disease caused by HIV. It is widely prevalent in the world and is still not thoroughly cured at present. The progress, problems and development prospects in inhibiting HIV infection through RNA interference are intro- duced in this paper.

  9. Lipid-based nutrient supplements do not affect efavirenz but lower plasma nevirapine concentrations in Ethiopian adult HIV patients

    DEFF Research Database (Denmark)

    Abdissa, A; Olsen, Mette Frahm; Yilma, D

    2015-01-01

    OBJECTIVES: Lipid-based nutrient supplements (LNSs) are increasingly used in HIV programmes in resource-limited settings. However, the possible effects of LNSs on the plasma concentrations of antiretroviral drugs have not been assessed. Here, we aimed to assess the effects of LNSs on plasma...... efavirenz and nevirapine trough concentrations in Ethiopian adult HIV-infected patients. METHODS: The effects of LNSs were studied in adults initiating antiretroviral therapy (ART) in a randomized trial. Patients with body mass index (BMI) > 17 kg/m(2) (n = 282) received daily supplementation of an LNS.......9; -0.9 μg/mL; P = 0.01), respectively, compared with the group not receiving supplements. There were no differences between groups with respect to efavirenz plasma concentrations. The CYP2B6 516 G>T polymorphism was associated with a 5 μg/mL higher plasma efavirenz concentration compared with the wild...

  10. Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

    Science.gov (United States)

    Jayaraman, Bhargavi; Mavor, David; Gross, John D; Frankel, Alan D

    2015-10-27

    The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.

  11. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients.

    Science.gov (United States)

    Meyer, C N; Svenson, M; Schade Larsen, C; Odum, N; Skinhøj, P; Bendtzen, K

    2000-02-01

    Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta, are uncommon in healthy individuals and HIV patients, and are apparently without prognostic significance. In contrast to earlier reports, IL-2 antibodies were found only in HIV patients treated with rIL-2.

  12. Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers.

    Science.gov (United States)

    Whatley, Angela S; Ditzler, Mark A; Lange, Margaret J; Biondi, Elisa; Sawyer, Andrew W; Chang, Jonathan L; Franken, Joshua D; Burke, Donald H

    2013-02-05

    RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs. The screen also identified a new, nonpseudoknot motif with a conserved unpaired UCAA element. High-throughput sequence (HTS) analysis identified 181 clusters capable of forming this novel element. Comparative sequence analysis, enzymatic probing and RT inhibition by aptamer variants established the essential requirements of the motif, which include two conserved base pairs (AC/GU) on the 5' side of the unpaired UCAA. Aptamers in this family inhibit RT in primer extension assays with IC(50) values in the low nmol/l range, and they suppress viral replication with a potency that is comparable with that of previously studied aptamers. All three known anti-RT aptamer families (pseudoknots, the UCAA element, and the recently described "(6/5)AL" motif) are therefore suitable for developing aptamer-based antiviral gene therapies.Molecular Therapy - Nucleic Acids (2013) 2, e71; doi:10.1038/mtna.2012.62; published online 5 February 2013.

  13. Blood-brain barrier integrity, intrathecal immunoactivation, and neuronal injury in HIV

    OpenAIRE

    Anesten, B.; YILMAZ, A.; Hagberg, L.; Zetterberg, H; Nilsson, S; Brew, B. J.; Fuchs, D.; Price, R W; Gisslén, M.

    2016-01-01

    Objective: Although blood?brain barrier (BBB) impairment has been reported in HIV-infected individuals, characterization of this impairment has not been clearly defined. Methods: BBB integrity was measured by CSF/plasma albumin ratio in this cross-sectional study of 631 HIV-infected individuals and 71 controls. We also analyzed CSF and blood HIV RNA and neopterin, CSF leukocyte count, and neurofilament light chain protein (NFL) concentrations. The HIV-infected participants included untreated ...

  14. A parallel genome-wide mRNA and microRNA profiling of the frontal cortex of HIV patients with and without HIV-associated dementia shows the role of axon guidance and downstream pathways in HIV-mediated neurodegeneration

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    Zhou Li

    2012-11-01

    Full Text Available Abstract Background HIV-associated dementia (HAD is the most common dementia type in young adults less than 40 years of age. Although the neurotoxins, oxidative/metabolic stress and impaired activity of neurotrophic factors are believed to be underlying reasons for the development of HAD, the genomic basis, which ultimately defines the virus-host interaction and leads to neurologic manifestation of HIV disease is lacking. Therefore, identifying HIV fingerprints on the host gene machinery and its regulation by microRNA holds a great promise and potential for improving our understanding of HAD pathogenesis, its diagnosis and therapy. Results A parallel profiling of mRNA and miRNA of the frontal cortex autopsies from HIV positive patients with and without dementia was performed using Illumina Human-6 BeadChip and Affymetrix version 1.0 miRNA array, respectively. The gene ontology and pathway analysis of the two data sets showed high concordance between miRNA and mRNAs, revealing significant interference with the host axon guidance and its downstream signalling pathways in HAD brains. Moreover, the differentially expressed (DE miRNAs identified in this study, in particular miR-137, 153 and 218, based on which most correlations were built cumulatively targeted neurodegeneration related pathways, implying their future potential in diagnosis, prognosis and possible therapies for HIV-mediated and possibly other neurodegenerative diseases. Furthermore, this relationship between DE miRNAs and DE mRNAs was also reflected in correlation analysis using Bayesian networks by splitting-averaging strategy (SA-BNs, which revealed 195 statistically significant correlated miRNA-mRNA pairs according to Pearson’s correlation test (P Conclusions Our study provides the first evidence on unambiguous support for intrinsic functional relationship between mRNA and miRNA in the context of HIV-mediated neurodegeneration, which shows that neurologic manifestation in HIV

  15. Lower Ribavirin Plasma Concentrations in HCV/HIV-Coinfected Patients Than in HCV-Monoinfected Patients Despite Similar Dosage

    NARCIS (Netherlands)

    Deenen, M.J.; Kanter, C.T.M.M. de; Dofferhoff, A.S.; Grintjes-Huisman, K.J.T.; Ven, A.J.A.M. van der; Fleuren, H.W.; Gisolf, E.H.; Koopmans, P.P.; Drenth, J.P.H.; Burger, D.M.

    2015-01-01

    BACKGROUND: Hepatitis C virus (HCV)/HIV-coinfected patients respond worse to dual therapy with ribavirin (RBV)/peginterferon compared with HCV-monoinfected patients. Several trials found that lower RBV plasma concentrations are associated with impaired virological response rates. The aim of this stu

  16. In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries.

    Science.gov (United States)

    Rouet, François; Ménan, Hervé; Viljoen, Johannes; Ngo-Giang-Huong, Nicole; Mandaliya, Kishor; Valéa, Diane; Lien, Truong Xuan; Danaviah, Sivapragashini; Rousset, Dominique; Ganon, Amandine; Nerrienet, Eric

    2008-09-01

    The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hépatites virales B et C. It exists now as a complete standardized commercial test.

  17. Pokeweed antiviral protein restores levels of cellular APOBEC3G during HIV-1 infection by depurinating Vif mRNA.

    Science.gov (United States)

    Krivdova, Gabriela; Hudak, Katalin A

    2015-10-01

    Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.

  18. HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

    Science.gov (United States)

    Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

    2014-06-01

    Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

  19. The impact of criminalization of HIV non-disclosure on the healthcare engagement of women living with HIV in Canada: a comprehensive review of the evidence

    OpenAIRE

    Patterson, Sophie E; Milloy M-J; Gina Ogilvie; Saara Greene; Valerie Nicholson; Micheal Vonn; Robert Hogg; Angela Kaida

    2015-01-01

    Introduction: In 2012, the Supreme Court of Canada ruled that people living with HIV (PLWH) must disclose their HIV status to sexual partners prior to sexual activity that poses a “realistic possibility” of HIV transmission for consent to sex to be valid. The Supreme Court deemed that the duty to disclose could be averted if a person living with HIV both uses a condom and has a low plasma HIV-1 RNA viral load during vaginal sex. This is one of the strictest legal standards criminalizing HIV n...

  20. Lack of indinavir-associated nephrological complications in HIV-infected adults (predominantly women) with high indinavir plasma concentration in Abidjan, Côte d'Ivoire.

    Science.gov (United States)

    Danel, Christine; Moh, Raoul; Peytavin, Gilles; Anzian, Amani; Minga, Albert; Gomis, Olivier Ba; Seri, Boga; Nzunettu, Gustave; Gabillard, Delphine; Salamon, Roger; Bissagnene, Emmanuel; Anglaret, Xavier

    2007-01-01

    To report the tolerance of indinavir combined with ritonavir (IDV/r 800/100 mg) twice daily (bid) in sub-Saharan African HIV-infected adults. HAART-naives patients started zidovudine plus lamivudine plus IDV/r 800/100 mg bid. Follow-up included standardized documentation of morbidity, CD4(+) cell count, creatininemia, plasma HIV-1 RNA, and IDV minimal plasma concentration (C(min)) measurements at month 1 (M1), M3, and M6. Seventy HIV-1-infected adults (68 women, median CD4 235/mm(3)) started HAART. At M6, 63% had undetectable viral load, and the median gain in CD4 since baseline was +128/mm(3). During the first 6 months, 21 patients experimented with 23 treatment modifications (reduction in IDV/r 400/100 mg bid, n = 11; switch to efavirenz, n = 11; zidovudine replaced by stavudine, n = 1), including 22 for digestive intolerance and 1 for severe anemia. At M1, M3, and M6, 67, 59, and 48 patients were still receiving IDV/r 800/100 mg bid, of whom 70%, 72%, and 60% had IDV Cmin above 5 ng/ml, respectively. In these patients, at M1, M3, and M6, the mean (+/- SD) IDV C(min) were 3431 +/- 3835 ng/ml, 2288 +/- 2116 ng/ml, and 1543 +/- 2398 ng/ml, respectively. There was no renal insufficiency of any grade, and no symptoms of urinary stones. The IDV/r 800/100 mg bid-containing regimen led to high IDV Cmin and a high rate of digestive intolerance. There was a surprising lack of nephrological side effects during the 6 months of follow-up, supporting the hypothesis that nephrological tolerance of IDV might be higher in sub-Saharan African individuals than in Americans or Europeans.

  1. Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients.

