Science.gov

Sample records for increases hiv dna

  1. HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Naïve Patients

    PubMed Central

    Valcour, Victor G.; Ananworanich, Jintanat; Agsalda, Melissa; Sailasuta, Napapon; Chalermchai, Thep; Schuetz, Alexandra; Shikuma, Cecilia; Liang, Chin-Yuan; Jirajariyavej, Supunee; Sithinamsuwan, Pasiri; Tipsuk, Somporn; Clifford, David B.; Paul, Robert; Fletcher, James L. K.; Marovich, Mary A.; Slike, Bonnie M.; DeGruttola, Victor; Shiramizu, Bruce

    2013-01-01

    Objectives Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14+ enriched monocytes predicted cognitive impairment and brain injury. Methods We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14+ enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF). Results The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14+ HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14+ HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions. Interpretation Reservoir burden of HIV DNA in monocyte-enriched (CD14+) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS. PMID:23936155

  2. Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy.

    PubMed

    Cossarizza, Andrea; Riva, Agostino; Pinti, Marcello; Ammannato, Silvia; Fedeli, Paolo; Mussini, Cristina; Esposito, Roberto; Galli, Massimo

    2003-08-01

    We have evaluated mitochondrial (mt) DNA content in CD4 and CD8 peripheral blood lymphocytes (PBLs) from HIV-infected patients taking highly active antiretroviral therapy (HAART) who display different types of adipose tissue alterations. A cross-sectional study was performed in a total of 23 patients with lipodystrophy (LD): nine patients with fat accumulation, six patients with fat loss, eight patients with combined form, who were compared to 11 individuals infected by HIV without LD (HIV-positive) and 10 seronegative controls (CTRL). PBLs were obtained by standard methods, that is, gradient density centrifugation on Ficoll, and CD4 or CD8 cells were positively isolated by magnetic sorting to eliminate the contamination of platelets. mtDNA content was then measured by an original assay based upon real-time PCR. mtDNA content was significantly increased in CD4 T cells from patients with LD, while no differences were present between CD4 and CD8 cells from HIV-positive and CTRL individuals. Nor were any differences found when comparing LD or HIV-positive patients treated with stavudine or zidovudine, or taking D-drugs or non D-drugs. Patients with fat accumulation had significantly higher mtDNA content compared to HIV-positive and CTRL, this phenomenon regarding both CD4 and CD8 PBLs. Considering all HIV-positive patients (including LD), mtDNA content showed a significant, positive correlation with cholesterolaemia but not with triglyceridaemia and glycaemia. Relatively high mtDNA content in LD patients, as well as the correlation between mtDNA content and cholesterol in all HIV-positive subjects, suggest the involvement of mitochondria in such a pathology. However, further studies are needed to confirm these initial observations and ascertain whether the quantification of mtDNA in PBL is a useful and reliable marker to investigate and monitor HAART-related changes in fat distribution.

  3. Increased presence of Epstein-Barr virus DNA in ocular fluid samples from HIV negative immunocompromised patients with uveitis

    PubMed Central

    Ongkosuwito, J.; Van der Lelij, A.; Bruinenberg, M.; Doorn, M. W.; Feron, E.; Hoyng, C.; de Keizer, R. J W; Klok, A.; Kijlstra, A.

    1998-01-01

    AIMS—To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis.
METHODS—Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n=46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract.
RESULTS—Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p<0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p=0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis.
CONCLUSIONS—When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.

 Keywords: Epstein-Barr virus; intraocular fluid; polymerase chain reaction; uveitis PMID:9602620

  4. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  5. HIV DNA Integration

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2012-01-01

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified. PMID:22762018

  6. China update: HIV increasing.

    PubMed

    Gil, V E

    1993-01-01

    The HIV/AIDS case rate in China increased 117% over the period 1990-1992, from 446 to 957 HIV infections. While the majority of cases in 1990 were localized in Yunnan among minority farmers and manual laborers, infection is now found in 19 provinces, counties, and urban areas over a wider spectrum of society. The concentration of cases among IV-drug users has decreased from 83.4% to 72.6%. HIV monitoring and prevention stations have been in place in the country since 1986. The government also encouraged special zones and semiautonomous cities as well as the World Health Organization to take steps to monitor and prevent the spread of HIV. While these effort have served to augment the degree of sex education previously provided, low budgets, bureaucracy, and ambivalence have impeded control efforts. Only 1.25 million of the 1.16 billion population has been serosampled over 8 years and 100,000 fewer serosamples were taken in 1992 compared to in 1991. Neither the general population nor health workers have sufficient knowledge about HIV/AIDS to prevent its continued spread. While gay men sampled in Beijing were better informed about transmission means and risk groups, over two thirds believed they were not at risk if they avoided having sex with foreigners. Recent economic reform measures allowing large movements of population from rural to urban areas, increased disposable income available for prostitutes, and greater exposure to alternative sexual norms and behaviors through the media and music further increase the risk of HIV transmission, especially among the younger generation. To counter these risks, an AIDS hotline for information and referrals has been established in Beijing which openly reaches out to the homosexual community and fields 8-12 calls/day. Training programs for doctors, counselors, professors, and social workers have been attended by people from more than 30 provinces and regions. In addition, modest research into sexual behavior is also being

  7. Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

    PubMed Central

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

  8. Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis.

    PubMed Central

    Ji, J; Hoffmann, J S; Loeb, L

    1994-01-01

    The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus. Images PMID:7510388

  9. Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates.

    PubMed

    John, Jubi; Kim, Youngju; Bennett, Nicholas; Das, Kalyan; Liekens, Sandra; Naesens, Lieve; Arnold, Eddy; Maguire, Anita R; Götte, Matthias; Dehaen, Wim; Balzarini, Jan

    2015-10-22

    Alpha-carboxynucleoside phosphonates (α-CNPs) are novel viral DNA polymerase inhibitors that do not need metabolic conversion for enzyme inhibition. The prototype contains a cyclopentyl linker between nucleobase and α-carboxyphosphonate and preferentially (50- to 100-fold) inhibits HIV-1 RT compared with herpetic DNA polymerases. A synthesis methodology involving three steps has been developed for the synthesis of a series of novel α-CNPs, including a Rh(II)-catalyzed O-H insertion that connects the carboxyphosphonate group to a linker moiety and an attachment of a nucleobase to the other end of the linker by a Mitsunobu reaction followed by final deprotection. Replacing the cyclopentyl moiety in the prototype α-CNPs by a more flexible entity results in a selectivity shift of ∼ 100-fold in favor of the herpetic DNA polymerases when compared to selectivity for HIV-1 RT. The nature of the kinetic interaction of the acyclic α-CNPs against the herpetic DNA polymerases differs from the nature of the nucleobase-specific kinetic interaction of the cyclopentyl α-CNPs against HIV RT. PMID:26450273

  10. Mitochondrial DNA mutations in ageing and disease: implications for HIV?

    PubMed

    Payne, Brendan A I; Gardner, Kristian; Chinnery, Patrick F

    2015-01-01

    Mitochondrial DNA (mtDNA) mutations cause neurological and multisystem disease. Somatic (acquired) mtDNA mutations are also associated with degenerative diseases and with normal human ageing. It is well established that certain nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drugs cause inhibition of the mtDNA polymerase, pol γ, leading to a reduction in mtDNA content (depletion). Given this effect of NRTI therapy on mtDNA replication, it is plausible that NRTI treatment may also lead to increased mtDNA mutations. Here we review recent evidence for an effect of HIV infection or NRTI therapy on mtDNA mutations, as well as discussing the methodological challenges in addressing this question. Finally, we discuss the possible implications for HIV-infected persons, with particular reference to ageing.

  11. HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

    PubMed Central

    Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L.; Stout, Richard R.; Robb, Merlin L.; Shattock, Robin J.; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

    2015-01-01

    Background We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. Methods HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. Results The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Conclusion Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. Trial Registration International Standard

  12. Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

    PubMed

    Weinberg, J B; Shugars, D C; Sherman, P A; Sauls, D L; Fyfe, J A

    1998-05-19

    Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase. PMID:9610370

  13. Regional Cortical Thinning Associated with Detectable Levels of HIV DNA

    PubMed Central

    Kirk, Gregory R.; Sailasuta, Napapon; Valcour, Victor; Shiramizu, Bruce; Nakamoto, Beau K.; Shikuma, Cecilia

    2012-01-01

    High levels of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs), and specifically within CD14+ blood monocytes, have been found in HIV-infected individuals with neurocognitive impairment and dementia. The failure of highly active antiretroviral therapy (HAART) to eliminate cognitive dysfunction in HIV may be secondary to persistence of HIV-infected PBMCs which cross the blood-brain barrier, leading to perivascular inflammation and neuronal injury. This study assessed brain cortical thickness relative to HIV DNA levels and identified, we believe for the first time, a neuroimaging correlate of detectable PBMC HIV DNA in subjects with undetectable HIV RNA. Cortical thickness was compared between age- and education-matched groups of older (>40 years) HIV-seropositive subjects on HAART who had detectable (N = 9) and undetectable (N = 10) PBMC HIV DNA. Statistical testing revealed highly significant (P < 0.001) cortical thinning associated with detectable HIV DNA. The largest regions affected were in bilateral insula, orbitofrontal and temporal cortices, right superior frontal cortex, and right caudal anterior cingulate. Cortical thinning correlated significantly with a measure of psychomotor speed. The areas of reduced cortical thickness are key nodes in cognitive and emotional processing networks and may be etiologically important in HIV-related neurological deficits. PMID:22016479

  14. DNA confinement drives uncoating of the HIV Virus

    NASA Astrophysics Data System (ADS)

    Rouzina, I.; Bruinsma, R.

    2014-09-01

    The enzyme reverse transcriptase converts single-stranded RNA molecules into double-stranded DNA molecules inside mature HIV viral capsids. We present a model for the uncoating of the HIV virus where the capsid uncoating process is driven by the confinement force exerted on the capsid wall porduced to the double-stranded DNA generated by reverse transcriptase.

  15. Quantitative HIV-1 proviral DNA detection: a multicentre analysis.

    PubMed

    De Rossi, Anita; Zanchetta, Marisa; Vitone, Francesca; Antonelli, Guido; Bagnarelli, Patrizia; Buonaguro, Luigi; Capobianchi, Maria Rosaria; Clementi, Massimo; Abbate, Isabella; Canducci, Filippo; Monachetti, Alessia; Riva, Elisabetta; Rozera, Gabriella; Scagnolari, Carolina; Tagliamonte, Maria; Re, Maria Carla

    2010-10-01

    Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients. PMID:21213587

  16. HIV DNA Vaccine: Stepwise Improvements Make a Difference

    PubMed Central

    Felber, Barbara K.; Valentin, Antonio; Rosati, Margherita; Bergamaschi, Cristina; Pavlakis, George N.

    2014-01-01

    Inefficient DNA delivery methods and low expression of plasmid DNA have been major obstacles for the use of plasmid DNA as vaccine for HIV/AIDS. This review describes successful efforts to improve DNA vaccine methodology over the past ~30 years. DNA vaccination, either alone or in combination with other methods, has the potential to be a rapid, safe, and effective vaccine platform against AIDS. Recent clinical trials suggest the feasibility of its translation to the clinic. PMID:26344623

  17. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  18. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects

    PubMed Central

    Gach, Johannes S.; Gorlani, Andrea; Dotsey, Emmanuel Y.; Becerra, Juan C.; Anderson, Chase T. M.; Berzins, Baiba; Felgner, Philip L.; Forthal, Donald N.; Deeks, Steven G.; Wilkin, Timothy J.; Casazza, Joseph P.; Koup, Richard A.; Katlama, Christine; Autran, Brigitte; Murphy, Robert L.; Achenbach, Chad J.

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  19. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    PubMed

    Gach, Johannes S; Gorlani, Andrea; Dotsey, Emmanuel Y; Becerra, Juan C; Anderson, Chase T M; Berzins, Baiba; Felgner, Philip L; Forthal, Donald N; Deeks, Steven G; Wilkin, Timothy J; Casazza, Joseph P; Koup, Richard A; Katlama, Christine; Autran, Brigitte; Murphy, Robert L; Achenbach, Chad J

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  20. DNA/MVA Vaccines for HIV/AIDS.

    PubMed

    Iyer, Smita S; Amara, Rama R

    2014-01-01

    Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous "prime-boost" vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA)-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.

  1. Quantitation of HIV-1 DNA with a sensitive TaqMan assay that has broad subtype specificity.

    PubMed

    van der Sluis, Renée M; van Montfort, Thijs; Centlivre, Mireille; Schopman, Nick C T; Cornelissen, Marion; Sanders, Rogier W; Berkhout, Ben; Jeeninga, Rienk E; Paxton, William A; Pollakis, Georgios

    2013-01-01

    The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form. PMID:23059551

  2. Increased DNA methylation in the suicide brain.

    PubMed

    Haghighi, Fatemeh; Xin, Yurong; Chanrion, Benjamin; O'Donnell, Anne H; Ge, Yongchao; Dwork, Andrew J; Arango, Victoria; Mann, J John

    2014-09-01

    Clinical studies find that childhood adversity and stressful life events in adulthood increase the risk for major depression and for suicide. The predispositions to either major depression or suicide are thought to depend on genetic risk factors or epigenetic effects. We investigated DNA methylation signatures postmortem in brains of suicides with diagnosis of major depressive disorder. DNA methylation levels were determined at single C-phosphate-G (CpG) resolution sites within ventral prefrontal cortex of 53 suicides and nonpsychiatric controls, aged 16 to 89 years. We found that DNA methylation increases throughout the lifespan. Suicides showed an 8-fold greater number of methylated CpG sites relative to controls (P < 2.2 x 10(-16)), with greater DNA methylation changes over and above the increased methylation observed in normal aging. This increased DNA methylation may be a significant contributor to the neuropathology and psychopathology underlying the risk of suicide in depression.

  3. Human macrophages support persistent transcription from unintegrated HIV-1 DNA

    SciTech Connect

    Kelly, Jeremy; Beddall, Margaret H.; Yu Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu Yuntao

    2008-03-15

    Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.

  4. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

    PubMed Central

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph

    2015-01-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838–3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective—the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)—and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  5. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    PubMed

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  6. Increased Epstein-Barr virus in breast milk occurs with subclinical mastitis and HIV shedding.