    Science.gov (United States)

    Li, Hongqiu; Wang, Zhe; Fu, Qin; Zhang, Jing

    2014-11-01

    In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR-146a) in the plasma of 120 Chinese postmenopausal women who were divided into three groups (normal, osteopenia and osteoporosis) according to the T-scores. Downregulation of miR-21, as well as upregulation of miR-133a, was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR-146a level in plasma among the three groups was not significant (p > 0.01). The circulating miRNA expression levels and bone mineral density (BMD) were examined during a multiple correlation analysis as a dependent variable after adjusting for age, weight and height. We have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. Our study suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis.

  2. EVALUATION OF THE PLASMA MICRO RNA EXPRESSION LEVELS IN SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

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    Ali Bay

    2013-11-01

    Full Text Available Background: Hemophagocytic lymphohistiocytosis (HLH is a life threatening hyper inflammatory disease. Micro RNAs (miRNA are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response. Objective: To determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease. Patients and Methods: Patients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics. Results: A total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01. We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009. miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031. Conclusion: We showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.

  3. Non-invasive aneuploidy detection using free fetal DNA and RNA in maternal plasma: recent progress and future possibilities.

    NARCIS (Netherlands)

    Go, A.T.; Vugt, J.M.G. van; Oudejans, C.B.

    2011-01-01

    BACKGROUND: Cell-free fetal DNA (cff DNA) and RNA can be detected in maternal plasma and used for non-invasive prenatal diagnostics. Recent technical advances have led to a drastic change in the clinical applicability and potential uses of free fetal DNA and RNA. This review summarizes the latest cl

  4. High-intensity endurance training improves adiponectin mRNA and plasma concentrations.

    Science.gov (United States)

    Moghadasi, Mehrzad; Mohebbi, Hamid; Rahmani-Nia, Farhad; Hassan-Nia, Sadegh; Noroozi, Hamid; Pirooznia, Nazanin

    2012-04-01

    Adiponectin is an anti-inflammatory protein that reduced in obesity. Exercise training may reduce the adipose tissue (AT), although it is not well known whether exercise-induced change in AT, increases the adiponectin mRNA expression and plasma concentrations or not; therefore, the purpose of this study was to investigate the adiponectin mRNA and plasma concentrations in middle-aged men after 12 weeks high-intensity exercise training and after a week detraining. Sixteen sedentary overweight and obese middle-aged men (age 41.18 ± 6.1 years; ± SD) volunteered to participate in this study. The subjects were randomly assigned to training group (n = 8) or control group (n = 8). The training group performed endurance training 4 days a week for 12 weeks at an intensity corresponding to 75-80% individual maximum oxygen consumption for 45 min. After 12 weeks of training, subjects underwent a week of detraining. The results showed that the BMI as well as central and peripheral AT volume were decreased in the training group compared to the control group (P training group resulted in a significant increase (P training compared to the control group (P training group. In conclusion, high-intensity endurance training caused an increase adiponectin mRNA in obese middle-aged men.

  5. The structure of HIV-1 genomic RNA in the gp120 gene determines a recombination hot spot in vivo.

    Science.gov (United States)

    Galetto, Román; Moumen, Abdeladim; Giacomoni, Véronique; Véron, Michel; Charneau, Pierre; Negroni, Matteo

    2004-08-27

    By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

  6. High-throughput sequencing of plasma microRNA in chronic fatigue syndrome/myalgic encephalomyelitis.

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    Ekua W Brenu

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME. The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. RESULTS: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p in the CFS/ME patients. CONCLUSION: Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.

  7. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

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    Clavel François

    2008-02-01

    Full Text Available Abstract Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1. Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70, and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799, soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5 was also evaluated for a subset of the recombinant viruses (n = 20. Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in

  8. Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

    Science.gov (United States)

    Dai, Z; Cai, W; Hu, F; Lan, Y; Li, L; Chung, C; Caughey, B; Zhang, K; Tang, X

    2015-01-01

    Lipodystrophy is a common complication in HIV-infected patients taking highly active antiretroviral therapy. Its early diagnosis is crucial for timely modification of antiretroviral therapy. We hypothesize that mitochondrial DNA in plasma may be a potential marker of LD in HIV-infected individuals. In this study, we compared plasma mitochondrial DNA levels in HIV-infected individuals and non-HIV-infected individuals to investigate its potential diagnostic value. Total plasma DNA was extracted from 67 HIV-infected patients at baseline and 12, 24 and 30 months after initiating antiretroviral therapy. Real-time quantitative PCR was used to determine the mitochondrial DNA levels in plasma. Lipodystrophy was defined by the physician-assessed presence of lipoatrophy or lipohypertrophy in one or more body regions. The mitochondrial DNA levels in plasma were significantly higher at baseline in HIV-infected individuals than in non-HIV-infected individuals (pmitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (pmitochondrial DNA level (with cut-off value mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk.

  9. Correlates of HIV-1 genital shedding in Tanzanian women.

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    Clare Tanton

    Full Text Available BACKGROUND: Understanding the correlates of HIV shedding is important to inform strategies to reduce HIV infectiousness. We examined correlates of genital HIV-1 RNA in women who were seropositive for both herpes simplex virus (HSV-2 and HIV-1 and who were enrolled in a randomised controlled trial of HSV suppressive therapy (aciclovir 400 mg b.i.d vs. placebo in Tanzania. METHODOLOGY: Samples, including a cervico-vaginal lavage, were collected and tested for genital HIV-1 and HSV and reproductive tract infections (RTIs at randomisation and 6, 12 and 24 months follow-up. Data from all women at randomisation and women in the placebo arm during follow-up were analysed using generalised estimating equations to determine the correlates of cervico-vaginal HIV-1 RNA detection and load. PRINCIPAL FINDINGS: Cervico-vaginal HIV-1 RNA was detected at 52.0% of 971 visits among 482 women, and was independently associated with plasma viral load, presence of genital ulcers, pregnancy, bloody cervical or vaginal discharge, abnormal vaginal discharge, cervical ectopy, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, an intermediate bacterial vaginosis score and HSV DNA detection. Similar factors were associated with genital HIV-1 RNA load. CONCLUSIONS: RTIs were associated with increased presence and quantity of genital HIV-1 RNA in this population. These results highlight the importance of integrating effective RTI treatment into HIV care services.

  10. Acute plasma biomarkers of T cell activation set-point levels and of disease progression in HIV-1 infection.

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    Anne-Sophie Liovat

    Full Text Available T cell activation levels, viral load and CD4(+ T cell counts at early stages of HIV-1 infection are predictive of the rate of progression towards AIDS. We evaluated whether the inflammatory profile during primary HIV-1 infection is predictive of the virological and immunological set-points and of disease progression. We quantified 28 plasma proteins during acute and post-acute HIV-1 infection in individuals with known disease progression profiles. Forty-six untreated patients, enrolled during primary HIV-1 infection, were categorized into rapid progressors, progressors and slow progressors according to their spontaneous progression profile over 42 months of follow-up. Already during primary infection, rapid progressors showed a higher number of increased plasma proteins than progressors or slow progressors. The plasma levels of TGF-β1 and IL-18 in primary HIV-1 infection were both positively associated with T cell activation level at set-point (6 months after acute infection and together able to predict 74% of the T cell activation variation at set-point. Plasma IP-10 was positively and negatively associated with, respectively, T cell activation and CD4(+ T cell counts at set-point and capable to predict 30% of the CD4(+ T cell count variation at set-point. Moreover, plasma IP-10 levels during primary infection were predictive of rapid progression. In primary infection, IP-10 was an even better predictor of rapid disease progression than viremia or CD4(+ T cell levels at this time point. The superior predictive capacity of IP-10 was confirmed in an independent group of 88 HIV-1 infected individuals. Altogether, this study shows that the inflammatory profile in primary HIV-1 infection is associated with T cell activation levels and CD4(+ T cell counts at set-point. Plasma IP-10 levels were of strong predictive value for rapid disease progression. The data suggest IP-10 being an earlier marker of disease progression than CD4(+ T cell counts or

  11. Nonlinear and Robust Control Strategy Based on Chemotherapy to Minimize the HIV Concentration in Blood Plasma

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    Ricardo Aguilar-López

    2016-01-01

    Full Text Available A nonlinear PI-type control strategy is designed in order to minimize the HIV concentration in blood plasma, via medical drug injection, under the framework of bounded uncertain input disturbances. For control design it is considered a simplified mathematical model of the virus infection as a benchmark. The model is based on mass balances of healthy cells, infected cells, and the virus concentrations. The proposed controller contains a nonlinear feedback PI structure of bounded functions of the regulation error. The closed-loop stability of the system is analyzed via Lyapunov technique, in which robustness against system disturbances is demonstrated. Numerical experiments show a satisfactory performance of the proposed methodology as a HIV therapy, in which the virion particles and the infected CD4+T cells are minimized and, as an interesting result, the drug dosage can be suspended, thus avoiding drug resistance from the virus. Finally, the proposed controller is compared to a standard sliding-mode and hyperbolic tangent controllers showing better performance.

  12. Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

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    Miwako Narita, Anri Saito, Aya Kojima, Minami Iwabuchi, Naoya Satoh, Takayoshi Uchiyama, Akie Yamahira, Tatsuo Furukawa, Hirohito Sone, Masuhiro Takahashi

    2012-01-01

    Full Text Available Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

  13. Cold Atmospheric Plasma Inhibits HIV-1 Replication in Macrophages by Targeting Both the Virus and the Cells

    Science.gov (United States)

    Volotskova, Olga; Dubrovsky, Larisa; Keidar, Michael; Bukrinsky, Michael

    2016-01-01

    Cold atmospheric plasma (CAP) is a specific type of partially ionized gas that is less than 104°F at the point of application. It was recently shown that CAP can be used for decontamination and sterilization, as well as anti-cancer treatment. Here, we investigated the effects of CAP on HIV-1 replication in monocyte-derived macrophages (MDM). We demonstrate that pre-treatment of MDM with CAP reduced levels of CD4 and CCR5, inhibiting virus-cell fusion, viral reverse transcription and integration. In addition, CAP pre-treatment affected cellular factors required for post-entry events, as replication of VSV-G-pseudotyped HIV-1, which by-passes HIV receptor-mediated fusion at the plasma membrane during entry, was also inhibited. Interestingly, virus particles produced by CAP-treated cells had reduced infectivity, suggesting that the inhibitory effect of CAP extended to the second cycle of infection. These results demonstrate that anti-HIV activity of CAP involves the effects on target cells and the virus, and suggest that CAP may be considered for potential application as an anti-HIV treatment. PMID:27783659

  14. Effect of HSV-2 Suppressive Therapy on Genital Tract HIV-1 RNA Shedding among Women on HAART: A Pilot Randomized Controlled Trial

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    A. E. Nijhawan

    2012-01-01

    Full Text Available Background. The role of suppressive HSV therapy in women coinfected with HSV-2 and HIV-1 taking highly active antiretroviral therapy (HAART is unclear. Methods. 60 women with HIV-1/HSV-2 coinfection on HAART with plasma HIV-1 viral load (PVL ≤75 copies/mL were randomized to receive acyclovir (N=30 or no acyclovir (N=30. PVL, genital tract (GT HIV-1, and GT HSV were measured every 4 weeks for one year. Results. Detection of GT HIV-1 was not significantly different in the two arms (OR 1.23, P=0.67, although this pilot study was underpowered to detect this difference. When PVL was undetectable, the odds of detecting GT HIV were 0.4 times smaller in the acyclovir arm than in the control arm, though this was not statistically significant (P=0.07. The odds of detecting GT HSV DNA in women receiving acyclovir were significantly lower than in women in the control group, OR 0.38, P<0.05. Conclusions. Chronic suppressive therapy with acyclovir in HIV-1/HSV-2-positive women on HAART significantly reduces asymptomatic GT HSV shedding, though not GT HIV shedding or PVL. PVL was strongly associated with GT HIV shedding, reinforcing the importance of HAART in decreasing HIV sexual transmission.

  15. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique.

    Science.gov (United States)

    Jani, Ilesh V; Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I; Loquiha, Osvaldo; Lehe, Jonathan D; Vojnov, Lara; Peter, Trevor F

    2016-08-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted.

  16. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique

    Science.gov (United States)

    Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E.; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I.; Loquiha, Osvaldo; Lehe, Jonathan D.; Vojnov, Lara; Peter, Trevor F.

    2016-01-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted. PMID:27252459

  17. Patterns of recombination in HIV-1M are influenced by selection disfavouring the survival of recombinants with disrupted genomic RNA and protein structures.

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    Michael Golden

    Full Text Available Genetic recombination is a major contributor to the ongoing diversification of HIV. It is clearly apparent that across the HIV-genome there are defined recombination hot and cold spots which tend to co-localise both with genomic secondary structures and with either inter-gene boundaries or intra-gene domain boundaries. There is also good evidence that most recombination breakpoints that are detectable within the genes of natural HIV recombinants are likely to be minimally disruptive of intra-protein amino acid contacts and that these breakpoints should therefore have little impact on protein folding. Here we further investigate the impact on patterns of genetic recombination in HIV of selection favouring the maintenance of functional RNA and protein structures. We confirm that chimaeric Gag p24, reverse transcriptase, integrase, gp120 and Nef proteins that are expressed by natural HIV-1 recombinants have significantly lower degrees of predicted folding disruption than randomly generated recombinants. Similarly, we use a novel single-stranded RNA folding disruption test to show that there is significant, albeit weak, evidence that natural HIV recombinants tend to have genomic secondary structures that more closely resemble parental structures than do randomly generated recombinants. These results are consistent with the hypothesis that natural selection has acted both in the short term to purge recombinants with disrupted RNA and protein folds, and in the longer term to modify the genome architecture of HIV to ensure that recombination prone sites correspond with those where recombination will be minimally deleterious.

  18. Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR RNA.

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    Matthew S Lalonde

    2011-05-01

    Full Text Available The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (- strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.

  19. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  20. HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia

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    Mishra Ritu

    2012-06-01

    Full Text Available Abstract Background HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. Methods We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. Results HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor–associated factor 3 TRAF3 is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3 and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. Conclusion HIV-1 Tat protein can modulate TRAF3 expression through

  1. DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain.

    Science.gov (United States)

    Fröhlich, Alvaro; Rojas-Araya, Bárbara; Pereira-Montecinos, Camila; Dellarossa, Alessandra; Toro-Ascuy, Daniela; Prades-Pérez, Yara; García-de-Gracia, Francisco; Garcés-Alday, Andrea; Rubilar, Paulina S; Valiente-Echeverría, Fernando; Ohlmann, Théophile; Soto-Rifo, Ricardo

    2016-05-01

    DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention.

  2. Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1

    NARCIS (Netherlands)

    Konstantinova, P.; de Haan, P.; Das, A.T.; Berkhout, B.

    2006-01-01

    Recombination due to template switching during reverse transcription is a major source of genetic variability in retroviruses. In the present study we forced a recombination event in human immunodeficiency virus type 1 (HIV-1) by electroporation of T cells with DNA from a molecular HIV-1 clone that

  3. Plasma micro-RNA biomarkers for diagnosis and prognosis after traumatic brain injury: A pilot study.

    Science.gov (United States)

    Mitra, Biswadev; Rau, Thomas F; Surendran, Nanda; Brennan, James H; Thaveenthiran, Prasanthan; Sorich, Edmond; Fitzgerald, Mark C; Rosenfeld, Jeffrey V; Patel, Sarjubhai A

    2017-04-01

    Prediction of post-concussive syndrome after apparent mild traumatic brain injury (TBI) and subsequent cognitive recovery remains challenging, with substantial limitations of current methods of cognitive testing. This pilot study aimed to determine if levels of micro ribonucleic acids (RNAs) circulating in plasma are altered following TBI, and if changes to levels of such biomarkers over time could assist in determination of prognosis after TBI. Patients were enrolled after TBI on presentation to the Emergency Department and allocated to three groups: A - TBI (physical trauma to the head), witnessed loss of consciousness, amnesia, GCS=15, a normal CT Brain and a recorded first pass after post-traumatic amnesia (PTA) scale; B TBI, witnessed LOC, amnesia, GCS=15, a normal CT brain and a PTA scale test fail and: C - TBI and initial GCS RNA was then assayed using a custom miRNA PCR array. Two micro-RNAs, mir142-3p and mir423-3p demonstrated potential clinical utility differentiating patients after mild head injury into those at greater risk of developing amnesia and therefore, post-concussive syndromes. In addition, these miRNA demonstrated a decrease in expression over time, possibly indicative of brain healing after the injury. Further evaluation of these identified miRNA markers with larger patient cohorts, correlation with clinical symptoms and analysis over longer time periods are essential next steps in developing objective markers of severity of TBI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD

    OpenAIRE

    Ménard, Catherine; Rezende, Flavio A.; Miloudi, Khalil; Wilson, Ariel; Tétreault, Nicolas; Hardy, Pierre; SanGiovanni, John Paul; De Guire, Vincent; Sapieha, Przemyslaw

    2016-01-01

    Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and...

  5. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

    Science.gov (United States)

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  6. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR

    Directory of Open Access Journals (Sweden)

    McCaffrey Timothy

    2007-07-01

    Full Text Available Abstract Background RNA interference (RNAi is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA or micro RNA (miRNA from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. Results In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. Conclusion HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.

  7. Effectiveness of first-line antiretroviral therapy based on NNRTIs vs ritonavir-boosted PIs in HIV-1 infected patients with high plasma viral load

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    A Imaz

    2012-11-01

    Full Text Available Purpose of the study: Few clinical trials have compared non-nucleoside reverse transcriptase inhibitors (NNRTI and ritonavir-boosted protease inhibitors (PI/r as initial combined antiretroviral therapy (cART for HIV-1-infected patients with high plasma viral load (pVL, and non-conclusive results have been reported. We compared the effectiveness between NNRTI and PI/r as first-line cART for HIV-1-infected patients with high pVL. Methods: Observational retrospective study of 664 consecutive treatment-naïve HIV-1-infected patients with pVL (HIV-1 RNA >100,000 copies/mL who initiated NNRTI or PI/r-based cART between 2000–2010 in three University hospitals. Only currently preferred or alternative regimens in clinical guidelines were included. Primary endpoint: percentage of therapeutic failures at week 48. Virologic failure was defined as: a lack of virologic response (<1 log RNA HIV-1 decrease in first 3 months; b RNA HIV-1 >50 c/mL at week 48; c confirmed rebound >50 c/ml after a previous value <50 c/mL. Intent-to-treat (ITT noncompleter=failure and on-treatment (OT analyses were performed. Results: 62% of patients initiated NNRTI-regimens (83% efavirenz and 38% PI/r-regimens (62% lopinavir/. Baseline characteristics: male 83%; median age 39 yrs; median CD4 count: 212/µL (NNRTI 232 vs PI/r 177, p=0.028; pVL 5.83 log10 c/mL (NNRTI 5.43 vs PI/r 5.55, p=0.007; AIDS 24% (NNRTI 21% vs PI/r 29%, p=0.015. NRTI backbones were tenofovir plus 3TC or FTC in 72%. The percentage of therapeutic failure was higher in the PI/r group (ITT NC=F 26% vs 18%, p=0.012 with no differences in virologic failures (PI/r 5%, NNRTI 6%, p=0.688. The rate of treatment changes due to toxicity and/or voluntary discontinuations was higher in the PI/r group (15% vs 8%, p=0.008. A multivariate analysis adjusted for age, gender, CD4 count, VL and AIDS showed NNRTI vs PI/r as the only variable associated with treatment response (OR 0.61, 95% CI 0.41–0.88. Median pVL and rate of

  8. Proviral HIV-1 DNA in gingival crevicular fluid of HIV-1-infected patients in various stages of HIV disease.

    Science.gov (United States)

    Maticic, M; Poljak, M; Kramar, B; Tomazic, J; Vidmar, L; Zakotnik, B; Skaleric, U

    2000-07-01

    The oral cavity is rarely reported to be a site of human immunodeficiency virus (HIV) transmission, despite detectable virus in saliva and relatively frequent prevalence of periodontal disease in HIV-infected persons yielding increased excretion of mononuclear-cell-enriched gingival fluid. To search for possible sources of HIV in saliva, and using the polymerase chain-reaction technique, we sought the presence and shedding patterns of proviral HIV-1 DNA in gingival crevicular fluid in a group of patients previously determined as HIV-1-seropositive. Periodontal status at the collection sites was monitored by several clinical parameters, including Plaque Index, Gingival Index, probing depth, and clinical attachment loss. Gingival crevicular fluid samples were collected by means of paper points. Proviral HIV-1 DNA was detected in the gingival fluid of 17 out of 35 HIV-1-infected patients. Its detection correlated significantly with higher plasma HIV-1 RNA viral load (p = 0.03) and not with peripheral blood CD4+ cell count, the presence of blood in gingival fluid, or oral lesions. There was a significant correlation between clinical attachment loss at the sites of fluid collection and plasma HIV-1 RNA viral load (p = 0.002), and borderline correlation between the latter and probing depth (p = 0.54) in the group of patients harboring proviral HIV-1 DNA in gingival crevicular fluid. The results of our study suggest that mononuclear cells present in gingival crevicular fluid and harboring proviral HIV-1 DNA could represent a potential source of HIV-1 in the presence or absence of local bleeding, especially in persons with advanced HIV infection and increased loss of clinical attachment.