    PubMed

    Sanosyan, Armen; Rutagwera, David G; Molès, Jean-Pierre; Bollore, Karine; Peries, Marianne; Kankasa, Chipepo; Mwiya, Mwiya; Tylleskär, Thorkild; Nagot, Nicolas; Van De Perre, Philippe; Tuaillon, Edouard

    2016-07-01

    Epstein-Barr virus (EBV) in breast milk and subclinical mastitis (SCM) are both associated with human immunodeficiency virus (HIV) shedding and possibly with postnatal HIV transmission. The objective of this nested case-control study was to investigate the interplay between SCM and EBV replication in breast milk of HIV-infected mothers.The relationships between EBV deoxyribonucleic acid (DNA) shedding, HIV-1 ribonucleic acid (RNA) level, and SCM were explored in breast milk samples of Zambian mothers participating in the ANRS 12174 trial. Mammary gland inflammation was defined as a breast milk sodium to potassium ratio (Na/K) greater than 0.6 and further subclassified as either "possible SCM" (Na/K ratio 0.6-1.0) or SCM (Na/K ratio ≥ 1.0). Breast milk interleukin 8 (IL-8) was measured as a surrogate marker of mammary gland inflammation.EBV DNA was detected in breast milk samples from 42 out of 83 (51%) participants and was associated with HIV-1 shedding in breast milk (P = 0.006). EBV DNA levels were higher in samples with SCM and "possible SCM" compared to non-SCM breast milk samples (P = 0.06; P = 0.007). An EBV DNA level of >200 copies/mL was independently associated with SCM and "possible SCM" (OR: 2.62; 95%: 1.13-6.10). In patients with SCM, higher EBV replication in the mammary gland was associated with a lower induction of IL-8 (P = 0.013). Resistance to DNase treatment suggests that EBV DNA in lactoserum is encapsidated.SCM and decreased IL-8 responses are associated with an increased EBV shedding in breast milk which may in turn facilitate HIV replication in the mammary gland. PMID:27399077

  7. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  8. Cheminformatics methods for novel nanopore analysis of HIV DNA termini

    PubMed Central

    Winters-Hilt, Stephen; Landry, Matthew; Akeson, Mark; Tanase, Maria; Amin, Iftekhar; Coombs, Amy; Morales, Eric; Millet, John; Baribault, Carl; Sendamangalam, Srikanth

    2006-01-01

    Background Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis. Results HMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus. PMID:17118144

  9. The increase in global HIV epidemics in MSM.

    PubMed

    Beyrer, Chris; Sullivan, Patrick; Sanchez, Jorge; Baral, Stefan D; Collins, Chris; Wirtz, Andrea L; Altman, Dennis; Trapence, Gift; Mayer, Kenneth

    2013-11-13

    Epidemics of HIV in MSM continue to expand in most low, middle, and upper income countries in 2013 and rates of new infection have been consistently high among young MSM. Current prevention and treatment strategies are insufficient for this next wave of HIV spread. We conducted a series of comprehensive reviews of HIV prevalence and incidence, risks for HIV, prevention and care, stigma and discrimination, and policy and advocacy options. The high per act transmission probability of receptive anal intercourse, sex role versatility among MSM, network level effects, and social and structural determinants play central roles in disproportionate disease burdens. HIV can be transmitted through large MSM networks at great speed. Molecular epidemiologic data show marked clustering of HIV in MSM networks, and high proportions of infections due to transmission from recent infections. Prevention strategies that lower biological risks, including those using antiretrovirals, offer promise for epidemic control, but are limited by structural factors including, discrimination, criminalization, and barriers to healthcare. Subepidemics, including among racial and ethnic minority MSM in the United States and UK, are particularly severe and will require culturally tailored efforts. For the promise of new and combined bio-behavioral interventions to be realized, clinically competent healthcare is necessary and community leadership, engagement, and empowerment are likely to be key. Addressing the expanding epidemics of HIV in MSM will require continued research, increased resources, political will, policy change, structural reform, community engagement, and strategic planning and programming, but it can and must be done.

  10. HBV-DNA levels predict overall mortality in HIV/HBV coinfected individuals.

    PubMed

    Nikolopoulos, Georgios K; Paraskevis, Dimitrios; Psichogiou, Mina; Hatzakis, Angelos

    2016-03-01

    The coinfection of Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has been associated with increased death rates. However, the relevant research has mostly relied on serologic HBV testing [HBV surface antigen (HBsAg)]. The aim of this work was to explore the relationship of HBV viraemia with overall mortality among HIV/HBV coinfected individuals. The analysis included 1,609 HIV seropositives of a previously described cohort (1984-2003) with limited exposure to tenofovir (12%) and a median follow-up of approximately 5 years. Those with persistent expression of HBsAg were further tested for HBV-DNA. The data were analyzed using Poisson regression models. Totally, 101 participants were chronic carriers of HBsAg (6.28%). Of these, 81 were tested for HBV-DNA. The median HBV-DNA levels were 3.81 log (base-10) International Units (IU)/ml. A third (31%) of those tested for HBV-DNA had received tenofovir. Before developing acquired immune deficiency syndrome (AIDS), the adjusted incidence rate ratio (IRR) for all-cause mortality of coinfected patients with HBV viraemia above the median value versus the HIV monoinfected group was 3.44 [95% confidence interval (CI): 1.05-11.27]. Multivariable regressions in the coinfected group only (n = 81) showed that one log-10 increase in HBV-DNA levels was associated with an elevated risk for death (IRR: 1.24, 95%CI: 1.03-1.49). HBV-DNA levels predict overall mortality in the setting of HIV/HBV coinfection, especially during the period before developing AIDS, and could thus help prioritize needs and determine the frequency of medical monitoring.

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

    PubMed

    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.

  12. Cytomegalovirus DNA in Semen and Blood Is Associated With Higher Levels of Proviral HIV DNA

    PubMed Central

    Gianella, Sara; Anderson, Christy M.; Vargas, Milenka V.; Richman, Douglas D.; Little, Susan J.; Morris, Sheldon R.; Smith, Davey M.

    2013-01-01

    Over three-fourths of human immunodeficiency virus (HIV)–infected men who have sex with men (MSM) have at least one herpesvirus detected in their semen, and cytomegalovirus (CMV) is the most prevalent. The presence of CMV is associated with higher T-cell immune activation and with HIV disease progression in treated and untreated individuals. In this study of 113 antiretroviral (ART)–naive HIV-infected MSM, we found that CMV replication in blood and semen was associated with higher levels of HIV DNA in peripheral blood mononuclear cells. These observations suggest that interventions aimed to reduce CMV replication and, thus, systemic immune activation could decrease the size of the latent HIV reservoir. PMID:23275608

  13. Increases in Condomless Sex in the Swiss HIV Cohort Study

    PubMed Central

    Kouyos, Roger D.; Hasse, Barbara; Calmy, Alexandra; Cavassini, Matthias; Furrer, Hansjakob; Stöckle, Marcel; Vernazza, Pietro L.; Bernasconi, Enos; Weber, Rainer; Günthard, Huldrych F.

    2015-01-01

    Condomless sex is a key driver of sexually transmitted diseases. In this study, we assess the long-term changes (2000–2013) of the occurrence of condomless sex among human immunodeficiency virus (HIV)-infected individuals enrolled in the Swiss HIV Cohort study. The frequencies with which HIV-infected individuals reported condomless sex were either stable or only weakly increasing for 2000–2008. For 2008–2013, these rates increased significantly for stable relationships among heterosexuals and men who have sex with men (MSM) and for occasional relationships among MSM. Our results highlight the increasing public health challenge posed by condomless sex and show that condomless sex has been increasing even in the most recent years. PMID:26180827

  14. Increased longevity in HIV: caring for older HIV-infected adults.

    PubMed

    Ball, Susan C

    2014-01-01

    The demographics of the HIV-infected population in the United States have shifted in a way that few would have predicted 30 years ago when the tide of sick and dying patients largely consisted of young men. Effective ART has allowed those infected to live long, productive lives and to grow old with their disease. With the increase in life expectancy afforded by HIV treatment, the cause of death among HIV-infected individuals is far more likely to be from an HIV-associated non-AIDS condition. Nonetheless, HIV seems to accelerate the aging process, and care providers involved in the treatment of older patients with HIV need to be aware that their patients are at increased risk of developing various common disorders, compared to uninfected same-age patients. Clinicians need to remain vigilant to the possibility of a new diagnosis of HIV among their older patients. Awareness of current or distant risk, frank discussions of sexual practices, and willingness to offer routine testing are crucial to making this diagnosis, with the recognition that longevity for patients with HIV is directly linked to how soon they enter care. HIV infection adds another challenge to the management of older patients; geriatricians and HIV specialists need to coordinate their efforts to provide patients with comprehensive multidisciplinary care. Older patients with HIV also have social and psychological needs that extend beyond the medical office. Maintaining independence, acknowledging limitations, reducing risk of adverse events such as falls or medication errors, and supporting self-acceptance and awareness are only a few of the many areas where care providers outside the medical office can be important for patients' ongoing well-being. Accessing family support, community outreach, church affiliation, or other outpatient support networks can be useful for patients. The remarkable change in prognosis brought about by effective ART in the mid-1990s has meant that HIV is now, for many, a

  15. HIV-1 DNA shedding in genital ulcers and its associated risk factors in Pune, India.

    PubMed

    Gadkari, D A; Quinn, T C; Gangakhedkar, R R; Mehendale, S M; Divekar, A D; Risbud, A R; Chan-Tack, K; Shepherd, M; Gaydos, C; Bollinger, R C

    1998-07-01

    HIV infection status was determined in 302 consecutive patients with genital ulcer disease (GUD) presenting to two sexually transmitted disease (STD) clinics in Pune, India. Of the 71 (24%) individuals with HIV infection, 67 (94%) were HIV antibody-positive, and 4 (6%) were HIV antibody-negative but p24 antigen-positive at the time of presentation. HIV-1 DNA was detected in 24 (34%) specimens. The genital ulcers of all four acutely infected p24-antigenemic subjects were HIV-1 DNA-positive by polymerase chain reaction (PCR) assay, compared with 20 of 67 (30%) seropositive patients (p = .01). Presence of chancroid, GUD symptoms for > 10 days, and concurrent diagnosis of cervicitis or urethritis were significantly associated risk factors for HIV-1 DNA shedding in ulcers. Early GUD diagnosis and aggressive treatment of HIV-infected patients may significantly reduce secondary transmission of HIV to other sex partners.

  16. HIV-1 Tat depresses DNA-PK{sub CS} expression and DNA repair, and sensitizes cells to ionizing radiation

    SciTech Connect

    Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun . E-mail: zhoupk@nic.bmi.ac.cn

    2006-07-01

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and {gamma}-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and {gamma}H2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy {gamma}-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G{sub 2}/M arrest as compared with parental cells. In addition, the G{sub 2}/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased

  17. Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications.

    PubMed

    Avettand-Fènoël, Véronique; Hocqueloux, Laurent; Ghosn, Jade; Cheret, Antoine; Frange, Pierre; Melard, Adeline; Viard, Jean-Paul; Rouzioux, Christine

    2016-10-01

    HIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis. PMID:27559075

  18. Autograft HIV-DNA load predicts HIV-1 peripheral reservoir after stem cell transplantation for AIDS-related lymphoma patients.

    PubMed

    Zanussi, Stefania; Bortolin, Maria Teresa; Pratesi, Chiara; Tedeschi, Rosamaria; Basaglia, Giancarlo; Abbruzzese, Luciano; Mazzucato, Mario; Spina, Michele; Vaccher, Emanuela; Tirelli, Umberto; Rupolo, Maurizio; Michieli, Mariagrazia; Di Mascio, Michele; De Paoli, Paolo

    2015-01-01

    Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/10(6) autograft mononuclear cells, range 13-706 vs. 82 HIV-DNA copies/10(6) peripheral blood mononuclear cells (PBMCs), range 13-479, p = 0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R(2) = 0.84, p = 0.01) to month 12 follow-up (R(2) = 0.99, p = 0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure.

  19. Prevalence of human herpesvirus-8 salivary shedding in HIV increases with CD4 count.

    PubMed

    Gandhi, M; Koelle, D M; Ameli, N; Bacchetti, P; Greenspan, J S; Navazesh, M; Anastos, K; Greenblatt, R M

    2004-08-01

    Human herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), which occurs in epidemic form in human immunodeficiency virus(HIV)-infected individuals. Saliva is the only mucosal fluid in which infectious HHV-8 has been identified, although factors associated with HHV-8 salivary shedding remain unclear. Our study performed PCR analysis for HHV-8 DNA in saliva (and other body fluids) in 66 HIV- and HHV-8-co-infected women without KS so that we could examine predictors for HHV-8 DNA detection. CD4 count was the most significant predictor of HHV-8 salivary shedding, with increased prevalence of HHV-8 salivary DNA at higher CD4 counts. The odds of salivary HHV8 shedding at CD4 counts > = 350 cells/microL was 63 times the odds of shedding at CD4 < 350 (95%CI, 1.3-3078), with an increase in effect size when the analysis was restricted to those with a CD4 nadir > 200. Analysis of these data suggests an increased potential for HHV-8 transmission early in HIV infection, with implications for HHV-8 prevention.

  20. HIV-1 DNA predicts disease progression and post-treatment virological control.

    PubMed

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan; Koelsch, Kersten K; Kelleher, Anthony D; Phillips, Rodney E; Frater, John

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. PMID:25217531

  1. HIV-1 DNA predicts disease progression and post-treatment virological control.

    PubMed

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan; Koelsch, Kersten K; Kelleher, Anthony D; Phillips, Rodney E; Frater, John

    2014-09-12

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.

  2. Increased antiretroviral therapy prescription and HIV viral suppression among persons receiving clinical care for HIV infection

    PubMed Central

    Bradley, Heather; Mattson, Christine L.; Beer, Linda; Huang, Ping; Shouse, R. Luke

    2016-01-01

    Objective To assess trends during 2009–2013 in antiretroviral therapy (ART) prescription and viral suppression among adults receiving HIV clinical care in the United States. Design We used data from the Medical Monitoring Project, a surveillance system producing national estimates of characteristics of HIV-infected adults receiving clinical care in the United States. Methods We estimated weighted proportions of persons receiving HIV medical care who were prescribed ART and achieved HIV viral suppression (<200 copies/ml) at both last test and at all tests in the previous 12 months during 2009–2013. We assessed trends overall and by gender, age, race/ethnicity, and sexual behavior/orientation. Results ART prescription and viral suppression increased significantly during 2009–2013, overall and in subgroups. ART prescription increased from 89 to 94% (P for trend <0.01). Viral suppression at last measurement increased from 72 to 80% (P for trend <0.01). The largest increases were among 18–29 year olds (56–68%), 30–39 year olds (62–75%), and non-Hispanic blacks (64–76%). Sustained viral suppression increased from 58 to 68% (P for trend <0.01). The largest increases were among 18–29 year olds (32–51%), 30–39 year olds (47–63%), and non-Hispanic blacks (49–61%). Conclusion Adults receiving HIV medical care are increasingly likely to be prescribed ART and achieve viral suppression. Recent efforts to promote early antiretroviral therapy use may have contributed to these increases, bringing us closer to realizing key goals of the National HIV/AIDS Strategy. PMID:27465279

  3. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  4. Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

    SciTech Connect

    Korber, Bette; Fischer, William; Wallstrom, Timothy

    2009-01-01

    An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.

  5. Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment

    PubMed Central

    Massanella, Marta; Richman, Douglas D.; Little, Susan J.; Spina, Celsa A.; Vargas, Milenka V.; Lada, Steven M.; Daar, Eric S.; Dube, Michael P.; Haubrich, Richard H.; Morris, Sheldon R.; Smith, Davey M.

    2014-01-01

    ABSTRACT Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in

  6. Safety and Immunogenicity Study of Multiclade HIV-1 Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA Vaccine in Africa

    PubMed Central

    Allen, Susan; Than, Soe; Adams, Elizabeth M.; Graham, Barney S.; Koup, Richard A.; Bailer, Robert T.; Smith, Carol; Dally, Len; Tarragona-Fiol, Tony; Bergin, Philip J.; Hayes, Peter; Ho, Martin; Loughran, Kelley; Komaroff, Wendy; Stevens, Gwynneth; Thomson, Helen; Boaz, Mark J.; Cox, Josephine H.; Schmidt, Claudia; Gilmour, Jill; Nabel, Gary J.; Fast, Patricia

    2010-01-01

    Background We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults. Methodology/Principal Findings Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone. Conclusions/Significance The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints. Trial

  7. Prostaglandin E₂ increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression.

    PubMed

    Huang, Steven K; Scruggs, Anne M; Donaghy, Jake; McEachin, Richard C; Fisher, Aaron S; Richardson, Bruce C; Peters-Golden, Marc

    2012-09-01

    Although alterations in DNA methylation patterns have been associated with specific diseases and environmental exposures, the mediators and signaling pathways that direct these changes remain understudied. The bioactive lipid mediator prostaglandin E(2) (PGE(2)) has been shown to exert a myriad of effects on cell survival, proliferation, and differentiation. Here, we report that PGE(2) also signals to increase global DNA methylation and DNA methylation machinery in fibroblasts. HumanMethylation27 BeadChip array analysis of primary fetal (IMR-90) and adult lung fibroblasts identified multiple genes that were hypermethylated in response to PGE(2). PGE(2), compared with nontreated controls, increased expression and activity (EC(50)∼10(7) M) of one specific isoform of DNA methyltransferase, DNMT3a. Silencing of DNMT3a negated the ability of PGE(2) to increase DNMT activity. The increase in DNMT3a expression was mediated by PGE(2) signaling via its E prostanoid 2 receptor and the second messenger cAMP. PGE(2), compared with the untreated control, increased the expression and activity of Sp1 and Sp3 (EC(50)∼3×10(7) M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE(2) increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE(2) decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is regulated by a ubiquitous bioactive endogenous mediator. Given that PGE(2) biosynthesis is modulated by environmental toxins, various disease states, and commonly used pharmacological agents, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.

  8. Cellular HIV type 1 DNA levels are equivalent among drug-sensitive and drug-resistant strains in newly diagnosed and antiretroviral naive patients.

    PubMed

    Antoniadou, Zoi-Anna; Hezka, Johana; Kousiappa, Ioanna; Mamais, Ioannis; Skoura, Lemonia; Pilalas, Dimitris; Metallidis, Simeon; Nicolaidis, Pavlos; Malisiovas, Nicolaos; Kostrikis, Leondios G

    2014-03-01

    The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5

  9. HIV-1 gene expression: lessons from provirus and non-integrated DNA

    PubMed Central

    Wu, Yuntao

    2004-01-01

    Replication of HIV-1 involves a series of obligatory steps such as reverse transcription of the viral RNA genome into double-stranded DNA, and subsequent integration of the DNA into the human chromatin. Integration is an essential step for HIV-1 replication; yet the natural process of HIV-1 infection generates both integrated and high levels of non-integrated DNA. Although proviral DNA is the template for productive viral replication, the non-integrated DNA has been suggested to be active for limited viral gene synthesis. In this review, the regulation of viral gene expression from proviral DNA will be summarized and issues relating to non-integrated DNA as a template for transcription will be discussed, as will the possible function of pre-integration transcription in HIV-1 replication cycle. PMID:15219234

  10. Ethical reflections on pharmacogenetics and DNA banking in a cohort of HIV-infected patients.

    PubMed

    de Montgolfier, Sandrine; Moutel, Grégoire; Duchange, Nathalie; Theodorou, Ioannis; Hervé, Christian; Leport, Catherine

    2002-12-01

    The aim of this study was to analyse ethical issues concerning the storage of human biological samples to be used in genetic analyses and pharmacogenetic research based on a French experience of DNA banking in a cohort of human immunodeficiency virus (HIV)-infected patients started on a protease inhibitor-containing treatment. We describe the ethical issues raised during the establishment of a DNA bank, including questions dealing with autonomy, benefit to the patient, information sharing and confidentiality as well as guarantees concerning the storage and use of DNA. The practical applications of themes illustrated theoretically in the literature are discussed. Most of the points raised are not specific to HIV, but some of them may be more accurate due to the characteristics of the HIV population, which is more involved in the social debate through the community life and the increased risk of stigmatization. Our results are summarized in the memorandum and consent form presented in the Appendices. One issue still open to discussion is the way the results of genetic data will be given to the patients. This work should allow other researchers and members of evaluation committees to enrich their considerations and should stimulate discussion on this topic.

  11. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

  12. Postnatal Depression Symptoms are Associated with Increased Diarrhea among Infants of HIV-Positive Ghanaian Mothers

    PubMed Central

    Okronipa, Harriet E.T.; Marquis, Grace S.; Lartey, Anna; Brakohiapa, Lucy; Perez-Escamilla, Rafael; Mazur, Robert E.

    2014-01-01

    HIV infection is linked to increased prevalence of depression which may affect maternal caregiving practices and place young infants at increased risk of illness. We examined the incidence and days ill with diarrhea among infants of HIV positive (HIV-P), HIV negative (HIV-N), and unknown HIV status (HIV-U) women, and determined if symptoms of maternal postnatal depression (PND) modulated the risk of diarrhea. Pregnant women (n=492) were recruited from 3 antenatal clinics; mothers and infants were followed for 12 mo postpartum. Diarrheal incidence was 0.6 episodes/100-d at risk. More HIV-P than HIV-N and HIV-U women tended to report PND symptoms (P=0.09). PND symptoms increased the risk of infantile diarrhea only for HIV-P and HIV-U but not HIV-N women (interaction term, P=0.02). Health care providers should be aware of the increased risk of infantile diarrhea when both maternal HIV and PND symptoms are present and take preventive action to reduce morbidity. PMID:22331392

  13. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-03-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication

  14. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

    PubMed Central

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-01-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

  15. Nucleocapsid Protein Annealing of a Primer-Template Enhances (+)-Strand DNA Synthesis and Fidelity by HIV-1 Reverse Transcriptase†

    PubMed Central

    Kim, Jiae; Roberts, Anne; Yuan, Hua; Xiong, Yong; Anderson, Karen S.

    2012-01-01

    Human immunodeficiency virus type-1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Using a pre-steady state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3-7 fold) in the rate of incorporation (kpol) by RT as compared to heat annealed primer-template with single nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (Kd) of the nucleotide. These differences in kpol and Kd were not through direct interactions between HIV-1 RT and NCp7. When examining extension by RT, the data suggests that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat annealed primer-template. This enhancement in rate is mediated through interactions with NCp7’s zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV. PMID:22210155

  16. Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial

    PubMed Central

    Joseph, Sarah; Geldmacher, Christof; Munseri, Patricia J.; Aboud, Said; Missanga, Marco; Mann, Philipp; Wahren, Britta; Ferrari, Guido; Polonis, Victoria R.; Robb, Merlin L.; Weber, Jonathan; Tatoud, Roger; Maboko, Leonard; Hoelscher, Michael; Lyamuya, Eligius F.; Biberfeld, Gunnel; Sandström, Eric; Kroidl, Arne; Bakari, Muhammad; Nilsson, Charlotta; McCormack, Sheena

    2016-01-01

    -MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07). Conclusion We report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA. Trial Registration International Clinical Trials Registry PACTR2010050002122368 PMID:27192151

  17. HIV-1 Infection Leads to Increased Transcription of Human Endogenous Retrovirus HERV-K (HML-2) Proviruses In Vivo but Not to Increased Virion Production

    PubMed Central

    Bhardwaj, Neeru; Maldarelli, Frank; Mellors, John

    2014-01-01

    ABSTRACT Recent studies suggest that human endogenous retrovirus group K (HERV-K) provirus expression plays a role in the pathogenesis of HIV-1 infection. In particular, RNA from the HML-2 subgroup of HERV-K proviruses has been reported to be highly expressed at the cellular level and detectable in the plasma of HIV-1-infected patients, suggestive of virion production and, perhaps, replication. In this study, we developed an HML-2-specific quantitative-PCR assay that detects 51 of the 89 known HML-2 proviruses in the human genome. Plasma and peripheral blood mononuclear cells (PBMCs) from HIV-negative controls and HIV-1-infected patients were collected for analysis of HML-2 RNA expression. Contrary to previous reports, we did not detect high levels of HML-2 RNA in the plasma of HIV-1-infected patients, but we did observe a significant increase of HML-2 RNA in total PBMCs compared to HIV-negative controls. The level of HML-2 expression in PBMCs does not appear to be related to patient use of antiretrovirals or to HIV-1 plasma RNA, cellular RNA, or cellular DNA levels. To investigate the source of HML-2 RNA expression, patient PBMCs were sorted into CD3+ CD4+, CD3+ CD8+, CD3− CD14+, and CD3− CD20+ cell subsets and then analyzed for HML-2 RNA levels. No single cell subset was enriched for HML-2 RNA expression in HIV-1-infected patients, but there appears to be substantial variability in the level of HML-2 expression depending on the cell type. IMPORTANCE Here, we report that human endogenous retrovirus group K (HERV-K) (HML-2) proviruses are expressed at significantly higher levels in peripheral blood mononuclear cells (PBMCs) from patients with HIV-1 infection than in those from uninfected individuals. However, contrary to previous reports, this expression did not lead to detectable virions in the plasma of these patients. In addition, we found that HML-2 proviruses were expressed in multiple blood cell types from HIV-1-infected individuals, and the magnitude of

  18. Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment.

    PubMed

    Corley, Michael J; Dye, Christian; D'Antoni, Michelle L; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M; Ndhlovu, Lishomwa C; Maunakea, Alika K

    2016-01-01

    Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation "signature" potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI. PMID:27629381

  19. Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment

    PubMed Central

    Corley, Michael J.; Dye, Christian; D’Antoni, Michelle L.; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M.; Ndhlovu, Lishomwa C.; Maunakea, Alika K.

    2016-01-01

    Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation “signature” potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI. PMID:27629381

  20. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  1. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  2. A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

    PubMed

    Rohrman, Brittany A; Richards-Kortum, Rebecca R

    2012-09-01

    Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

  3. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression.

    PubMed

    Koczor, Christopher A; Fields, Earl; Jedrzejczak, Mark J; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A; Lewis, William

    2015-11-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. PMID:26307267

  4. Characterization of the minimal DNA-binding domain of the HIV integrase protein.

    PubMed Central

    Lutzke, R A; Vink, C; Plasterk, R H

    1994-01-01

    The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity. Images PMID:7937137

  5. PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

    PubMed

    Knuchel, M C; Graf, B; Schlaepfer, E; Kuster, H; Fischer, M; Weber, R; Cone, R W

    2000-02-01

    We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)

  6. Short communication: Methamphetamine treatment increases in vitro and in vivo HIV replication.

    PubMed

    Toussi, Sima Shelly; Joseph, Aviva; Zheng, Jian Hua; Dutta, Monica; Santambrogio, Laura; Goldstein, Harris

    2009-11-01

    To delineate the mechanistic basis for the epidemiological association between methamphetamine use and accelerated progression to AIDS, we evaluated the direct in vitro and in vivo effects of methamphetamine on HIV-1 replication. Methamphetamine administration significantly increased HIV-1 production by both HIV-infected monocytes and CD4 T lymphocytes in vitro. In addition, in vivo methamphetamine treatment increased HIV production and viremia in mice transgenic for a replication-competent HIV provirus and human cyclin T1. Methamphetamine activated transcription of the HIV long terminal repeat (LTR) regulatory region, was associated with nuclear translocation of NF-kappaB. Our results provide further insights into the mechanisms by which methamphetamine accelerates disease course in HIV-infected individuals.