  9. Plasma levels of microRNA in chronic kidney disease: patterns in acute and chronic exercise.

    Science.gov (United States)

    Van Craenenbroeck, Amaryllis H; Ledeganck, Kristien J; Van Ackeren, Katrijn; Jürgens, Angelika; Hoymans, Vicky Y; Fransen, Erik; Adams, Volker; De Winter, Benedicte Y; Verpooten, Gert A; Vrints, Christiaan J; Couttenye, Marie M; Van Craenenbroeck, Emeline M

    2015-12-15

    Exercise training is an effective way to improve exercise capacity in chronic kidney disease (CKD), but the underlying mechanisms are only partly understood. In healthy subjects (HS), microRNA (miRNA or miR) are dynamically regulated following exercise and have, therefore, been suggested as regulators of cardiovascular adaptation to exercise. However, these effects were not studied in CKD before. The effect of acute exercise (i.e., an acute exercise bout) was assessed in 32 patients with CKD and 12 age- and sex-matched HS (study 1). miRNA expression in response to chronic exercise (i.e., a 3-mo exercise training program) was evaluated in 40 CKD patients (study 2). In a subgroup of study 2, the acute-exercise induced effect was evaluated at baseline and at follow-up. Plasma levels of a preselected panel miRNA, involved in exercise adaptation processes such as angiogenesis (miR-126, miR-210), inflammation (miR-21, miR-146a), hypoxia/ischemia (miR-21, miR-210), and progenitor cells (miR-150), were quantified by RT-PCR. Additionally, seven miRNA involved in similar biological processes were quantified in the subgroup of study 2. Baseline, studied miRNA were comparable in CKD and HS. Following acute exercise, miR-150 levels increased in both CKD (fold change 2.12 ± 0.39, P = 0.002; and HS: fold change 2.41 ± 0.48 P = 0.018, P for interaction > 0.05). miR-146a acutely decreased in CKD (fold change 0.92 ± 0.13, P = 0.024), whereas it remained unchanged in HS. Levels of miR-21, miR-126, and miR-210 remained unaltered. Chronic exercise did not elicit a significant change in the studied miRNA levels. However, an acute exercise-induced decrease in miR-210 was observed in CKD patients, only after training (fold change 0.76 ± 0.15). The differential expression in circulating miRNA in response to acute and chronic exercise may point toward a physiological role in cardiovascular adaptation to exercise, also in CKD.

  10. Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors.

    Science.gov (United States)

    Radi, Marco; Falchi, Federico; Garbelli, Anna; Samuele, Alberta; Bernardo, Vincenzo; Paolucci, Stefania; Baldanti, Fausto; Schenone, Silvia; Manetti, Fabrizio; Maga, Giovanni; Botta, Maurizio

    2012-03-01

    Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.

  11. Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA.

    Science.gov (United States)

    Boutimah-Hamoudi, Fatima; Leforestier, Erwan; Sénamaud-Beaufort, Catherine; Nielsen, Peter E; Giovannangeli, Carine; Saison-Behmoaras, Tula Ester

    2007-01-01

    DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.

  12. Relationship between innate immunity, soluble markers and metabolic-clinical parameters in HIV+ patients ART treated with HIV-RNA<50 cp/mL

    Directory of Open Access Journals (Sweden)

    Chiara Dentone

    2014-11-01

    Full Text Available Introduction: The persistence of immune activation and inflammation in HIV patients with HIV-RNA (VL undetectable causes many co-morbidities [1–3]. The aim of this study is to correlate monocytes (m and NK cell activation levels, soluble markers and oxidative stress with clinical, biochemical and metabolic data in HIV-1 infected patients with VL≤50 copies (cp/mL on antiretroviral therapy. Materials and Methods: Multicentre, cross-sectional study in patients with VL≤50 cp/mL and on antiretroviral therapy by at least six months. We studied: activation/homing markers (CD38, HLA-DR, CCR-2, PDL-1 on inflammatory, intermediate, proinflammatory m; activatory receptors NKp30, NKp46 and HLA-DR on NK cells; soluble inflammatory (sCD14, adiponectina, MCP-1 and stress oxidative markers (dRoms, antiRoms. Univariate analyses are performed with non-parametric and Pearson tests. The significant correlations were adjusted for possible known confounding factors (smoking, Cytomegalovirus IgG serology, Raltegravir, Protease Inhibitor [PI] therapy and HCV-RNA with multivariate analysis. Results: In the 68 patients the positive correlation between age and antiRoms was significant also after adjustment for PI use (p=0.05. The% CD8+T was associated with% proinflammatory m (p=0.043 and with their expression of CCR2 mean fluorescence intensity (MFI (p=0.012. The% NKp46+ was positively correlated with CD4+T count (p=0.001. The fibrinogen was positively associated with dRoms (p=0.052 and the positive correlation between triglycerides and antiRoms has been confirmed (p<0.001; the impact of antiRoms on HDL/triglycerides ratio (p=0.006 was observed after adjustment for PI use. The BMI was associated with smoking (p=0.011. Only the maraviroc-treated patients showed minimal arterial pressure, fibrinogen and antiRoms lower (p=0.001, 0.004 e 0.006 and sCD14 values higher (p=0.029. Conclusions: Patients with long history of HIV infection and stable immunological and

  13. Plasma microRNA-186 and proteinuria in focal segmental glomerulosclerosis.

    Science.gov (United States)

    Zhang, Changming; Zhang, Wanfen; Chen, Hui-Mei; Liu, Chunbei; Wu, Junnan; Shi, Shaolin; Liu, Zhi-Hong

    2015-02-01

    MicroRNAs (miRNAs) are stable in circulation, and their unique expression profiles can serve as fingerprints for various diseases. This study explored whether plasma miRNAs could be used as biomarkers to evaluate disease activity in patients with focal segmental glomerulosclerosis (FSGS). Retrospective and prospective cohorts. 78 patients with FSGS with nephrotic proteinuria (protein excretion > 3.5g/24 h), 35 patients with FSGS in complete remission, 63 patients with membranous nephropathy, 59 patients with diabetic nephropathy, and 69 apparently healthy controls were recruited. Plasma samples from 51 other patients with FSGS with nephrotic proteinuria were collected prospectively before and after steroid treatment. Plasma miRNA concentration. Complete remission (protein excretion 3.5g/24 h after 8 weeks of steroid treatment). Quantitative reverse transcription-polymerase chain reaction analysis of plasma miRNAs. Increases in miR-125b, miR-186, and miR-193a-3p levels were identified in a pooled plasma sample of 9 patients with FSGS compared with that of 9 healthy controls and were confirmed with individual samples from patients with FSGS (n=32) and healthy controls (n=30). Areas under the receiver operating characteristic curves of miR-125b, miR-186, miR-193a-3p, and the 3 miRNAs in combination were 0.882, 0.789, 0.910, and 0.963, respectively. miR-125b and miR-186 concentrations were significantly lower in patients with FSGS in complete remission (n=35) than those with nephrotic proteinuria (n=37). In a prospective study, miR-125b and miR-186 levels declined markedly in patients with FSGS with complete remission (n=29), but not those with no response (n=22), after steroid treatment. Plasma miR-125b and miR-186 levels were not elevated in patients with membranous nephropathy (n=63) and diabetic nephropathy (n=59) regardless of degree of proteinuria. Last, plasma miR-186, but not miR-125b, level was correlated with degree of proteinuria in patients with FSGS (151

  14. Plasma Concentration of the Neurofilament Light Protein (NFL) is a Biomarker of CNS Injury in HIV Infection: A Cross-Sectional Study.

    Science.gov (United States)

    Gisslén, Magnus; Price, Richard W; Andreasson, Ulf; Norgren, Niklas; Nilsson, Staffan; Hagberg, Lars; Fuchs, Dietmar; Spudich, Serena; Blennow, Kaj; Zetterberg, Henrik

    2016-01-01

    Cerebrospinal fluid (CSF) neurofilament light chain protein (NFL) is a sensitive marker of neuronal injury in a variety of neurodegenerative conditions, including the CNS dysfunction injury that is common in untreated HIV infection. However, an important limitation is the requirement for lumbar puncture. For this reason, a sensitive and reliable blood biomarker of CNS injury would represent a welcome advance in both clinical and research settings. To explore whether plasma concentrations of NFL might be used to detect CNS injury in HIV infection, an ultrasensitive Single molecule array (Simoa) immunoassay was developed. Using a cross-sectional design, we measured NFL in paired CSF and plasma samples from 121 HIV-infected subjects divided into groups according to stage of their systemic disease, presence of overt HIV-associated dementia (HAD), and after antiretroviral treatment (ART)-induced viral suppression. HIV-negative controls were also examined. Plasma and CSF NFL concentrations were very highly correlated (r = 0.89, P NFL was more than 50-fold lower plasma than CSF it was within the quantifiable range of the new plasma assay in all subjects, including the HIV negatives and the HIV positives with normal CSF NFL concentrations. The pattern of NFL changes were almost identical in plasma and CSF, both exhibiting similar age-related increases in concentrations along with highest values in HAD and substantial elevations in ART-naïve neuroasymptomatic subjects with low blood CD4(+) T cells. These results show that plasma NFL may prove a valuable tool to evaluate ongoing CNS injury in HIV infection that may be applied in the clinic and in research settings to assess the presence if active CNS injury. Because CSF NFL is also elevated in a variety of other CNS disorders, sensitive measures of plasma NFL may similarly prove useful in other settings.

  15. HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

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    Akkina Ramesh

    2005-01-01

    Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

  16. Comparison Analysis of Dysregulated LncRNA Profile in Mouse Plasma and Liver after Hepatic Ischemia/Reperfusion Injury.