  7. HIV-1 DNA predicts disease progression and post-treatment virological control

    PubMed Central

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

  8. Systematic review of strategies to increase men's HIV-testing in sub-Saharan Africa

    PubMed Central

    Hensen, Bernadette; Taoka, Sachiko; Lewis, James J.; Weiss, Helen A.; Hargreaves, James

    2014-01-01

    Objective: This systematic review summarizes evidence on the effectiveness of strategies to increase men's HIV-testing in sub-Saharan Africa. Methods: Medline, EmBase, Africa-Wide Information and Global Health were searched. Cluster and individually randomized trials evaluating interventions to increase the proportion of adults (≥15 years) testing for HIV were eligible if they were conducted in sub-Saharan Africa, included men in the study population, and reported HIV-testing data by sex. References were independently screened. Results: Of the 1852 references, 15 papers including 16 trials were eligible. Trials were judged too heterogeneous to combine in meta-analysis. Three interventions invited men to attend antenatal care-based HIV-testing via pregnant partners, of which two showed a significant effect on partner-testing. One intervention invited men to HIV-test through pregnant partners and showed an increase in HIV-testing when it was offered in bars compared with health facilities. A trial of notification to partners of newly diagnosed HIV-positive patients showed an increase in testing where notification was by healthcare providers compared with notification by the patient. Three interventions reached men already at health facilities and eight reported the effects of community-based HIV-testing. Mobile-testing had a significant effect on HIV-testing compared with standard voluntary counselling and testing. Home-based testing also had a significant effect, but reached smaller numbers of men than mobile-testing. Discussion: Interventions to encourage HIV-testing can increase men's levels of HIV-testing. Community-based programmes in particular had a large effect on population levels of HIV-testing. More data on costs and potential population impact of these approaches over different time-horizons would aid policy-makers in planning resource allocation to increase male HIV-testing. PMID:25062091

  9. Nano-Delivery Vehicles/Adjuvants for DNA Vaccination Against HIV.

    PubMed

    Dong, Yaqiong; Yang, Jun; Zhang, Jinchao; Zhang, Xin

    2016-03-01

    More than 75 million people has been infected HIV and it is responsible for nearly 36 million deaths on a global scale. As one of the deadliest infectious diseases, HIV is becoming the urgent issue of the global epidemic to tackle. In order to settle this problem from the source, some effective prevention strategies should be developed to control the pandemic of HIV. Vaccines, especially DNA vaccines, could be the optimal way to control the spread of HIV due to the unparalleled superiority that DNA vaccines could generate long-term humoral and cellular immune responses which could provide protective immunity for HIV. But the naked DNA could hardly enter into cells and is easily degraded by DNases and lysosomes, so designing effective delivery system is a promising strategy. Since delivery system could be constructed to promote efficient delivery of DNA into mammalian cells, protect them from degradation, and also could be established to be a target system to arrive at certain position of expectation. The current review discusses the potential of various nano-delivery vehicles/adjuvants such as polymer, lipid, liposome, peptide and inorganic material in improving efficiency of diverse modalities available for HIV DNA vaccines.

  10. Nano-Delivery Vehicles/Adjuvants for DNA Vaccination Against HIV.

    PubMed

    Dong, Yaqiong; Yang, Jun; Zhang, Jinchao; Zhang, Xin

    2016-03-01

    More than 75 million people has been infected HIV and it is responsible for nearly 36 million deaths on a global scale. As one of the deadliest infectious diseases, HIV is becoming the urgent issue of the global epidemic to tackle. In order to settle this problem from the source, some effective prevention strategies should be developed to control the pandemic of HIV. Vaccines, especially DNA vaccines, could be the optimal way to control the spread of HIV due to the unparalleled superiority that DNA vaccines could generate long-term humoral and cellular immune responses which could provide protective immunity for HIV. But the naked DNA could hardly enter into cells and is easily degraded by DNases and lysosomes, so designing effective delivery system is a promising strategy. Since delivery system could be constructed to promote efficient delivery of DNA into mammalian cells, protect them from degradation, and also could be established to be a target system to arrive at certain position of expectation. The current review discusses the potential of various nano-delivery vehicles/adjuvants such as polymer, lipid, liposome, peptide and inorganic material in improving efficiency of diverse modalities available for HIV DNA vaccines. PMID:27455611

  11. Dose-dependent inhibition of Gag cellular immunity by Env in SIV/HIV DNA vaccinated macaques

    PubMed Central

    Valentin, Antonio; Li, Jinyao; Rosati, Margherita; Kulkarni, Viraj; Patel, Vainav; Jalah, Rashmi; Alicea, Candido; Reed, Steven; Sardesai, Niranjan; Berkower, Ira; Pavlakis, George N; Felber, Barbara K

    2015-01-01

    The induction of a balanced immune response targeting the major structural proteins, Gag and Env of HIV, is important for the development of an efficacious vaccine. The use of DNA plasmids expressing different antigens offers the opportunity to test in a controlled manner the influence of different vaccine components on the magnitude and distribution of the vaccine-induced cellular and humoral immune responses. Here, we show that increasing amounts of env DNA results in greatly enhanced Env antibody titers without significantly affecting the levels of anti-Env cellular immune responses. Co-immunization with Env protein further increased antibody levels, indicating that vaccination with DNA only is not sufficient for eliciting maximal humoral responses against Env. In contrast, under high env:gag DNA plasmid ratio, the development of Gag cellular responses was significantly reduced by either SIV or HIV Env, whereas Gag humoral responses were not affected. Our data indicate that a balanced ratio of the 2 key HIV/SIV vaccine components, Gag and Env, is important to avoid immunological interference and to achieve both maximal humoral responses against Env to prevent virus acquisition and maximal cytotoxic T cell responses against Gag to prevent virus spread. PMID:26125521

  12. Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA.

    PubMed

    Gross, Andrew M; Jaeger, Philipp A; Kreisberg, Jason F; Licon, Katherine; Jepsen, Kristen L; Khosroheidari, Mahdieh; Morsey, Brenda M; Swindells, Susan; Shen, Hui; Ng, Cherie T; Flagg, Ken; Chen, Daniel; Zhang, Kang; Fox, Howard S; Ideker, Trey

    2016-04-21

    HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression.

  13. Human herpes virus-6 increases HIV-1 expression in co-infected T cells via nuclear factors binding to the HIV-1 enhancer.

    PubMed Central

    Ensoli, B; Lusso, P; Schachter, F; Josephs, S F; Rappaport, J; Negro, F; Gallo, R C; Wong-Staal, F

    1989-01-01

    Human Herpes virus-6 (HHV-6) can co-infect with HIV-1 human CD4+ T-cells, leading to accelerated cell death, and factors in HHV-6-infected cells stimulate HIV-1 LTR directed gene expression. In this study, we have examined the mechanism of HIV-1 activation by HHV-6 and localized the cis-acting sequences of HIV-1 LTR responsive to trans-activation. Increased HIV-1 LTR directed gene expression is obtained in HIV-1 infected cells co-infected with HHV-6, or in HHV-6 infected cells co-transfected with the HIV-1 tat gene. Parallel increases of HIV-1-specific transcripts are seen by in situ hybridization in HHV-6/HIV-1 doubly infected cells as compared to single HIV-1 infection. Similarly, infection by HHV-6 increases the steady-state level of HIV-1 LTR mRNA that parallels CAT enzymatic activity, suggesting a transcriptional and/or post-transcriptional activation. Sequences necessary for HIV-1 LTR activation by HHV-6 are distinct from those required for that tat response and map to a region of the HIV-1 LTR from -103 to -48. The HIV-1 enhancer sequence (-105 to -80) is sufficient to confer HHV-6 inducibility to a heterologous promoter, and nuclear protein(s) activated or induced by HHV-6 infection specifically bind to the NF kappa B motifs of the HIV-1 enhancer region.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2573513

  14. Evaluation of a Community Health Worker Intervention to Reduce HIV/AIDS Stigma and Increase HIV Testing Among Underserved Latinos in the Southwestern U.S.

    PubMed Central

    Becker, Davida; Espinoza, Lilia; Nguyen-Rodriguez, Selena; Diaz, Gaby; Carricchi, Ana; Galvez, Gino; Garcia, Melawhy

    2015-01-01

    Objectives Latinos are at an elevated risk for HIV infection. Continued HIV/AIDS stigma presents barriers to HIV testing and affects the quality of life of HIV-positive individuals, yet few interventions addressing HIV/AIDS stigma have been developed for Latinos. Methods An intervention led by community health workers (promotores de salud, or promotores) targeting underserved Latinos in three southwestern U.S. communities was developed to decrease HIV/AIDS stigma and increase HIV knowledge and perception of risk. The intervention was led by HIV-positive and HIV-affected (i.e., those who have, or have had, a close family member or friend with HIV/AIDS) promotores, who delivered interactive group-based educational sessions to groups of Latinos in Spanish and English. To decrease stigma and motivate behavioral and attitudinal change, the educational sessions emphasized positive Latino cultural values and community assets. The participant pool comprised 579 Latino adults recruited in El Paso, Texas (n=204); San Ysidro, California (n=175); and Los Angeles, California (n=200). Results From pretest to posttest, HIV/AIDS stigma scores decreased significantly (p<0.001). Significant increases were observed in HIV/AIDS knowledge (p<0.001), willingness to discuss HIV/AIDS with one's sexual partner (p<0.001), and HIV risk perception (p=0.006). Willingness to test for HIV in the three months following the intervention did not increase. Women demonstrated a greater reduction in HIV/AIDS stigma scores when compared with their male counterparts, which may have been related to a greater increase in HIV/AIDS knowledge scores (p=0.016 and p=0.007, respectively). Conclusion Promotores interventions to reduce HIV/AIDS stigma and increase HIV-related knowledge, perception of risk, and willingness to discuss sexual risk with partners show promise in reaching underserved Latino communities. PMID:26327724

  15. Project VOGUE: A partnership for increasing HIV knowledge and HIV vaccine trial awareness among House Ball leaders in Western New York

    PubMed Central

    Alio, Amina P.; Fields, Sheldon D.; Humes, Damon L.; Bunce, Catherine A.; Wallace, Stephaun E.; Lewis, Cindi; Elder, Heather; Wakefield, Steven; Keefer, Michael C.

    2014-01-01

    Men who sleep with men (MSM) and transgender individuals of color, the largest demographic in the House Ball community (HBC) are amongst the group at highest risk for HIV infection in the United States. The HBC have limited access to culturally appropriate HIV education. This study aimed to develop a partnership with HBC leaders to uncover strategies for increasing HIV prevention knowledge, including participation in HIV vaccine trials. To this end a research institution-community-HBC partnership was established. In-depth qualitative and quantitative data were collected from the 14 HBC leaders in western New York, revealing that knowledge of HIV and related vaccine trials was limited. Barriers to increasing HIV knowledge included fear of peer judgment, having inaccurate information about HIV, and lack of education. Among the HBC, community partnerships will further aid in the development of future HIV prevention programs and increase individuals’ willingness to participate in future HIV vaccine trials. PMID:25642120

  16. Performance Characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System

    PubMed Central

    Kuritzkes, Daniel R.; Grant, Robert M.; Feorino, Paul; Griswold, Marshal; Hoover, Marie; Young, Russell; Day, Stephen; Lloyd, Jr., Robert M.; Reid, Caroline; Morgan, Gillian F.; Winslow, Dean L.

    2003-01-01

    The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a “gold standard” reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of ≥1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples. PMID:12682150

  17. Mitochondrial DNA content increase in response to cigarette smoking.

    PubMed

    Masayesva, Brett G; Mambo, Elizabeth; Taylor, Rodney J; Goloubeva, Olga G; Zhou, Shaoyu; Cohen, Yoram; Minhas, Khalid; Koch, Wayne; Sciubba, James; Alberg, Anthony J; Sidransky, David; Califano, Joseph

    2006-01-01

    An increase in mitochondrial DNA (mtDNA) content and decline in mitochondrial function occurs with aging and in response to DNA-damaging agents, including tobacco smoke. We did a cross-sectional study and quantified changes in mtDNA content in a population of individuals with varied smoking and alcohol exposure. Age, smoking history, ethanol intake, and other demographic data were characterized for 604 individuals participating in a screening study for smoking-related upper aerodigestive malignancy. Total DNA was extracted from exfoliated cells in saliva. DNA from a nuclear gene, beta-actin, and two mitochondrial genes, cytochrome c oxidase I and II (Cox I and Cox II), were quantified by real-time PCR. mtDNA content was correlated with age, exposure history, and other variables using multivariate regression analyses. A significant increase (P<0.001) in mtDNA content was noted in smokers (31% and 29% increase for Cox I and Cox II, respectively) and former smokers (31% and 34%) when compared with never smokers. This association persisted after adjustment for other significant factors including age, alcohol drinking, and income (P<0.001). Increased mtDNA content was positively associated with pack-years of smoking (P=0.02). Despite an average smoking cessation interval of 21 years in former smokers, tobacco cessation interval was not statistically significantly associated with mtDNA content. Smoking is associated with increased mtDNA content in a dose-dependent fashion. Mitochondrial DNA alterations in response to smoking persist for several decades after smoking cessation, consistent with long-term, smoking-related damage.

  18. Positively charged surfaces increase the flexibility of DNA.

    PubMed

    Podestà, Alessandro; Indrieri, Marco; Brogioli, Doriano; Manning, Gerald S; Milani, Paolo; Guerra, Rosalinda; Finzi, Laura; Dunlap, David

    2005-10-01

    Many proteins "bind" DNA through positively charged amino acids on their surfaces. However, to overcome significant energetic and topological obstacles, proteins that bend or package DNA might also modulate the stiffness that is generated by repulsions between phosphates within DNA. Much previous work describes how ions change the flexibility of DNA in solution, but when considering macromolecules such as chromatin in which the DNA contacts the nucleosome core each turn of the double helix, it may be more appropriate to assess the flexibility of DNA on charged surfaces. Mica coated with positively charged molecules is a convenient substrate upon which the flexibility of DNA may be directly measured with a scanning force microscope. In the experiments described below, the flexibility of DNA increased as much as fivefold depending on the concentration and type of polyamine used to coat mica. Using theory that relates charge neutralization to flexibility, we predict that phosphate repulsions were attenuated by approximately 50% in the most flexible DNA observed. This simple method is an important tool for investigating the physiochemical causes and molecular biological effects of DNA flexibility, which affects DNA biochemistry ranging from chromatin stability to viral encapsulation.

  19. A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity.

    PubMed

    Ammar, Farah F; Hobaika, Zeina; Abdel-Azeim, Safwat; Zargarian, Loussinée; Maroun, Richard G; Fermandjian, Serge

    2016-04-01

    We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg(2+) (5 mm) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg(2+) significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg(2+)-assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase-derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL. PMID:27239438

  20. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity

    PubMed Central

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao

    2015-01-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell–cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  1. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    Introduction In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). Conclusions 2-LTR circles

  2. HIV Drug Resistance Mutations in Proviral DNA from a Community Treatment Program

    PubMed Central

    Derache, Anne; Shin, Hyoung-Shik; Balamane, Maya; White, Elizabeth; Israelski, Dennis; Klausner, Jeffrey D.; Freeman, Alexandra H.; Katzenstein, David

    2015-01-01

    Background Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations. Methods The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu. Results Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively. Conclusions The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence. PMID:25635815

  3. Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA

    PubMed Central

    Aga, Evgenia; Cillo, Anthony R.; Yates, Aarika L.; Besson, Guillaume; Fyne, Elizabeth; Koontz, Dianna L.; Jennings, Cheryl; Zheng, Lu; Mellors, John W.

    2016-01-01

    Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1 pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4+ T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10 copies/106 PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10 copies/106 PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r = 0.77 to 1; P = 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4+ T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription. PMID:26763968

  4. Does HIV increase the risk of spousal violence in sub-Saharan Africa?

    PubMed

    Chin, Yoo-Mi

    2013-09-01

    Although a positive association is found between HIV prevalence and intimate partner violence, a causal interpretation is hard to establish due to the endogeneity of HIV prevalence. Using the distance from the origin of the virus as an instrument, I find that an exogenous increase in HIV prevalence in a cluster has a sizable positive effect on the risk of physical and sexual violence against women within marriage. The results of this study confirm a gender-specific negative externality of the disease and encourage policy efforts to incorporate services for violence against women into existing HIV programs.

  5. Efficacy of an Educational Intervention to Increase Consent for HIV Testing in Rural Appalachia

    PubMed Central

    Basta, Tania B.; Stambaugh, Teena; Fisher, Celia B.

    2016-01-01

    This study sought to assess barriers and enhance readiness to consent to home and Planned Parenthood HIV testing among 60 out-patients from a mental health and substance abuse clinic in rural Appalachia. Testing barriers included not knowing where to get tested, lack of confidentiality, and loss of partners if one tested sero-positive. The intervention yielded lowered HIV stigma, increase in HIV knowledge, and agreement to take the HIV home test. These results are encouraging because they suggest that a brief educational intervention is a critical pathway to the success of the National Institutes on Drug Abuse’s Seek, Test, Treat, and Retain initiative in poor rural counties.

  6. Monocytes from HIV+ individuals show impaired cholesterol efflux and increased foam cell formation after transendothelial migration

    PubMed Central

    MAISA, Anna; HEARPS, Anna C.; ANGELOVICH, Thomas A.; PEREIRA, Candida F.; ZHOU, Jingling; SHI, Margaret D.Y.; PALMER, Clovis S.; MULLER, William A.; CROWE, Suzanne M.; JAWOROWSKI, Anthony

    2016-01-01

    Design HIV+ individuals have an increased risk of atherosclerosis and cardiovascular disease which is independent of antiretroviral therapy and traditional risk factors. Monocytes play a central role in the development of atherosclerosis, and HIV-related chronic inflammation and monocyte activation may contribute to increased atherosclerosis, but the mechanisms are unknown. Methods Using an in vitro model of atherosclerotic plaque formation, we measured the transendothelial migration of purified monocytes from age-matched HIV+ and uninfected donors and examined their differentiation into foam cells. Cholesterol efflux and the expression of cholesterol metabolism genes were also assessed. Results Monocytes from HIV+ individuals showed increased foam cell formation compared to controls (18.9% vs 0% respectively, p=0.004) and serum from virologically suppressed HIV+ individuals potentiated foam cell formation by monocytes from both uninfected and HIV+ donors. Plasma TNF levels were increased in HIV+ vs control donors (5.9 vs 3.5 pg/ml, p=0.02) and foam cell formation was inhibited by blocking antibodies to TNF receptors, suggesting a direct effect on monocyte differentiation to foam cells. Monocytes from virologically suppressed HIV+ donors showed impaired cholesterol efflux and decreased expression of key genes regulating cholesterol metabolism, including the cholesterol transporter ABCA1 (p=0.02). Conclusions Monocytes from HIV+ individuals show impaired cholesterol efflux and are primed for foam cell formation following trans-endothelial migration. Factors present in HIV+ serum, including elevated TNF levels, further enhance foam cell formation. The pro-atherogenic phenotype of monocytes persists in virologically suppressed HIV+ individuals and may contribute mechanistically to increased atherosclerosis in this population. PMID:26244384

  7. Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.

    PubMed

    Boyle, David S; Lehman, Dara A; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

    2013-04-02

    Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

  8. HIV

    PubMed Central

    Chawla, Sumit; Sahoo, Soumya Swaroop; Jain, Rambilas; Khanna, Pardeep; Mehta, Bharti; Singh, Inderjeet

    2014-01-01

    Getting to zero: zero new HIV infections, zero deaths from AIDS-related illness, zero discrimination is the theme of World AIDS Day 2012. Given the spread of the epidemic today, getting to zero may sound difficult, but significant progress is underway. The total annual loss for the entire country due to HIV is 7% of GDP, which exceeds India’s annual health expenditure in 2004. The additional loss due to loss of labor income and increased medical expenditure as measured by the external transfers, account for 5% of the country’s health expenditure and 0.23% of GDP. Given that the HIV incidence rate is only 0.27% in India, these losses are quite staggering. Despite the remarkable achievements in development of anti-retroviral therapies against HIV and the recent advances in new prevention technologies, the rate of new HIV infections continue to outpace efforts on HIV prevention and control. Thus, the development of a safe and effective vaccine for prevention and control of AIDS remains a global public health priority and the greatest opportunity to eventually end the AIDS pandemic. PMID:24056755

  9. Electroporation-mediated administration of candidate DNA vaccines against HIV-1.

    PubMed

    Vasan, Sandhya

    2014-01-01

    Vaccines to prevent HIV remain desperately needed, but a number of challenges, including retroviral integration, establishment of anatomic reservoir sites, high sequence diversity, and heavy envelope glycosylation. have precluded development of a highly effective vaccine. DNA vaccines have been utilized as candidate HIV vaccines because of their ability to generate cellular and humoral immune responses, the lack of anti-vector response allowing for repeat administration, and their ability to prime the response to viral-vectored vaccines. Because the HIV epidemic has disproportionately affected the developing world, the favorable thermostability profile and relative ease and low cost of manufacture of DNA vaccines offer additional advantages. In vivo electroporation (EP) has been utilized to improve immune responses to DNA vaccines as candidate HIV-1 vaccines in standalone or prime-boost regimens with both proteins and viral-vectored vaccines in several animal models and, more recently, in human clinical trials. This chapter describes the preclinical and clinical development of candidate DNA vaccines for HIV-1 delivered by EP, including challenges to bringing this technology to the developing world.

  10. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

    PubMed

    Klotman, Mary E; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A; Lu, Wuyuan; Porter, Edith; Chang, Theresa L

    2008-05-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

  11. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

    PubMed

    Klotman, Mary E; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A; Lu, Wuyuan; Porter, Edith; Chang, Theresa L

    2008-05-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection. PMID:18424739

  12. Methionine increases BDNF DNA methylation and improves memory in epilepsy

    PubMed Central

    Parrish, R Ryley; Buckingham, Susan C; Mascia, Katherine L; Johnson, Jarvis J; Matyjasik, Michal M; Lockhart, Roxanne M; Lubin, Farah D

    2015-01-01

    Objective Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus. Methods Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model. Results We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade. Interpretation Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE. PMID:25909085

  13. Potent Functional Antibody Responses Elicited by HIV-I DNA Priming and Boosting with Heterologous HIV-1 Recombinant MVA in Healthy Tanzanian Adults

    PubMed Central

    Joachim, Agricola; Nilsson, Charlotta; Aboud, Said; Bakari, Muhammad; Lyamuya, Eligius F.; Robb, Merlin L.; Marovich, Mary A.; Earl, Patricia; Moss, Bernard; Ochsenbauer, Christina; Wahren, Britta; Mhalu, Fred; Sandström, Eric; Biberfeld, Gunnel; Ferrari, Guido; Polonis, Victoria R.

    2015-01-01

    Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA) virus boosting (HIVIS03). The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC) assay using luciferase reporter-infectious molecular clones (LucR-IMC) was employed. The serum neutralizing activity was significantly (but not completely) reduced upon depletion of natural killer (NK) cells from PBMC (p=0.006), indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development. Trial Registration Controlled-Trials ISRCTN90053831 The Pan African Clinical Trials Registry ATMR2009040001075080 (currently PACTR2009040001075080) PMID:25874723

  14. Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral-Treated HIV Infection

    PubMed Central

    Jiang, Wei; Lederman, Michael M.; Hunt, Peter; Sieg, Scott F.; Haley, Kathryn; Rodriguez, Benigno; Landay, Alan; Martin, Jeffrey; Sinclair, Elizabeth; Asher, Ava I.; Deeks, Steven G.; Douek, Daniel C.; Brenchley, Jason M.

    2009-01-01

    The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection. PMID:19265479

  15. Hepatitis C virus coinfection independently increases the risk of cardiovascular disease in HIV-positive patients.

    PubMed

    Fernández-Montero, J V; Barreiro, P; de Mendoza, C; Labarga, P; Soriano, V

    2016-01-01

    Patients infected with HIV are at increased risk for cardiovascular disease despite successful antiretroviral therapy. Likewise, chronic hepatitis C virus (HCV) infection is associated with extrahepatic complications, including cardiovascular disease. However the risk of cardiovascular disease has not been formally examined in HIV/HCV-coinfected patients. A retrospective study was carried out to assess the influence of HCV coinfection on the risk of cardiovascular events in a large cohort of HIV-infected patients recruited since year 2004. A composite event of cardiovascular disease was used as an endpoint, including myocardial infarction, angina pectoris, stroke or death due to any of them. A total of 1136 patients (567 HIV-monoinfected, 70 HCV-monoinfected and 499 HIV/HCV-coinfected) were analysed. Mean age was 42.7 years, 79% were males, and 46% were former injection drug users. Over a mean follow-up of 79.4 ± 21 months, 3 patients died due to cardiovascular disease, whereas 29 suffered a first episode of coronary ischaemia or stroke. HIV/HCV-coinfected patients had a greater incidence of cardiovascular disease events and/or death than HIV-monoinfected individuals (4% vs 1.2%, P = 0.004) and HCV-monoinfected persons (4% vs 1.4%, P = 0.5). After adjusting for demographics, virological parameters and classical cardiovascular disease risk factors (smoking, hypertension, diabetes, high LDL cholesterol), both HIV/HCV coinfection (HR 2.91; CI 95%: 1.19-7.12; P = 0.02) and hypertension (HR 3.65; CI 95%: 1.34-9.94; P = 0.01) were independently associated with cardiovascular disease events and/or death in HIV-infected patients. Chronic hepatitis C and hypertension are independently associated with increased cardiovascular disease risk in HIV-infected patients. Therefore, treatment of chronic hepatitis C should be prioritized in HIV/HCV-coinfected patients regardless of any liver fibrosis staging.

  16. Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Santos, Fernanda da Costa; Batalha, Pedro Netto; Brozeguini, Leonardo; Seidl, Peter R.; de Alencastro, Ricardo Bicca; Cunha, Anna Cláudia; de Souza, Maria Cecília B. V.; Ferreira, Vitor F.; Giongo, Viveca A.; Cirne-Santos, Cláudio; Paixão, Izabel C. P.

    2014-09-01

    Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

  17. LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

    PubMed

    Jones, R Brad; Song, Haihan; Xu, Yang; Garrison, Keith E; Buzdin, Anton A; Anwar, Naveed; Hunter, Diana V; Mujib, Shariq; Mihajlovic, Vesna; Martin, Eric; Lee, Erika; Kuciak, Monika; Raposo, Rui André Saraiva; Bozorgzad, Ardalan; Meiklejohn, Duncan A; Ndhlovu, Lishomwa C; Nixon, Douglas F; Ostrowski, Mario A

    2013-12-01

    Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.

  18. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    NASA Astrophysics Data System (ADS)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  19. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

    PubMed

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  20. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    PubMed Central

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  1. Advantage of a rapid extraction method of HIV1 DNA suitable for polymerase chain reaction.

    PubMed

    Vignoli, C; de Lamballerie, X; Zandotti, C; Tamalet, C; de Micco, P

    1995-01-01

    We describe a new protocol for extraction of DNA suitable for HIV1 gene amplification from clinical samples using "Chelex-100" chelating resin. Comparison was made with the classic proteinase K extraction method; 154 specimens were extracted with both methods and subjected to PCR (polymerase chain reaction). The Chelex-100 procedure optimized the yield of DNA recovery and minimized contamination due to sample manipulation. It decreased false negative results due to PCR inhibitors. A DNA sample suitable for use in PCR was obtained in 30 minutes. Chelex-100 treatment is a simple, rapid and low-cost method for DNA extraction in clinical laboratories.

  2. Cryopreservation increases DNA fragmentation in spermatozoa of smokers.

    PubMed

    Aydin, Mehmet Serif; Senturk, Gozde Erkanli; Ercan, Feriha

    2013-05-01

    Smoking causes subfertility due to deterioration of spermatozoa including decreased concentration and abnormal morphology. Although evidence on the deleterious effects of smoking on spermatozoa parameters is well known, its interference with cryopreservation is not clear. This study aimed to investigate the effects of cryopreservation on sperm parameters and DNA fragmentation in non-smokers and smokers. Semen samples were obtained from 40 normospermic male volunteers of whom 20 were non-smokers and 20 smokers. Samples were analyzed in terms of motility, concentration, morphology, and DNA fragmentation before freezing and 1 and 3 months after freezing and thawing. Ultrastructural alterations were investigated by transmission electron microscopy. Sperm morphology seemed to be more affected after cryopreservation in samples obtained from smokers. Ultrastructural examination showed alterations in the integrity of the membranes and increased subacrosomal swelling. Before freezing, the increase in DNA fragmentation rate in smokers was not statistically significant compared to that of non-smokers. However, after thawing, the DNA fragmentation rates were significantly high in both non-smokers and smokers compared to their respective rates before freezing. The extent of the increase in DNA fragmentation rate was significantly higher in smokers after thawing compared to that of non-smokers. In conclusion, cryopreservation causes alterations in membrane integrity and increases DNA fragmentation, thus triggering relatively negative effects on the sperm samples of smokers compared to that of non-smokers.