    Science.gov (United States)

    Chen, Zhenzhen; Luo, Yanjin; Yang, Weili; Ding, Liwei; Wang, Junpei; Tu, Jian; Geng, Bin; Cui, Qinghua; Yang, Jichun

    2015-01-01

    Long noncoding RNAs (LncRNAs) have been believed to be the major transcripts in various tissues and organs, and may play important roles in regulation of many biological processes. The current study determined the LncRNA profile in mouse plasma after liver ischemia/reperfusion injury (IRI) using microarray technology. Microarray assays revealed that 64 LncRNAs were upregulated, and 244 LncRNAs were downregulated in the plasma of liver IRI mouse. Among these dysregulated plasma LncRNAs, 59-61% were intergenic, 22-25% were antisense overlap, 8-12% were sense overlap and 6-7% were bidirectional. Ten dysregulated plasma LncRNAs were validated by quantitative PCR assays, confirming the accuracy of microarray analysis result. Comparison analysis between dysregulated plasma and liver LncRNA profile after liver IRI revealed that among the 308 dysregulated plasma LncRNAs, 245 LncRNAs were present in the liver, but remained unchanged. In contrast, among the 98 dysregulated liver LncRNAs after IRI, only 19 were present in the plasma, but remained unchanged. LncRNA AK139328 had been previously reported to be upregulated in the liver after IRI, and silencing of hepatic AK139328 ameliorated liver IRI. Both microarray and RT-PCR analyses failed to detect the presence of AK139328 in mouse plasma. In summary, the current study compared the difference between dysregulated LncRNA profile in mouse plasma and liver after liver IRI, and suggested that a group of dysregulated plasma LncRNAs have the potential of becoming novel biomarkers for evaluation of ischemic liver injury.

  17. CRF01_AE-specific neutralizing activity observed in plasma derived from HIV-1-infected Thai patients residing in northern Thailand: comparison of neutralizing breadth and potency between plasma derived from rapid and slow progressors.

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    Sompong Sapsutthipas

    Full Text Available BACKGROUND: Development of a protective vaccine against human immunodeficiency virus type 1 (HIV-1 is an important subject in the field of medical sciences; however, it has not yet been achieved. Potent and broadly neutralizing antibodies are found in the plasma of some HIV-1-infected patients, whereas such antibody responses have failed to be induced by currently used vaccine antigens. In order to develop effective vaccine antigens, it is important to reveal the molecular mechanism of how strong humoral immune responses are induced in infected patients. As part of such studies, we examined the correlation between the anti-HIV-1 neutralizing antibody response and disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the anti-HIV-1 neutralizing activity of plasma derived from 33 rapid and 34 slow progressors residing in northern Thailand. The level of neutralizing activity varied considerably among plasmas, and no statistically significant differences in the potency and breadth of neutralizing activities were observed overall between plasma derived from rapid and slow progressors; however, plasma of 4 slow progressors showed neutralizing activity against all target viruses, whereas none of the plasma of rapid progressors showed such neutralizing activity. In addition, 21% and 9% of plasmas derived from slow and rapid progressors inhibited the replication of more than 80% of CRF01_AE Env-recombinant viruses tested, respectively. Neutralization of subtype B and C Env-recombinant viruses by the selected plasma was also examined; however, these plasma samples inhibited the replication of only a few viruses tested. CONCLUSIONS/SIGNIFICANCE: Although no statistically significant differences were observed in the potency and breadth of anti-HIV-1 neutralizing activities between plasma derived from rapid and slow progressors, several plasma samples derived from slow progressors neutralized CRF01_AE Env-recombinant viruses more frequently than

  18. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C;

    2000-01-01

    of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  19. Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

    Directory of Open Access Journals (Sweden)

    Clerzius Guerline

    2005-10-01

    Full Text Available Abstract Increasing evidence indicates that RNA interference (RNAi may be used to provide antiviral immunity in mammalian cells. Human micro (miRNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN-induced protein kinase R (PKR but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si and micro (miRNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

  20. Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

    Science.gov (United States)

    Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

    2016-03-01

    The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding.

  1. Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

    Science.gov (United States)

    Jacquenet, S; Ropers, D; Bilodeau, P S; Damier, L; Mougin, A; Stoltzfus, C M; Branlant, C

    2001-01-15

    The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

  2. Impact of HIV type 1 DNA levels on spontaneous disease progression: a meta-analysis.

    Science.gov (United States)

    Tsiara, Chrissa G; Nikolopoulos, Georgios K; Bagos, Pantelis G; Goujard, Cecile; Katzenstein, Terese L; Minga, Albert K; Rouzioux, Christine; Hatzakis, Angelos

    2012-04-01

    Several studies have reported the prognostic strength of HIV-1 DNA with variable results however. The aims of the current study were to estimate more accurately the ability of HIV-1 DNA to predict progression of HIV-1 disease toward acquired immunodeficiency syndrome (AIDS) or death, and to compare the prognostic information obtained by HIV-1 DNA with that derived from plasma HIV-1 RNA. Eligible articles were identified through a comprehensive search of Medline, ISI Web of Science, Scopus, and Google Scholar. The analysis included univariate and bivariate random-effects models. The univariate meta-analysis of six studies involving 1074 participants showed that HIV-1 DNA was a strong predictive marker of AIDS [relative risk (RR): 3.01, 95% confidence interval (CI): 1.88-4.82] and of all-cause mortality (RR: 3.49, 95% CI: 2.06-5.89). The bivariate model using the crude estimates of primary studies indicated that HIV-1 DNA was a significantly better predictor than HIV-1 RNA of either AIDS alone (ratio of RRs=1.47, 95% CI: 1.05-2.07) or of combined (AIDS or death) progression outcomes (ratio of RRs=1.51, 95% CI: 1.11-2.05). HIV-1 DNA is a strong predictor of HIV-1 disease progression. Moreover, there is some evidence that HIV-1 DNA might have better predictive value than plasma HIV-1 RNA.

  3. Altered subcellular localization of the NeuN/Rbfox3 RNA splicing factor in HIV-associated neurocognitive disorders (HAND).

    Science.gov (United States)

    Lucas, Calixto-Hope; Calvez, Mathilde; Babu, Roshni; Brown, Amanda

    2014-01-13

    The anti-NeuN antibody has been widely used for over 15 years to unambiguously identify post-mitotic neurons in the central nervous system of a wide variety of vertebrates including mice, rats and humans. In contrast to its widely reported nuclear localization, we found significantly higher NeuN reactivity in the cytoplasm of neurons in brain sections from HIV-infected individuals with cognitive impairment compared to controls. The protein target of anti-NeuN antisera was recently identified as the neuron-specific RNA splicing factor, Rbfox3, but its significance in diseases affecting the brain has not been previously reported. RNA splicing occurs in the nucleus hence, the altered localization of RbFox3 to the cytoplasm may lead to the downregulation of neuronal gene expression.

  4. Potential of RNA aptamers in the prevention of HIV-1 subtype C infections

    CSIR Research Space (South Africa)

    London, GM

    2014-10-01

    Full Text Available Compounds that have been used to prevent human immunodeficiency virus type-I (HIV-1) infections include synthetic chemicals, plant extras and monoclonal antibodies. Although most of these compounds have potent antiviral activity, they often fail...

  5. An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA.

    Science.gov (United States)

    Widera, Marek; Erkelenz, Steffen; Hillebrand, Frank; Krikoni, Aikaterini; Widera, Darius; Kaisers, Wolfgang; Deenen, René; Gombert, Michael; Dellen, Rafael; Pfeiffer, Tanya; Kaltschmidt, Barbara; Münk, Carsten; Bosch, Valerie; Köhrer, Karl; Schaal, Heiner

    2013-03-01

    Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

  6. Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction

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    Larance Ronsard

    2017-04-01

    Full Text Available HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans-activator of transcription (Tat, a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans-activation response (TAR RNA. In this study, HIV-1 infected patients (n = 120 from North India revealed Ser46Phe (20% and Ser61Arg (2% mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

  7. MicroRNA Profile in CD8+ T-Lymphocytes from HIV-Infected Individuals: Relationship with Antiviral Immune Response and Disease Progression.

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    Lander Egaña-Gorroño

    Full Text Available The relationship between host microRNAs (miRNA, viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response.miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11, Elite Controllers (EC, n = 15, Viremic Controllers (VC, n = 15, Viremic Progressors (VP, n = 13 and HIV-infected patients on therapy (ART, n = 14 was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms.A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response.Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA

  8. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

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    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  9. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Iordanskiy, Sergey [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Van Duyne, Rachel [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Romerio, Fabio [Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Kashanchi, Fatah, E-mail: fkashanc@gmu.edu [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States)

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  10. Elevated plasma soluble CD14 and skewed CD16+ monocyte distribution persist despite normalisation of soluble CD163 and CXCL10 by effective HIV therapy: a changing paradigm for routine HIV laboratory monitoring?

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    Alison Castley

    Full Text Available OBJECTIVE: We investigated plasma and flow cytometric biomarkers of monocyte status that have been associated with prognostic utility in HIV infection and other chronic inflammatory diseases, comparing 81 HIV+ individuals with a range of treatment outcomes to a group of 21 healthy control blood donors. Our aim is to develop and optimise monocyte assays that combine biological relevance, clinical utility, and ease of adoption into routine HIV laboratory practice. DESIGN: Cross-sectional evaluation of concurrent plasma and whole blood samples. METHODS: A flow cytometry protocol was developed comprising single-tube CD45, CD14, CD16, CD64, CD163, CD143 analysis with appropriately matched isotype controls. Plasma levels of soluble CD14 (sCD14, soluble CD163 (sCD163 and CXCL10 were measured by ELISA. RESULTS: HIV status was associated with significantly increased expression of CD64, CD143 and CD163 on CD16+ monocytes, irrespective of the virological response to HIV therapy. Plasma levels of sCD14, sCD163 and CXCL10 were also significantly elevated in association with viremic HIV infection. Plasma sCD163 and CXCL10 levels were restored to healthy control levels by effective antiretroviral therapy while sCD14 levels remained elevated despite virological suppression (p<0.001. CONCLUSIONS: Flow cytometric and plasma biomarkers of monocyte activation indicate an ongoing systemic inflammatory response to HIV infection, characterised by persistent alterations of CD16+ monocyte expression profiles and elevated sCD14 levels, that are not corrected by antiretroviral therapy and likely to be prognostically significant. In contrast, sCD163 and CXCL10 levels declined on antiretroviral therapy, suggesting multiple activation pathways revealed by these biomarkers. Incorporation of these assays into routine clinical care is feasible and warrants further consideration, particularly in light of emerging therapeutic strategies that specifically target innate immune

  11. HIV and schistosomiasis in rural Zimbabwe

    DEFF Research Database (Denmark)

    Kotzé, Sebastian Ranzi; Zinyama-Gutsire, Rutendo; Kallestrup, Per

    2015-01-01

    BACKGROUND: Vitamin A has widespread effects on immune function and is therefore interesting in HIV-infection. Retinol-binding protein (RBP or RBP4) is a negative acute-phase protein and a marker of vitamin A status. Our aim was to investigate the association of RBP with HIV progression, infection...... with schistosomiasis, inflammatory cytokines, and mortality. METHODS: The study included 192 HIV-infected and 177 HIV-uninfected individuals from Mupfure in rural Zimbabwe. Of these, 208 were infected with Schistosoma haematobium, 27 with S. mansoni and 48 with both. Plasma RBP, HIV-RNA, CD4 cell count, haemoglobin......, cytokines, clinical staging (CDC category), self-reported level of function (Karnoffsky Performance Score, KPS) and schistosomiasis status were assessed at baseline. Participants were followed up for survival 3-4 years post-enrolment. RESULTS: RBP levels were lower in HIV-infected individuals(p

  12. Plasma levels of soluble CD14 independently predict mortality in HIV infection

    DEFF Research Database (Denmark)

    Sandler, Netanya G; Wand, Handan; Roque, Annelys

    2011-01-01

    Chronic human immunodeficiency virus (HIV) infection is associated with intestinal permeability and microbial translocation that contributes to systemic immune activation, which is an independent predictor of HIV disease progression. The association of microbial translocation with clinical outcom...