  3. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

  4. CCL2 Increases X4-tropic HIV-1 Entry into Resting CD4+ T Cells*

    PubMed Central

    Campbell, Grant R.; Spector, Stephen A.

    2008-01-01

    During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4+ T cells, we infected purified resting CD4+ T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4+ T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection. PMID:18784079

  5. Using provider performance incentives to increase HIV testing and counseling services in Rwanda.

    PubMed

    de Walque, Damien; Gertler, Paul J; Bautista-Arredondo, Sergio; Kwan, Ada; Vermeersch, Christel; de Dieu Bizimana, Jean; Binagwaho, Agnès; Condo, Jeanine

    2015-03-01

    Paying for performance provides financial rewards to medical care providers for improvements in performance measured by utilization and quality of care indicators. In 2006, Rwanda began a pay for performance scheme to improve health services delivery, including HIV/AIDS services. Using a prospective quasi-experimental design, this study examines the scheme's impact on individual and couples HIV testing. We find a positive impact of pay for performance on HIV testing among married individuals (10.2 percentage points increase). Paying for performance also increased testing by both partners by 14.7 percentage point among discordant couples in which only one of the partners is an AIDS patient.

  6. Antiretroviral Treatment is Associated with Increased Attentional Load-Dependent Brain Activation in HIV Patients

    PubMed Central

    Chang, L.; Yakupov, R.; Nakama, H.; Stokes, B.; Ernst, T.

    2009-01-01

    Objective The purpose of this paper was to determine whether antiretroviral medications, especially the nucleoside analogue reverse transcriptase inhibitors, lead to altered brain activation due to their potential neurotoxic effects in patients with human immunodeficiency virus (HIV) infection. Methods Forty-two right-handed men were enrolled in three groups: seronegative controls (SN, n=18), HIV subjects treated with antiretroviral medications (HIV+ ARV, n=12), or not treated with antiretroviral medications (HIV+NARV, n=12). Each subject performed a set of visual attention tasks with increasing difficulty or load (tracking two, three or four balls) during functional magnetic resonance imaging. Results HIV subjects, both groups combined, showed greater load-dependent increases in brain activation in the right frontal regions compared to SN (p-corrected=0.006). HIV+ARV additionally showed greater load-dependent increases in activation compared to SN in bilateral superior frontal regions (p-corrected=0.032) and a lower percent accuracy on the performance of the most difficult task (tracking four balls). Region of interest analyses further demonstrated that SN showed load-dependent decreases (with repeated trials despite increasing difficulty), while HIV subjects showed load-dependent increases in activation with the more difficult tasks, especially those on ARVs. Interpretation These findings suggest that chronic ARV treatments may lead to greater requirement of the attentional network reserve and hence less efficient usage of the network and less practice effects in these HIV patients. As the brain has a limited reserve capacity, exhausting the reserve capacity in HIV+ARV would lead to declined performance with more difficult tasks that require more attention. PMID:18247124

  7. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    PubMed

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  8. What role can gender-transformative programming for men play in increasing men's HIV testing and engagement in HIV care and treatment in South Africa?

    PubMed

    Fleming, Paul J; Colvin, Chris; Peacock, Dean; Dworkin, Shari L

    2016-11-01

    Men are less likely than women to test for HIV and engage in HIV care and treatment. We conducted in-depth interviews with men participating in One Man Can (OMC) - a rights-based gender equality and health programme intervention conducted in rural Limpopo and Eastern Cape, South Africa - to explore masculinity-related barriers to HIV testing/care/treatment and how participation in OMC impacted on these. Men who participated in OMC reported an increased capability to overcome masculinity-related barriers to testing/care/treatment. They also reported increased ability to express vulnerability and discuss HIV openly with others, which led to greater willingness to be tested for HIV and receive HIV care and treatment for those who were living with HIV. Interventions that challenge masculine norms and promote gender equality (i.e. gender-transformative interventions) represent a promising new approach to address men's barriers to testing, care and treatment. PMID:27267890

  9. What role can gender-transformative programming for men play in increasing men's HIV testing and engagement in HIV care and treatment in South Africa?

    PubMed

    Fleming, Paul J; Colvin, Chris; Peacock, Dean; Dworkin, Shari L

    2016-11-01

    Men are less likely than women to test for HIV and engage in HIV care and treatment. We conducted in-depth interviews with men participating in One Man Can (OMC) - a rights-based gender equality and health programme intervention conducted in rural Limpopo and Eastern Cape, South Africa - to explore masculinity-related barriers to HIV testing/care/treatment and how participation in OMC impacted on these. Men who participated in OMC reported an increased capability to overcome masculinity-related barriers to testing/care/treatment. They also reported increased ability to express vulnerability and discuss HIV openly with others, which led to greater willingness to be tested for HIV and receive HIV care and treatment for those who were living with HIV. Interventions that challenge masculine norms and promote gender equality (i.e. gender-transformative interventions) represent a promising new approach to address men's barriers to testing, care and treatment.

  10. Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

    PubMed

    Samoilova, E O; Krasheninnikov, I A; Vinogradova, E N; Kamenski, P A; Levitskii, S A

    2016-07-01

    Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome. PMID:27449618

  11. Increases in HIV Testing among Men Who Have Sex with Men — National HIV Behavioral Surveillance System, 20 U.S. Metropolitan Statistical Areas, 2008 and 2011

    PubMed Central

    Cooley, Laura A.; Oster, Alexandra M.; Rose, Charles E.; Wejnert, Cyprian; Le, Binh C.; Paz-Bailey, Gabriela

    2014-01-01

    In 2011, 62% of estimated new HIV diagnoses in the United States were attributed to male-to-male sexual contact (men who have sex with men, MSM); 39% of these MSM were black or African American. HIV testing, recommended at least annually by CDC for sexually active MSM, is an essential first step in HIV care and treatment for HIV-positive individuals. A variety of HIV testing initiatives, designed to reach populations disproportionately affected by HIV, have been developed at both national and local levels. We assessed changes in HIV testing behavior among MSM participating in the National HIV Behavioral Surveillance System in 2008 and 2011. We compared the percentages tested in the previous 12 months in 2008 and 2011, overall and by race/ethnicity and age group. In unadjusted analyses, recent HIV testing increased from 63% in 2008 to 67% in 2011 overall (P<0.001), from 63% to 71% among black MSM (P<0.001), and from 63% to 75% among MSM of other/multiple races (P<0.001); testing did not increase significantly for white or Hispanic/Latino MSM. Multivariable model results indicated an overall increase in recent HIV testing (adjusted prevalence ratio [aPR] = 1.07, P<0.001). Increases were largest for black MSM (aPR = 1.12, P<0.001) and MSM of other/multiple races (aPR = 1.20, P<0.001). Among MSM aged 18–19 years, recent HIV testing was shown to increase significantly among black MSM (aPR = 1.20, P = 0.007), but not among MSM of other racial/ethnic groups. Increases in recent HIV testing among populations most affected by HIV are encouraging, but despite these increases, improved testing coverage is needed to meet CDC recommendations. PMID:25180514

  12. Residual soil DNA extraction increases the discriminatory power between samples.

    PubMed

    Young, Jennifer M; Weyrich, Laura S; Clarke, Laurence J; Cooper, Alan

    2015-06-01

    Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

  13. Increasing global participation in genetics research through DNA barcoding.

    PubMed

    Adamowicz, Sarah J; Steinke, Dirk

    2015-12-01

    DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources. PMID:26642251

  14. Engineering RENTA, a DNA prime-MVA boost HIV vaccine tailored for Eastern and Central Africa.

    PubMed

    Nkolola, J P; Wee, E G-T; Im, E-J; Jewell, C P; Chen, N; Xu, X-N; McMichael, A J; Hanke, T

    2004-07-01

    For the development of human immunodeficiency virus type 1 (HIV-1) vaccines, traditional approaches inducing virus-neutralizing antibodies have so far failed. Thus the effort is now focused on elicitation of cellular immunity. We are currently testing in clinical trials in the United Kingdom and East Africa a T-cell vaccine consisting of HIV-1 clade A Gag-derived immunogen HIVA delivered in a prime-boost regimen by a DNA plasmid and modified vaccinia virus Ankara (MVA). Here, we describe engineering and preclinical development of a second immunogen RENTA, which will be used in combination with the present vaccine in a four-component DNA/HIVA-RENTA prime-MVA/HIVA-RENTA boost formulation. RENTA is a fusion protein derived from consensus HIV clade A sequences of Tat, reverse transcriptase, Nef and gp41. We inactivated the natural biological activities of the HIV components and confirmed immunogenicities of the pTHr.RENTA and MVA.RENTA vaccines in mice. Furthermore, we demonstrated in mice and rhesus monkeys broadening of HIVA-elicited T-cell responses by a parallel induction of HIVA- and RENTA-specific responses recognizing multiple HIV epitopes.

  15. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

    PubMed Central

    Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

    2016-01-01

    We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

  16. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

    PubMed

    Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

    2016-01-01

    We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. PMID:27644592

  17. Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.

    PubMed Central

    Esposito, D; Craigie, R

    1998-01-01

    HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction. PMID:9755183

  18. Increased Mutagen Sensitivity and DNA Damage in Pulmonary Arterial Hypertension

    PubMed Central

    Federici, Chiara; Drake, Kylie M.; Rigelsky, Christina M.; McNelly, Lauren N.; Meade, Sirena L.; Comhair, Suzy A. A.; Erzurum, Serpil C.

    2015-01-01

    Rationale: Pulmonary arterial hypertension (PAH) is a serious lung condition characterized by vascular remodeling in the precapillary pulmonary arterioles. We and others have demonstrated chromosomal abnormalities and increased DNA damage in PAH lung vascular cells, but their timing and role in disease pathogenesis is unknown. Objectives: We hypothesized that if DNA damage predates PAH, it might be an intrinsic cell property that is present outside the diseased lung. Methods: We measured DNA damage, mutagen sensitivity, and reactive oxygen species (ROS) in lung and blood cells from patients with Group 1 PAH, their relatives, and unrelated control subjects. Measurements and Main Results: Baseline DNA damage was significantly elevated in PAH, both in pulmonary artery endothelial cells (P < 0.05) and peripheral blood mononuclear cells (PBMC) (P < 0.001). Remarkably, PBMC from unaffected relatives showed similar increases, indicating this is not related to PAH treatments. ROS levels were also higher (P < 0.01). DNA damage correlated with ROS production and was suppressed by antioxidants (P < 0.001). PBMC from patients and relatives also showed markedly increased sensitivity to two chemotherapeutic drugs, bleomycin and etoposide (P < 0.001). Results were consistent across idiopathic, heritable, and associated PAH groups. Conclusions: Levels of baseline and mutagen-induced DNA damage are intrinsically higher in PAH cells. Similar results in PBMC from unaffected relatives suggest this may be a genetically determined trait that predates disease onset and may act as a risk factor contributing to lung vascular remodeling following endothelial cell injury. Further studies are required to fully characterize mutagen sensitivity, which could have important implications for clinical management. PMID:25918951

  19. Increasing HIV-1 molecular complexity among men who have sex with men in Bangkok.

    PubMed

    Leelawiwat, Wanna; Rutvisuttinunt, Wiriya; Arroyo, Miguel; Mueanpai, Famui; Kongpechsatit, Oranuch; Chonwattana, Wannee; Chaikummao, Supaporn; de Souza, Mark; vanGriensven, Frits; McNicholl, Janet M; Curlin, Marcel E

    2015-04-01

    In Thailand, new HIV-1 infections are largely concentrated in certain risk groups such as men who have sex with men (MSM), where annual incidence may be as high as 12% per year. The paucity of information on the molecular epidemiology of HIV-1 in Thai MSM limits progress in understanding the epidemic and developing new prevention methods. We evaluated HIV-1 subtypes in seroincident and seroprevalent HIV-1-infected men enrolled in the Bangkok MSM Cohort Study (BMCS) between 2006 and 2011. We characterized HIV-1 subtype in 231 seroprevalent and 194 seroincident subjects using the multihybridization assay (MHA). Apparent dual infections, recombinant strains, and isolates found to be nontypeable by MHA were further characterized by targeted genomic sequencing. Most subjects were infected with HIV-1 CRF01_AE (82%), followed by infections with recombinants (11%, primarily CRF01_AE/B recombinants), subtype B (5%), and dual infections (2%). More than 11 distinct chimeric patterns were observed among CRF01B_AE/B recombinants, most involving recombination within integrase. A significant increase in the proportion of nontypeable strains was observed among seroincident MSM between 2006 and 2011. CRF01_AE and subtype B were the most and least common infecting strains, respectively. The predominance of CRF01_AE among HIV-1 infections in Thai MSM participating in the BMCS parallels trends observed in Thai heterosexuals and injecting drug users. The presence of complex recombinants and a significant rise in nontypeable strains suggest ongoing changes in the genetic makeup of the HIV-1 epidemic in Thailand, which may pose challenges for HIV-1 prevention efforts and vaccine development.

  20. Self-testing as strategy to increase the uptake of HIV testing in South Africa.

    PubMed

    van Dyk, Alta C

    2013-03-01

    With better health care and greater access to antiretrovirals (ARVs) it has become important to increase the uptake of HIV testing. Major testing campaigns have been launched in South Africa, but no official provision exists for clients who prefer self-testing for HIV. The purpose of this study was to investigate which HIV-testing model (client-initiated, provider-initiated or self-testing) a sample of South Africans preferred and why. A semi-structured questionnaire was completed by 466 participants and 9 provinces were represented by the sample. The results indicated that 22.3% of the participants preferred self-testing for HIV (while 66.1% preferred client-initiated and 11.6% provider-initiated counselling and testing). Participants who preferred self-testing for HIV were predominantly (p < 0.05) male, white, lived in cities or towns, did not know their HIV status, and had no intention of being tested at a medical facility in future. The underlying themes of why participants preferred self-testing for HIV above testing in a medical facility were the need for privacy and confidentiality; the need to keep testing as well as the results secret; fear of discrimination and stigma; distrust in the health care system to keep results confidential; and an aversion to face-to-face counselling. Participants indicated that they would consider testing if self-testing for HIV with telephone counselling becomes readily available in their communities. HIV testing models in South Africa should be revisited and revised to offer people who would otherwise not be tested, with an alternative method of testing. PMID:25871310

  1. Increasing HIV-1 molecular complexity among men who have sex with men in Bangkok.

    PubMed

    Leelawiwat, Wanna; Rutvisuttinunt, Wiriya; Arroyo, Miguel; Mueanpai, Famui; Kongpechsatit, Oranuch; Chonwattana, Wannee; Chaikummao, Supaporn; de Souza, Mark; vanGriensven, Frits; McNicholl, Janet M; Curlin, Marcel E

    2015-04-01

    In Thailand, new HIV-1 infections are largely concentrated in certain risk groups such as men who have sex with men (MSM), where annual incidence may be as high as 12% per year. The paucity of information on the molecular epidemiology of HIV-1 in Thai MSM limits progress in understanding the epidemic and developing new prevention methods. We evaluated HIV-1 subtypes in seroincident and seroprevalent HIV-1-infected men enrolled in the Bangkok MSM Cohort Study (BMCS) between 2006 and 2011. We characterized HIV-1 subtype in 231 seroprevalent and 194 seroincident subjects using the multihybridization assay (MHA). Apparent dual infections, recombinant strains, and isolates found to be nontypeable by MHA were further characterized by targeted genomic sequencing. Most subjects were infected with HIV-1 CRF01_AE (82%), followed by infections with recombinants (11%, primarily CRF01_AE/B recombinants), subtype B (5%), and dual infections (2%). More than 11 distinct chimeric patterns were observed among CRF01B_AE/B recombinants, most involving recombination within integrase. A significant increase in the proportion of nontypeable strains was observed among seroincident MSM between 2006 and 2011. CRF01_AE and subtype B were the most and least common infecting strains, respectively. The predominance of CRF01_AE among HIV-1 infections in Thai MSM participating in the BMCS parallels trends observed in Thai heterosexuals and injecting drug users. The presence of complex recombinants and a significant rise in nontypeable strains suggest ongoing changes in the genetic makeup of the HIV-1 epidemic in Thailand, which may pose challenges for HIV-1 prevention efforts and vaccine development. PMID:25366819

  2. Increased immunoglobulin G production by short term cultured duodenal biopsy samples from HIV infected patients

    PubMed Central

    Schneider, T; Zippel, T; Schmidt, W; Pauli, G; Wahnschaffe, U; Chakravarti, S; Heise, W; Riecken, E; Zeitz, M; Ullrich, R

    1998-01-01

    Background—Secretory immunity is a major defence mechanism against infections at mucosal surfaces which are common in HIV infected patients. 
Aims—To analyse intestinal immunoglobulin production in HIV infection in comparison with that in saliva and serum. 
Patients and methods—Immunoglobulin G (IgG), A (IgA), and M (IgM) concentrations were determined in supernatants of short term cultured duodenal biopsy samples, serum, and saliva from HIV infected patients (n = 28) and controls (n = 14) by radial immunodiffusion. 
Results—IgG was increased in the supernatants of short term cultured biopsy samples and saliva from HIV infected patients compared with controls (p<0.01), but IgA and IgM levels were normal. In contrast, both IgG and IgA concentrations in serum were higher in HIV infected patients than in controls (p<0.002). No correlation was found between IgA produced by duodenal biopsy specimens and serum IgA. 
Conclusion—Abnormalities in mucosal immunoglobulin production in HIV infection were suprisingly small, indicating that specific secretory immunity rather than quantitative immunoglobulin production may be impaired. However, increased production of IgG could contribute to mucosal inflammation by complement activation. Our findings of normal mucosal IgA production and the lack of correlation between serum and mucosal IgA argues against an intestinal origin for the increased serum IgA levels in HIV infected patients. 

 Keywords: mucosal immunity; HIV infection; intestinal antibodies PMID:9577341

  3. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.

    PubMed

    Chen, Kuan-Ming; Harjes, Elena; Gross, Phillip J; Fahmy, Amr; Lu, Yongjian; Shindo, Keisuke; Harris, Reuben S; Matsuo, Hiroshi

    2008-03-01

    The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

  4. Changing spatial patterns and increasing rurality of HIV prevalence in the Democratic Republic of the Congo between 2007 and 2013.

    PubMed

    Carrel, Margaret; Janko, Mark; Mwandagalirwa, Melchior Kashamuka; Morgan, Camille; Fwamba, Franck; Muwonga, Jérémie; Tshefu, Antoinette K; Meshnick, Steven; Emch, Michael

    2016-05-01

    The Democratic Republic of the Congo (DRC) has one of the lowest HIV prevalence in sub-Saharan Africa, estimated at 1.1% [0.9-1.3] of adults aged 15-49 in 2013 (UNAIDS). Within the 2 million km(2) country, however, there exists spatial variation in HIV prevalence, with the highest HIV prevalence observed in the large cities of Kinshasa and Lubumbashi. Globally, HIV is an increasingly rural disease, diffusing outwards from urban centers of high HIV prevalence to places where HIV was previously absent or present at very low levels. Utilizing data collected during Demographic and Health Surveillance (DHS) in 2007 and 2013 in the DRC, we sought to update the map of HIV prevalence in the DRC as well as to explore whether HIV in the DRC is an increasingly rural disease or remains confined to urban areas. Bayesian kriging and regression indicate that HIV prevalence in rural areas of the DRC is higher in 2013 than in 2007 and that increased distance to an urban area is no longer protective against HIV as it was in 2007. These findings suggest that HIV education, testing and prevention efforts need to diffuse from urban to rural areas just as HIV is doing. PMID:26974234

  5. Increased HIV Testing among Men Who Have Sex with Men from 2008 to 2012, Nanjing, China

    PubMed Central

    Yan, Hongjing; Li, Jianjun; Raymond, H. Fisher; Huan, Xiping; Guan, Wenhui; Hu, Haiyang; Yang, Haitao; McFarland, Willi; Wei, Chongyi

    2016-01-01

    Background HIV testing is the first point of HIV treatment entry for HIV-infected individuals and an avenue to engage persons at risk in prevention. In China, where the prevalence of HIV among men who have sex with men (MSM) has been rising over the last decade, uptake of HIV testing has been low. Methods We examined changes in HIV testing in the preceding 12 months through two cross-sectional surveys conducted among MSM in Nanjing, Jiangsu province, China in 2008 and 2012. Respondent-driven sampling (RDS) was used to recruit participants. Questionnaire interviews and venous blood were collected to measure HIV testing, risk behaviors, and prevalence of HIV, syphilis, and HSV-2. Results A total of 430 and 589 MSM were surveyed in 2008 and 2012, respectively, with comparable samples in each round with respect to demographic characteristics. HIV testing in the past 12 months increased significantly from 20.1% (95% CI 13.3–26.8) in 2008 to 46.0% (95% CI 39.3–51.4, p < 0.001) in 2012. HIV prevalence was stable, at 6.6% (95% CI 2.5–11.3) in 2008 and 10.1% (95% CI 6.6–13.9, p = 0.240) in 2012, as was syphilis (14.3% in 2008 vs. 9.9% in 2012, p = 0.240). HSV-2 prevalence (18.6% in 2008 vs. 10.2% in 2012, p = 0.040) and self-reported STI in the last year (24.3% in 2008 vs. 14.3% in 2012, p = 0.020) significantly decreased. Changes in reported sexual behaviors were mixed and the profiles of who did and did not test varied between 2008 and 2012. Conclusions HIV testing uptake more than doubled among MSM in Nanjing from 2008 to 2012 –a period of massive promotion and scale up of testing programs for MSM. However, additional efforts are still needed to further increase the proportion of men being not only tested but also undergoing repeat testing if they engage in continued risk taking behavior. PMID:27111540

  6. In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75.

    PubMed

    McNeely, Melissa; Hendrix, Jelle; Busschots, Katrien; Boons, Eline; Deleersnijder, Angélique; Gerard, Melanie; Christ, Frauke; Debyser, Zeger

    2011-07-29

    Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.

  7. Apolipoprotein E4 genotype does not increase risk of HIV-associated neurocognitive disorders.

    PubMed

    Morgan, E E; Woods, S P; Letendre, S L; Franklin, D R; Bloss, C; Goate, A; Heaton, R K; Collier, A C; Marra, C M; Gelman, B B; McArthur, J C; Morgello, S; Simpson, D M; McCutchan, J A; Ellis, R J; Abramson, I; Gamst, A; Fennema-Notestine, C; Smith, D M; Grant, I; Vaida, F; Clifford, D B

    2013-04-01

    This is a cross-sectional, observational study to evaluate the hypothesis that HIV-seropositive (HIV+) apolipoprotein E4 (APOE4) carriers are at increased risk for HIV-associated neurocognitive disorders (HAND) compared to APOE4 noncarriers with HIV in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) Group sample. APOE genotype was determined in 466 CHARTER participants with varying disease stages and histories of antiretroviral treatment who did not have severe psychiatric or medical comorbid conditions that preclude diagnosis of HAND. HAND diagnoses were based on results of comprehensive neurobehavioral evaluation and use of current neuroAIDS diagnostic criteria. HAND status consists of two levels: neuropsychologically normal status (i.e., no HAND) and any HAND diagnosis (i.e., asymptomatic neurocognitive impairment, minor neurocognitive disorder, HIV-associated dementia). Logistic regression analyses revealed no association between APOE4 carrier status and HAND, and there were no interactions between APOE4 carrier status and ethnicity, age, substance use disorders, duration of infection, or nadir CD4. Results did not differ when analysis was restricted to symptomatic HAND, and no APOE4 gene dose-dependent relationship to HAND emerged. APOE4 status was not associated with concurrent HAND in this large, well-characterized sample. This does not preclude emergence of an association between APOE4 status and HAND as this population ages. Prospective, longitudinal studies are needed to examine APOE4 as a risk factor for neurocognitive decline, incident HAND at older ages, and potential associations with cerebrospinal fluid amyloid.

  8. Increasing heterosexual transmission of HIV-1 subtype C in Inland Central Western Brazil.

    PubMed

    Alcântara, Keila Correia; Reis, Monica Nogueira Guarda; Cardoso, Ludimila Paula Vaz; Bello, Gonzalo; Stefani, Mariane Martins Araújo

    2013-03-01

    The molecular epidemiology of HIV-1 in Brazil is complex and heterogeneous because several subtypes co-circulate with some important regional differences. This study evaluated HIV-1 subtypes amongst pregnant women living in the metropolitan area and in the interior cities from central western Brazil. From June 2008 to June 2010, 86.9% of confirmed cases of HIV-1 infection amongst pregnant women (172 out of 198 cases) were recruited in Goiania/Goias state. The HIV-1 pol gene was sequenced after nested-PCR. HIV-1 subtypes were assigned by REGA, phylogenetic, and bootscan analyses. The median age of participants was 26 years (15-41 years range); 58.7% of participants were diagnosed during prenatal care and 51.7% of participants came from >50 interior cities within Goias state. Amongst the 131 HIV-1 pol sequences, 64.9% were subtype B, 13.0% were BF1 recombinant, 11.4% were subtype C, 7.6% were subtype F1, and 2.3% were BC recombinant. According to the HIV-1 diagnosis date (1994-2010), a significant increase in subtype C and a decrease of BF1 mosaics were observed over time. All subtype C patients lived in interior cities where the highest prevalence of subtype C outside southern Brazil was observed (18.4%). Phylogenetic analysis revealed multiple independent introductions of the Brazilian subtype C clade from the southern/southeastern regions of Brazil. The HIV-1 epidemic in women from central western Brazil infected by the heterosexual route is characterized by an unexpectedly high prevalence of subtype C viruses highly related to those circulating in southern/southeastern Brazil. These findings highlight the importance of molecular surveillance programs outside large metropolitan regions in Brazil.

  9. NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses.

    PubMed

    Guo, Haitao; König, Renate; Deng, Meng; Riess, Maximilian; Mo, Jinyao; Zhang, Lu; Petrucelli, Alex; Yoh, Sunnie M; Barefoot, Brice; Samo, Melissa; Sempowski, Gregory D; Zhang, Aiping; Colberg-Poley, Anamaris M; Feng, Hui; Lemon, Stanley M; Liu, Yong; Zhang, Yanping; Wen, Haitao; Zhang, Zhigang; Damania, Blossom; Tsao, Li-Chung; Wang, Qi; Su, Lishan; Duncan, Joseph A; Chanda, Sumit K; Ting, Jenny P-Y

    2016-04-13

    Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1(-/-) mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses.

  10. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  11. Human papillomavirus DNA and mRNA prevalence and association with cervical cytological abnormalities in the Irish HIV population.

    PubMed

    Loy, Aisling; McInerney, Jamie; Pilkington, Loretto; Keegan, Helen; Delamere, Sandra; Martin, Cara M; Sheils, Orla; O'Leary, John J; Mulcahy, Fiona

    2015-10-01

    The complex interplay between HIV and human papillomavirus and its link to cervical dysplasia is poorly understood. This is the first study to assess the prevalence of oncogenic human papillomavirus mRNA in HIV-positive women, its relationship to HIV and its potential use in the triage of cervical cancer screening in HIV-positive women. In this cross-sectional study, we included 321 HIV-positive women. In all, 28.7% had abnormal cervical cytology, 51.1% were human papillomavirus DNA-positive and 21.8% tested positive for human papillomavirus mRNA. Women with a CD4 count of <200 × 10(6)/L were more likely to test positive for human papillomavirus DNA and mRNA. Virally suppressed women were less likely to be human papillomavirus DNA-positive; however, the same did not hold true for human papillomavirus mRNA. We found the human papillomavirus mRNA screening to be more specific when screening for low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion than human papillomavirus DNA at 84.53% compared to 57.36%. However, the sensitivity was less at 51.59% versus 91.07% for human papillomavirus DNA. It may be possible in the future to use human papillomavirus mRNA/DNA testing within a triage algorithm for the screening and management of cervical cancer in the HIV-positive patient.