  13. Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro.

    Science.gov (United States)

    Lanchy, Jean-Marc; Rentz, Casey A; Ivanovitch, John D; Lodmell, J Stephen

    2003-03-11

    An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.

  14. Impact of HIV Type 1 DNA Levels on Spontaneous Disease Progression: A Meta-Analysis

    DEFF Research Database (Denmark)

    Tsiara, Chrissa G; Nikolopoulos, Georgios K; Bagos, Pantelis G

    2012-01-01

    , and to compare the prognostic information obtained by HIV-1 DNA with that derived from plasma HIV-1 RNA. Eligible articles were identified through a comprehensive search of Medline, ISI Web of Science, Scopus, and Google Scholar. The analysis included univariate and bivariate random-effects models...

  15. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    Science.gov (United States)

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  16. High levels of plasma interferon gamma and +874T/A gene polymorphism is associated with HIV-TB co-infection.

    Science.gov (United States)

    Gutlapalli, V R; Sykam, Aparna; Tenali, Sandeep P; Suneetha, Sujai; Suneetha, Lavanya M

    2016-12-01

    Tuberculosis (TB) is one of the most frequent opportunistic infections in HIV patients leading to increased morbidity and death rate. This study was carried out to investigate the role of the cytokines IFN-γ and TNF-α level and their single nucleotide polymorphisms (SNPs) in HIV-TB co-infection. 247 HIV-TB (124 HIV-pulmonary TB, 123 HIV-extra pulmonary TB), 126 HIV positive individuals without tuberculosis and 129 healthy subjects (HS) were included to measure plasma levels of IFN-γ and TNF-α by sandwich ELISA and One way ANOVA statistical analysis was carried out among the groups. The SNPs of TNF-α-308 G/A, -238 G/A and IFN-γ+874 T/A were also investigated using amplification refractory mutation system polymerase chain reaction (ARMS-PCR). The frequencies between the groups were compared by Pearson's chi square statistical analysis. Plasma IFN-γ and TNF-α were significantly elevated in HIV-TB and TB (pTB groups compared to HS and HIV (pTB co-infection indicating 1.6 times increased risk for TB susceptibility. Elevated TNF-α levels in TB and HIV-TB suggest its involvement in TB pathogenesis. Copyright © 2016. Published by Elsevier Inc.

  17. Ultra-fast analysis of plasma and intracellular levels of HIV protease inhibitors in children: a clinical application of MALDI mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Jeroen J A van Kampen

    Full Text Available HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs, and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI-triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 microL plasma and 1x10(6 PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.

  18. microRNA在人类免疫缺陷病毒感染中的作用%The Function of miRNA in the HIV Infection

    Institute of Scientific and Technical Information of China (English)

    王羽欧; 桑伟伟; 张红胜

    2011-01-01

    microRNA(miRNA) play a significant role in the rugulation of gene expression, the “cross-talk” between the host and viruses on the level of miRNA is rather complicated. Emerging data suggest that viral miRNA (vmiRNA) derived from human immunodeficiency virus(HIV) may have a key role in regulating viral pathogenesis in vivo through various ways, such as helping maintain the latent state, and protecting the infected cells from apoptosis or stress induced cell death. Host's miRNA are involved in the antiviral defence mechanisms, but are encountered with diverse resistance at the same time, including the viral up- and downregulation of cellular miRNA and the RNA-silencing suppressors encoded by HIV. Deep researches into the dynamic strike-counterstrike interplay between HIV and the host on the level of miRNA help us understand pathogenic mechanisms of HIV further and develop new therapeutic strategies.%microRNA(miRNA)对调控基因表达有着重要作用,病毒与宿主在miRNA水平存在着复杂的"对话".研究显示,人类免疫缺陷病毒(HIV)可能编码病毒miRNA(vmiRNA),通过维持病毒潜伏、保护感染细胞免于凋亡或外力刺激下的细胞死亡等方式,在HIV病理过程中发挥重要作用.宿主miRNA则参与到了抗HIV的防御性机制中,但也面临HIV调控相应宿主miRNA、编码RNA沉默抑制物等多种阻力.深入研究HIV与宿主间miRNA水平上的动态相互作用,有助于进一步了解HIV的致病机理,开发新的治疗策略.

  19. A plasma microRNA signature as a biomarker for acquired aplastic anemia.

    Science.gov (United States)

    Hosokawa, Kohei; Kajigaya, Sachiko; Feng, Xingmin; Desierto, Marie J; Fernandez Ibanez, Maria Del Pilar; Rios, Olga; Weinstein, Barbara; Scheinberg, Phillip; Townsley, Danielle M; Young, Neal S

    2017-01-01

    Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.

  20. Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples

    DEFF Research Database (Denmark)

    Binderup, Helle Glud; Houlind, Kim; Madsen, Jonna Skov;

    2016-01-01

    was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0-1×109/L. Conclusion: We found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even......Background: In the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content...

  1. Molecular characterization of the HIV type 1 vpr gene in infected Chinese former blood/plasma donors at different stages of diseases.

    Science.gov (United States)

    Shen, Chengli; Gupta, Phalguni; Wu, Hao; Chen, Xinyue; Huang, Xiaojie; Zhou, Yusen; Chen, Yue

    2008-04-01

    The HIV-1 vpr regions were PCR amplified and sequenced from eight long-term nonprogressors' (LTNP) and seven AIDS patients' DNA samples of a cohort of HIV-1-infected Chinese plasma/blood donors (PBDs). Sequence analysis revealed that the patients' HIV-1 vpr sequences belong to HIV-1 subtype B and there were no differences in the divergence of vpr sequences between these two groups of patients. Similarly, in the deduced amino acid sequences, no significant differences have been detected in vpr functional domains from patients at different stages of the disease. Moreover, the predicted binding motifs of HLA A2 and A11 were highly conserved among patients' vpr amino acid sequences. These results show that vpr may not play an important role in HIV-1 pathogenesis in different stages of Chinese patients and may have important implications in developing vpr-related treatments suitable for HIV-1-infected PBDs.

  2. Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

    Science.gov (United States)

    Elliott, Julian H; Wightman, Fiona; Solomon, Ajantha; Ghneim, Khader; Ahlers, Jeffrey; Cameron, Mark J; Smith, Miranda Z; Spelman, Tim; McMahon, James; Velayudham, Pushparaj; Brown, Gregor; Roney, Janine; Watson, Jo; Prince, Miles H; Hoy, Jennifer F; Chomont, Nicolas; Fromentin, Rémi; Procopio, Francesco A; Zeidan, Joumana; Palmer, Sarah; Odevall, Lina; Johnstone, Ricky W; Martin, Ben P; Sinclair, Elizabeth; Deeks, Steven G; Hazuda, Daria J; Cameron, Paul U; Sékaly, Rafick-Pierre; Lewin, Sharon R

    2014-10-01

    Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. ClinicalTrials.gov NCT01365065.

  3. Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Julian H Elliott

    2014-10-01

    Full Text Available Human immunodeficiency virus (HIV persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART. The primary endpoint was change in cell associated unspliced (CA-US HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065. Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90% with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1. CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.ClinicalTrials.gov NCT01365065.

  4. Plasma Concentration of the Neurofilament Light Protein (NFL is a Biomarker of CNS Injury in HIV Infection: A Cross-Sectional Study

    Directory of Open Access Journals (Sweden)

    Magnus Gisslén

    2016-01-01

    Interpretation: These results show that plasma NFL may prove a valuable tool to evaluate ongoing CNS injury in HIV infection that may be applied in the clinic and in research settings to assess the presence if active CNS injury. Because CSF NFL is also elevated in a variety of other CNS disorders, sensitive measures of plasma NFL may similarly prove useful in other settings.

  5. A Single-Nucleotide Polymorphism in CYP2B6 Leads to >3-Fold Increases in Efavirenz Concentrations in Plasma and Hair Among HIV-Infected Women

    OpenAIRE

    Gandhi, Monica; Greenblatt, Ruth M.; Bacchetti, Peter; Jin, Chengshi; Yong HUANG; Anastos, Kathryn; Cohen, Mardge; DeHovitz, Jack A.; Sharp, Gerald B; Gange, Stephen J.; Liu, Chenglong; Hanson, Susan C.; Aouizerat, Bradley

    2012-01-01

    Background. Efavirenz exhibits marked interindividual variability in plasma levels and toxicities. Prior pharmacogenetic studies usually measure exposure via single plasma levels, examine limited numbers of polymorphisms, and rarely model multiple contributors. We analyzed numerous genetic and nongenetic factors impacting short-term and long-term exposure in a large heterogeneous population of human immunodeficiency virus (HIV)–infected women.

  6. Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

    Directory of Open Access Journals (Sweden)

    Sylvie Rato

    Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

  7. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  8. Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1.

    Science.gov (United States)

    Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Huidobro-Toro, Juan Pablo; Sargueil, Bruno; López-Lastra, Marcelo

    2012-01-01

    The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.

  9. Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1.

    Directory of Open Access Journals (Sweden)

    Maricarmen Vallejos

    Full Text Available The 5'untranslated regions (UTR of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES. In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.

  10. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

    Directory of Open Access Journals (Sweden)

    Orit Wolstein

    2014-01-01

    Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

  11. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  12. Distinct cytokine/chemokine network in semen and blood characterize different stages of HIV infection.

    Science.gov (United States)

    Vanpouille, Christophe; Introini, Andrea; Morris, Sheldon R; Margolis, Leonid; Daar, Eric S; Dube, Michael P; Little, Susan J; Smith, David M; Lisco, Andrea; Gianella, Sara

    2016-01-01

    The cytokine/chemokine network is used by the innate and adaptive immune system to orchestrate effective immune responses. Here, we describe the cross-sectional association between cytokine levels and stage of HIV infection to gain novel insights into HIV-1 immunopathogenesis and identify novel therapeutic targets. Concentrations of 31 cytokine/chemokines were retrospectively measured in blood and seminal plasma collected from 252 individuals enrolled in four well characterized cohorts: HIV-uninfected, untreated HIV-infected in early phase of infection, untreated HIV-infected in late phase of infection, and HIV-infected on antiretroviral therapy with undetectable HIV RNA levels in blood (<50 copies/ml). Cytokine/chemokine levels were measured by multiplex-bead array. Comparisons between groups were performed by Mann-Whitney U-test and P values were adjusted for multiple comparisons using the Benjamini-Hochberg method. Presence of HIV-infection skewed the cytokine/chemokine network towards a pro-inflammatory response in both blood and semen compared to HIV-uninfected controls. Such changes emerged within the first weeks of infection and were maintained thereafter: Among untreated HIV-infected individuals, none of the 31 measured cytokines were significantly different between early and later stages of infection. Suppression of plasma HIV RNA with ART did not result in normalization of the levels of pro-inflammatory cytokines in blood. In semen, several pro-inflammatory cytokines were even further upregulated in ART-treated compared with HIV-uninfected and HIV-untreated individuals. A profound disruption in the cytokine/chemokine network is evident in blood and semen from the earliest stage of HIV infection shortly after the first detection of systemic viremia. These changes are maintained throughout the chronic phase of the infection and do not normalize despite ART and suppression of plasma HIV RNA.

  13. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

    Directory of Open Access Journals (Sweden)

    Gene-Errol E Ringpis

    Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

  14. Impact of chemotherapy for HIV-1 related lymphoma on residual viremia and cellular HIV-1 DNA in patients on suppressive antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Anthony R Cillo

    Full Text Available The first cure of HIV-1 infection was achieved through complex, multimodal therapy including myeloablative chemotherapy, total body irradiation, anti-thymocyte globulin, and allogeneic stem cell transplantation with a CCR5 delta32 homozygous donor. The contributions of each component of this therapy to HIV-1 eradication are unclear. To assess the impact of cytotoxic chemotherapy alone on HIV-1 persistence, we longitudinally evaluated low-level plasma viremia and HIV-1 DNA in PBMC from patients in the ACTG A5001/ALLRT cohort on suppressive antiretroviral therapy (ART who underwent chemotherapy for HIV-1 related lymphoma without interrupting ART. Plasma HIV-1 RNA, total HIV-1 DNA and 2-LTR circles (2-LTRs in PBMC were measured using sensitive qPCR assays. In the 9 patients who received moderately intensive chemotherapy for HIV-1 related lymphoma with uninterrupted ART, low-level plasma HIV-1 RNA did not change significantly with chemotherapy: median HIV-1 RNA was 1 copy/mL (interquartile range: 1.0 to 20 pre-chemotherapy versus 4 copies/mL (interquartile range: 1.0 to 7.0 post-chemotherapy. HIV-1 DNA levels also did not change significantly, with median pre-chemotherapy HIV-1 DNA of 355 copies/106 CD4+ cells versus 228 copies/106 CD4+ cells post-chemotherapy. 2-LTRs were detectable in 2 of 9 patients pre-chemotherapy and in 3 of 9 patients post-chemotherapy. In summary, moderately intensive chemotherapy for HIV-1 related lymphoma in the context of continuous ART did not have a prolonged impact on HIV-1 persistence. Clinical trials registration unique identifier: NCT00001137.

  15. Impact of chemotherapy for HIV-1 related lymphoma on residual viremia and cellular HIV-1 DNA in patients on suppressive antiretroviral therapy.

    Science.gov (United States)

    Cillo, Anthony R; Krishnan, Supriya; McMahon, Deborah K; Mitsuyasu, Ronald T; Para, Michael F; Mellors, John W

    2014-01-01

    The first cure of HIV-1 infection was achieved through complex, multimodal therapy including myeloablative chemotherapy, total body irradiation, anti-thymocyte globulin, and allogeneic stem cell transplantation with a CCR5 delta32 homozygous donor. The contributions of each component of this therapy to HIV-1 eradication are unclear. To assess the impact of cytotoxic chemotherapy alone on HIV-1 persistence, we longitudinally evaluated low-level plasma viremia and HIV-1 DNA in PBMC from patients in the ACTG A5001/ALLRT cohort on suppressive antiretroviral therapy (ART) who underwent chemotherapy for HIV-1 related lymphoma without interrupting ART. Plasma HIV-1 RNA, total HIV-1 DNA and 2-LTR circles (2-LTRs) in PBMC were measured using sensitive qPCR assays. In the 9 patients who received moderately intensive chemotherapy for HIV-1 related lymphoma with uninterrupted ART, low-level plasma HIV-1 RNA did not change significantly with chemotherapy: median HIV-1 RNA was 1 copy/mL (interquartile range: 1.0 to 20) pre-chemotherapy versus 4 copies/mL (interquartile range: 1.0 to 7.0) post-chemotherapy. HIV-1 DNA levels also did not change significantly, with median pre-chemotherapy HIV-1 DNA of 355 copies/106 CD4+ cells versus 228 copies/106 CD4+ cells post-chemotherapy. 2-LTRs were detectable in 2 of 9 patients pre-chemotherapy and in 3 of 9 patients post-chemotherapy. In summary, moderately intensive chemotherapy for HIV-1 related lymphoma in the context of continuous ART did not have a prolonged impact on HIV-1 persistence. Clinical trials registration unique identifier: NCT00001137.

  16. HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

    Directory of Open Access Journals (Sweden)

    Katherine L. Williams

    2015-10-01

    -cell lineages in the same individual. Further, these mAbs mediate potent plasma IgG-specific ADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

  17. Progression of HIV in haemophilia.

    Science.gov (United States)

    Ragni, M V

    1998-07-01

    The recent elucidation of the life cycle and dynamics of the human immunodeficiency virus (HIV) and technological advances in development of the HIV RNA PCR assay for sensitive detection of viral load have revolutionized the diagnosis, management, and treatment of HIV infection. Beginning with initial infection, there is unremitting, high-level viral replication that persists throughout the course of HIV infection. The measure of the amount of virus present in plasma, HIV viral load, is the single most important predictor of HIV progression, the best indicator of immune system decline, and the best guide for initiating and monitoring antiviral treatment. Further, HIV viral load has become the new yardstick against which other markers, including CD4 number, age, chemokine receptor mutations, cytotoxic T-cell responses, and neutralizing antibody titers are assessed. For individuals with haemophilia, additional 'markers' may have significant impact on the outcome of HIV disease. Chronic factor concentrate treatment has led to transfusion-associated hepatitis, co-infection with hepatitis C (HCV), and chronic liver disease. The latter may become accelerated with HIV progression and may lead to hepatotoxicity with antiviral drug therapy. Chronic factor concentrate treatment has also been associated with immunosuppression, including both B- and T-cell immune defects. In HIV(+) haemophilic men, this immune deficit has led to lower CD4 counts with HIV progression and poorer CD4 response to antiviral drugs than in gay men. The underlying haemophilic bleeding tendency may result in significant haemorrhage with HIV-associated immune thrombocytopenia and with protease inhibitor antiretroviral therapy. Although AIDS is the leading cause of death in this group, the reduction in the size of the haemophilia population over the next two centuries is estimated to be small, and survival should improve as better antiviral therapeutics are identified.

  18. Plasma microRNA profiling of pediatric patients with immune thrombocytopenic purpura.

    Science.gov (United States)

    Bay, Ali; Coskun, Enes; Oztuzcu, Serdar; Ergun, Sercan; Yilmaz, Fatih; Aktekin, Elif

    2014-06-01

    Immune thrombocytopenic purpura (ITP) is a commonly acquired autoimmune bleeding disorder in children. MicroRNAs (miRNAs) are small RNAs which are found in cells and circulation, and play a role in protein synthesis and regulation. In this study, we aimed to determine a biomarker for childhood ITP comparing the plasma miRNA levels of children having ITP with healthy children. A total of 86 patients with ITP and 56 healthy children followed up by the Department of Pediatric Hematology and Oncology in University of Gaziantep since July 2011 were enrolled in the study. The 86 patients with ITP were evaluated in two groups as 43 acute ITP (aITP) and 43 chronic ITP (cITP) patients. Plasma expression levels of 379 miRNAs were investigated by RT-PCR (quantitative RT-PCR) technique and they were compared between aITP, cITP, and control groups. For all miRNAs, the average of raw quantification cycle values of three groups separately in the analysis chip was accepted as the reference gene value, and normalization was done according to this value. Statistically significant differences were detected in seven miRNAs (miR-302c-3p, miR-483-5p, miR-410, miR-544a, miR-302a-3p, miR-223-3p, and miR-597) investigated between the groups with respect to the expression levels. The expression rates were found to be over 95% in miR-302c-3p and miR-483-5p, over 75% in miR-410, and over 40% in miR-544, miR-302a-3p, and miR-223-3p in all three groups. The detection of significant differences between plasma miRNA levels of aITP and cITP patients and healthy children may provide useful information in the prediction of the course of disease, determination of disease etiopathogenesis, and the development of new therapeutic modalities.

  19. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Science.gov (United States)

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  20. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Weiming Wang

    Full Text Available CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN or Transcription activator-like effector nuclease (TALEN. In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9 and single guided RNAs (sgRNAs. We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  1. Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation

    DEFF Research Database (Denmark)

    Meerson, Ari; Ploug, Thorkil

    2016-01-01

    of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could...

  2. Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs

    DEFF Research Database (Denmark)

    Brix-Christensen, V.; Vestergaard, C.; Chew, M.

    2003-01-01

    Background: Plasma concentrations of inflammatory markers are increased in response to the trauma of cardiac surgery and cardiopulmonary bypass (CPB). It is, however, unknown whether the plasma cytokine levels and cytokine mRNA expression at organ level reflect each other. Methods: Twenty-six pig...... poorly reflected mRNA expression of the pro- and anti-inflammatory cytokines....

  3. Neurocognitive Impairment in HIV-Infected Naïve Patients with Advanced Disease: The Role of Virus and Intrathecal Immune Activation

    Directory of Open Access Journals (Sweden)

    Monica Airoldi

    2012-01-01

    Full Text Available Objective. To investigate intrathecal immune activation parameters and HIV-RNA in HIV-associated neurocognitive disorders (HAND of advanced naïve HIV-infected patients and to evaluate their dynamics before and after initiation of antiretroviral therapy (ART. Methods. Cross-sectional and longitudinal analysis of HIV RNA, proinflammatory cytokines (IL-6, IL-10, INF-γ, TNF-α, TGF-β1, and TGF-β2 and chemokines (MIP-1α, MIP-1β, and MCP-1 in plasma and cerebrospinal fluid (CSF of HIV-infected patients with CD4 <200/μL. Results. HAND was diagnosed at baseline in 6/12 patients. Baseline CSF HIV-RNA was comparable in patients with or without HAND, whereas CSF concentration of IL-6 and MIP-1β, proinflammatory cytokines, was increased in HAND patients. CSF evaluation at 12 weeks was available in 10/12 cases. ART greatly reduced HIV-RNA in all patients. Nevertheless, IL-6 and MIP-1β remained elevated after 12 weeks of therapy in HAND patients, in whom CSF HIV RNA decay was slower than the plasmatic one as well. Conclusion. Immune activation, as indicated by inflammatory cytokines, but not higher levels of HIV-RNA is observed in advanced naïve HIV-infected patients with HAND. In HAND patients, ART introduction resulted in a less rapid clearance of CSF viremia compared to plasma and no modifications of intratechal immune activation.

  4. Comparison of immunologic restoration and virologic response in plasma, tonsillar tissue, and cerebrospinal fluid in HIV-1-infected patients treated with double versus triple antiretroviral therapy in very early stages: The Spanish EARTH-2 Study. Early Anti-Retroviral Therapy Study.

    Science.gov (United States)

    García, F; Alonso, M M; Romeu, J; Knobel, H; Arrizabalaga, J; Ferrer, E; Dalmau, D; Ruiz, I; Vidal, F; Frances, A; Segura, F; Gomez-Sirvent, J L; Cruceta, A; Clotet, B; Pumarola, T; Gallart, T; O'Brien, W A; Miró, J M; Gatell, J M

    2000-09-01

    The objective of antiretroviral therapy is to obtain an almost complete and durable suppression of viral replication in all compartments to facilitate recovery of the immune system. We assessed the virologic effect in plasma, tonsillar tissue, and cerebrospinal fluid (CSF) in 94 HIV-1-infected patients with CD4 counts >500 x 106 cells per liter and viral load >5000 copies/ml randomly assigned to triple antiretroviral therapy (two nucleoside reverse transcriptase inhibitors (NRTIs) plus one protease inhibitor) versus double therapy (two NRTIs). We also analyzed the immunologic recovery in this cohort of patients. Lymphoid tissue and cerebrospinal fluid viral load, development of genotypic resistance, proliferative responses to HIV-1 specific antigens, and other immunophenotypic markers were analyzed. The proportion of patients who achieved a decrease in HIV RNA levels to <200 copies/ml was significantly greater in the triple therapy group than in the two drug groups (p =.0002 for each pair-wise difference). At week 52, tonsillar tissue HIV RNA from 5 patients treated with triple therapy was lower than the limit of detection, whereas the mean +/- standard error in patients with double therapy (n = 5) was 5.03 +/- 0.34 copies/mg/tissue. In all 10 patients, CSF viral load (VL) was <20 HIV-1 RNA copies/ml at week 52. CSF cell counts and protein levels tended to decrease after 52 weeks of antiretroviral therapy. After 1 year of therapy, 13 of 21 patients (62%) in the double-therapy groups (zidovudine plus lamivudine [n = 9] and stavudine plus lamivudine [n = 12]) had evidence of M184V mutation. None of the 10 samples of patients receiving triple therapy could be amplified because of low HIV RNA levels. The mean increase in CD4 cells at week 52 was greater in the stavudine and lamivudine and indinavir group than in the double-treatment arms (186 versus 67 and 102, respectively; p =.03). In patients treated with triple therapy, the increase in naive T cells (CD4 and CD8

  5. Effect of antiretroviral drugs on maternal CD4 lymphocyte counts, HIV-1 RNA levels, and anthropometric parameters of their neonates Efeito das drogas anti-retrovirais sobre os valores dos linfócitos TCD4, RNA do HIV-1 e parâmetros antropométricos de neonatos de gestantes portadoras do HIV-1

    Directory of Open Access Journals (Sweden)

    Patrícia El Beitune

    2005-06-01

    Full Text Available PURPOSE: To study the effect of antiretroviral drugs administered during pregnancy on CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women and on the anthropometric parameters of their neonates. METHODS: A prospective study was conducted on 57 pregnant women and their neonates divided into 3 groups: ZDV Group, HIV-infected mothers taking zidovudine (n = 20; triple therapy (TT Group, mothers taking zidovudine + lamivudine + nelfinavir (n = 25, and Control Group, normal women (n = 12. CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women were analyzed during two periods of pregnancy. The perinatal prognosis took into account preterm rates, birth weight, intrauterine growth restriction, perinatal death, and vertical transmission of HIV-1. Data were analyzed statistically using the nonparametric chi-square, Mann-Whitney, Friedman, Kruskal-Wallis, and Wilcoxon matched pairs tests, with the level of significance set at P OBJETIVOS: Estudar o efeito das drogas anti-retrovirais sobre a quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV-1 e parâmetros antropométricos de seus neonatos. MÉTODOS: Estudo prospectivo avaliando 57 gestantes e seus neonatos em três grupos: Grupo AZT, gestantes portadoras do HIV utilizando zidovudina (n=20; Grupo TT, mães utilizando zidovudina+lamivudina+nelfinavir (n=25, e Grupo Controle, mulheres saudáveis (n=12. A quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV foi analisada em dois períodos durante a gestação. O prognóstico perinatal levou em consideração as taxas de pré-termos, restrição de crescimento intra-útero, mortalidade perinatal e transmissão vertical do HIV-1. Os dados foram analisados utilizando-se testes não paramétricos de qui-quadrado, Mann-Whitney, Friedman, Kruskal-Wallys e Wilcoxon para amostras pareadas, considerando-se significativos valores associados a p<0,05. RESULTADOS: Observou-se homogeneidade entre

  6. Design and synthesis of á,á-trehalose derivatives bearing guanidino groups as inhibitors for the Tat Protein-TAR RNA interaction of HIV-1

    Institute of Scientific and Technical Information of China (English)

    Wang Min; Xu Zhidong; Tu Pengfei; Xiao Sulong; Yu Xiaolin; Yang Ming

    2004-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6′-diamino-6,6′ -dideoxy-á,á-trehalose was obtained from selective bromination of a,a-trehalose at C-6,6′, followed by acetylation, azide displacement, deacetylation and reduction.Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated 6,6′-bis(L-arginylamino)-6,6′-dideoxy-á,á-trehalose,6,6′-bisguanidino-6,6′-dideoxy-á ,á-trehalose, 6,6′-bis [((a)-guanidinobutyryl)amino]-6,6′-dideoxy-á,á-trehalose and 6,6′-bis(guanidinoacetylamino)-6,6′-dideoxy-á,á-trehalose, respectively.Their abilities to inhibit Tat-TAR RNA interaction were determined by a Tat-dependent HIV-1LTR-driven CAT assays.

  7. Effect of rosiglitazone on visfatin and retinol-binding protein-4 plasma concentrations in HIV-positive patients.

    Science.gov (United States)

    Haider, D G; Schindler, K; Mittermayer, F; Müller, M; Nowotny, P; Rieger, A; Luger, A; Ludvik, B; Wolzt, M

    2007-04-01

    Thiazolidinediones (TZD) may improve insulin resistance in patients with diabetes and HIV. The novel adipocytokines visfatin and retinol-binding protein-4 (RBP-4) have been proposed to influence the development of impaired glucose tolerance. The impact of TZD on these cytokines is yet unknown. In this randomized, double-blind, placebo-controlled parallel group study, 37 lean HIV-positive subjects aged 19-50 years were treated with 8 mg/day rosiglitazone (n=20) or placebo (n=17) for 6 months. Insulin sensitivity was estimated from the homeostasis model assessment (HOMA) index. Fasting visfatin, RBP-4, leptin, and adiponectin plasma concentrations were analyzed by immunoassays. Rosiglitazone had no effect on impaired insulin sensitivity, but increased median plasma visfatin from 6.2 ng/ml (95% CI: 5.9; 6.5) to 13.7 ng/ml (12.6; 19.1) (P<0.001) and adiponectin from 3.2 ng/ml (2.2; 4.0) to 4.0 ng/ml (3.3; 8.5; P<0.001). RBP-4 was lowered from 21.0 ng/ml (19.6; 23.1) to 16.3 ng/ml (15.2; 17.0; P<0.001), and leptin concentrations were unchanged. Adipocytokine concentrations were stable in subjects receiving placebo, where a deterioration in insulin sensitivity was detectable (P<0.05). Changes in visfatin and RBP-4 were correlated in subjects receiving rosiglitazone (r=-0.64, P<0.01) but not placebo (r=0.12, P=0.15). TZD treatment affects circulating adipocytokine concentrations in subjects with HIV. Reductions in RBP-4 and increases in visfatin may contribute to the pharmacodynamic action of TZD on glucose homeostasis. Quantification of adipocytokines might be useful to assess TZD treatment effectiveness in insulin-resistant subjects with HIV.

  8. Emtricitabine seminal plasma and blood plasma population pharmacokinetics in HIV-infected men in the EVARIST ANRS-EP 49 study.

    Science.gov (United States)

    Valade, Elodie; Tréluyer, Jean-Marc; Illamola, Silvia M; Bouazza, Naïm; Foissac, Frantz; De Sousa Mendes, Maïlys; Lui, Gabrielle; Chenevier-Gobeaux, Camille; Suzan-Monti, Marie; Rouzioux, Christine; Assoumou, Lambert; Viard, Jean-Paul; Hirt, Déborah; Urien, Saïk; Ghosn, Jade

    2015-11-01

    We aimed to describe bl