  12. HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus

    PubMed Central

    Chamontin, Célia; Rassam, Patrice; Ferrer, Mireia; Racine, Pierre-Jean; Neyret, Aymeric; Lainé, Sébastien; Milhiet, Pierre-Emmanuel; Mougel, Marylène

    2015-01-01

    HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism. PMID:25488808

  13. A trend towards increasing viral load in newly diagnosed HIV-infected inpatients in southeast China.

    PubMed

    Chen, Y; Wang, Z; Huang, A; Yuan, J; Wei, D; Ye, H

    2016-06-01

    Peripheral blood viral load is an important indicator of viral production and clearance. Previous studies have suggested that viral load might predict the rate of decrease in CD4+ cell count and progression to AIDS and death. Here, we conducted a retrospective analysis of the trends in HIV-1 viral load in southeast China. Among inpatients newly diagnosed with HIV infection, we found that viral load has increased over the past decade from 4·20 log10 copies/ml in 2002 to 6·61 log10 copies/ml in 2014, with a mean increase of 0·19 log10 copies/ml each year. However, the CD4+ cell count was stable and insensitive to changes in viral load. Thus, increasing viral load appears to be an emerging trend in newly diagnosed HIV-infected inpatients.

  14. Chronic opioid use is associated with increased DNA methylation correlating with increased clinical pain.

    PubMed

    Doehring, Alexandra; Oertel, Bruno Georg; Sittl, Reinhard; Lötsch, Jörn

    2013-01-01

    Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenotypic changes by altering gene transcription are summarized as epigenetics. A major epigenetic mechanism is methylation or demethylation at CpG-rich DNA islands. DNA methylation triggered by drugs has largely unexplored therapeutic consequences. Here we report increased methylation at a CpG rich island in the OPRM1 gene coding for μ-opioid receptors and at a global methylation site (LINE-1) in leukocytes of methadone-substituted former opiate addicts compared with matched healthy controls. Higher DNA methylation associated with chronic opioid exposure was reproduced in an independent cohort of opioid-treated as compared to non-opioid-treated pain patients. This suggests that opioids may stimulate DNA methylation. The OPRM1 methylation had no immediate effect on μ-opioid receptor transcription and was not associated with opioid dosing requirements. However, the global DNA methylation at LINE-1 was significantly correlated with increased chronic pain. This suggests inhibitory effects on the transcription of still unspecified nocifensive gene products. It further implies that opioids may be causally associated with increased genome-wide DNA methylation, although currently there is no direct evidence of this. This has phenotypic consequences for pain and may provide a new, epigenetics-associated mechanism of opioid-induced hyperalgesia. The results indicate a potential influence of opioid analgesics on the patients' epigenome. They emphasize the need for reliable and cost-effective screening tools and may imply that high-throughput screening for lead compounds in artificial expression systems may not provide the best tools for identifying new pain medications.

  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

    PubMed Central

    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. Integrin α4β7 expression increases HIV susceptibility in activated cervical CD4+ T cells via an HIV attachment-independent mechanism

    PubMed Central

    Ding, Jian; Tasker, Carley; Lespinasse, Pierre; Dai, Jihong; Fitzgerald-Bocarsly, Patricia; Lu, Wuyuan; Heller, Debra; Chang, Theresa L.

    2015-01-01

    Background CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. The integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immune phenotypes of human cervical T cells and examined HIV preference in integrin α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells (at-RA differentiated cells) were included as a comparison. Results In both peripheral and cervical cells, the majority of HIV p24+ cells were activated CD4+ T cells expressing integrin α4β7. Among infected at-RA differentiated cells, the frequency of CCR5 expression was higher in HIV p24+ cells than in HIV p24- cells; no such difference was observed in cervical cells. Neither the cyclic hexapeptide CWLDVC nor a monoclonal antibody against integrin α4β7 blocked HIV attachment or gp120 binding to target cells regardless of the presence of CD4, indicating that integrin α4β7 did not facilitate HIV capture by target cells. Conclusion Integrin α4β7 expression increases HIV susceptibility, but the mechanism is not through promoting HIV binding to target cells. PMID:26167616

  17. Optimizing HIV/AIDS resources in Armenia: increasing ART investment and examining HIV programmes for seasonal migrant labourers

    PubMed Central

    Kelly, Sherrie L; Shattock, Andrew J; Kerr, Cliff C; Stuart, Robyn M; Papoyan, Arshak; Grigoryan, Trdat; Hovhannisyan, Ruben; Grigoryan, Samvel; Benedikt, Clemens; Wilson, David P

    2016-01-01

    Introduction HIV prevalence is declining in key populations in Armenia including in people who inject drugs (PWID), men who have sex with men, prison inmates, and female sex workers (FSWs); however, prevalence is increasing among Armenians who seasonally migrate to work in countries with higher HIV prevalence, primarily to the Russian Federation. Methods We conducted a modelling study using the Optima model to assess the optimal resource allocation to meet targets from the 2013 to 2016 national strategic plan to minimize HIV incidence and AIDS-related deaths by 2020. Demographic, epidemiological, behavioural, and programme cost data from 2000 through 2014 were used to inform the model. The levels of coverage that could be attained among targeted populations with different investments, as well as their expected outcomes, were determined. In the absence of evidence of the efficacy of HIV programmes targeted at seasonal labour migrants, we conducted a sensitivity analysis to determine the cost-effective funding threshold for the seasonal labour migrant programme. Results The optimization analysis revealed that shifts in funding allocations could further minimize incidence and deaths by 2020 within the available resource envelope. The largest emphasis should be on antiretroviral therapy (ART), with the optimal investment to increase treatment coverage by 40%. Optimal investments also involve increases in opiate substitution therapy and FSW programmes, as well as maintenance of other prevention programmes for PWID and prevention of mother-to-child transmission. Additional funding for these increases should come from budgets for general population programmes. This is projected to avert 17% of new infections and 29% of AIDS-related deaths by 2020 compared to a baseline scenario of maintaining 2013 spending. Our sensitivity analysis demonstrated that, at current spending, coverage of annual testing among migrants of at least 43% should be achieved to warrant continuation

  18. Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency.

    PubMed Central

    Bednarik, D P; Cook, J A; Pitha, P M

    1990-01-01

    Infection of cells by HIV can result in a period of quiescence or latency which may be obviated by treatment with inducing agents such as 5-azacytidine. Evidence from these experiments demonstrate the existence of two CpG sites in the HIV LTR which can silence transcription of both reporter genes (CAT) and infectious proviral DNA when enzymatically methylated. This transcriptional block was consistently overcome by the presence of the trans-activator tat without significant demethylation of the HIV LTR. These results suggest that DNA hypermethylation of the HIV LTR may change the binding characteristics between LTR sequences and cellular proteins, thereby suppressing HIV LTR transcription and modulating viral expression. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2323336

  19. Ethical reflections on pharmacogenetics and DNA banking in a cohort of HIV-infected patients

    PubMed Central

    de Montgolfier, Sandrine; Moutel, Grégoire; Duchange, Nathalie; Theodorou, Ioannis; Hervé, Christian; Leport, Catherine

    2002-01-01

    The aim of this study was to analyze ethical questions concerning the storage of human biological samples to be used in genetic analyses and pharmacogenetic research based on a French experience of DNA banking in a cohort of HIV-infected patients receiving protease inhibitor treatment (APROCO). We describe the ethical issues raised during the establishment of a DNA bank, including questions dealing with autonomy, benefit to the patient, information sharing and confidentiality as well as guarantees concerning the storage and use of DNA. We describe the practical applications of themes illustrated theoretically in the literature. Most of the points raised are not specific to HIV, but some of them may be more accurate due to the characteristics of the HIV population. The questions raised are not exhaustive and we conclude with specific points that remain to be defined. Our results are summarized in the memorandum and consent form presented in the appendices. This work should allow other researchers and members of evaluation committees to enrich their considerations and should stimulate discussion on this subject. PMID:12464796

  20. Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.

    PubMed Central

    Sarafianos, S G; Kortz, U; Pope, M T; Modak, M J

    1996-01-01

    The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules. PMID:8912703

  1. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  2. Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay.

    PubMed

    Haugan, I R; Nilsen, B M; Worland, S; Olsen, L; Helland, D E

    1995-12-26

    Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evaluate the binding of DNA to HIV-1 integrase. In this assay the DNA-binding was found to be independent of Mn2+ concentration, inhibited by concentrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibited by monoclonal antibodies reacting with either the N-terminal or C-terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is concluded that both the N-terminal and the C-terminal regions of integrase are involved in DNA-binding and that the reported double-layered dot blot radio assay is well suited for further characterization of the integrase.

  3. Aortic endothelium in HIV-1 infection: chronic injury, activation, and increased leukocyte adherence.

    PubMed Central

    Zietz, C.; Hotz, B.; Stürzl, M.; Rauch, E.; Penning, R.; Löhrs, U.

    1996-01-01

    Clinical and serological studies provide evidence for a pathogenetically relevant vasculopathy in acquired immune deficiency syndrome (AIDS); however, the morphological status of the endothelium under conditions of human immunodeficiency virus (HIV)-1 infection is only sparsely documented. In this study we adapted an en face preparation technique of endothelium for use in immunohistochemistry and investigated the aortic endothelium of pre-AIDS and AIDS patients (n = 32) in comparison with an HIV-negative group (n = 17). The control group showed a regular pattern of evenly distributed aortic endothelial cells, whereas the endothelial cell pattern in the HIV-1-infected patients was clearly disturbed. Simultaneously, the degree of leukocyte adherence on the aortic endothelium increased significantly. These changes were accompanied by an up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (ELAM-1). The endothelium turnover increased, and one-half of the HIV-1-infected patients exhibited HLA-DR (major histocompatibility complex class II) antigen in the aortic endothelium. Our results provide evidence for a profound and repeated injury with regeneration and activation of the endothelium in HIV-1 infection. Injury as well as activation of the endothelium impairs its normal regulatory properties. This could have consequences for the maintenance of the blood-brain barrier; it might influence the immunologically important interaction of the endothelium with T cells; and it might trigger Kaposi's sarcoma. Images Figure 1 Figure 2 Figure 3 PMID:8952525

  4. rDNA-directed integration by an HIV-1 integrase--I-PpoI fusion protein.

    PubMed

    Schenkwein, Diana; Turkki, Vesa; Ahlroth, Mervi K; Timonen, Oskari; Airenne, Kari J; Ylä-Herttuala, Seppo

    2013-03-01

    Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.

  5. HIV Acquisition Is Associated with Increased Antimicrobial Peptides and Reduced HIV Neutralizing IgA in the Foreskin Prepuce of Uncircumcised Men

    PubMed Central

    Hirbod, Taha; Kong, Xiangrong; Kigozi, Godgrey; Ndyanabo, Anthony; Serwadda, David; Prodger, Jessica L.; Tobian, Aaron A.; Nalugoda, Fred; Wawer, Maria J.; Shahabi, Kamnoosh; Rojas, Olga L.; Gommerman, Jennifer L.; Broliden, Kristina

    2014-01-01

    Background The foreskin is the site of most HIV acquisition in uncircumcised heterosexual men. Although HIV-exposed, seronegative (HESN) uncircumcised men demonstrate HIV-neutralizing IgA and increased antimicrobial peptides (AMPs) in the foreskin prepuce, no prospective studies have examined the mucosal immune correlates of HIV acquisition. Methods To assess the association of foreskin immune parameters with HIV acquisition, antimicrobial peptides and IgA with the capacity to neutralize a primary clade C HIV strain were quantified by blinded investigators, using sub-preputial swabs collected longitudinally during a randomized trial of male circumcision for HIV prevention in Rakai, Uganda. Results Participants were 99 men who acquired HIV (cases) and 109 randomly selected controls who remained HIV seronegative. At enrollment, 44.4% of cases vs. 69.7% of controls demonstrated IgA neutralization (adjusted OR = 0.31; 95% CI, 0.16–0.61). IgA neutralization was detected in 38.7% of cases and 70.7% of controls at the last seronegative case visit prior to HIV acquisition and the comparable control visit (adjusted OR 0.21; 95% CI, 0.11–0.39). Levels of the α-defensins and secretory leukocyte protease inhibitor (SLPI) were over ten-fold higher in the foreskin prepuce of cases who acquired HIV, both at enrollment (mean 4.43 vs. 3.03 and 5.98 vs. 4.61 logn pg/mL, P = 0.005 and 0.009, respectively), and at the last seronegative visit (mean 4.81 vs. 3.15 and 6.46 vs. 5.20 logn pg/mL, P = 0.0002 and 0.013). Conclusions This prospective, blinded analysis is the first to assess the immune correlates of HIV acquisition in the foreskin. HIV-neutralizing IgA, previously associated with the HESN phenotype, was a biomarker of HIV protection, but other HESN associations correlated with increased HIV acquisition. This emphasizes the importance of prospective epidemiological studies or in vitro tissue studies to define the impact of mucosal parameters on HIV risk. PMID

  6. Increase in body cell mass and decrease in wasting are associated with increasing potency of antiretroviral therapy for HIV infection.

    PubMed

    Ferrando, Stephen J; Rabkin, Judith G; Lin, Shu-Hsing; McElhiney, Martin

    2005-04-01

    With the advent of potent combination antiretroviral therapy (ART), there has been a reduction in the incidence of wasting. However, few studies have investigated specific body composition changes associated with these treatments. This study aimed to investigate longitudinally the association of increasingly potent ART with changes in body cell mass and wasting utilizing bioelectric impedance analysis (BIA). In this longitudinal cohort study, 159 HIV-positive men were assessed semiannually from 1995 to 1997 for body composition utilizing BIA, CD4 lymphocyte count, HIV viral load, medical and depressive symptoms. Wasting was defined as body cell mass/height below the 90th percentile based on HIV positive norms. ART potency at each visit was scored utilizing published clinical guidelines, ranging from 1 (0-1 antiretrovirals) to 5 (3 or more antiretrovirals including a potent protease inhibitor). Viral resistance testing was not used. The mixed-effects model and the generalized estimating equations approaches were used to determine longitudinal correlates of body cell mass and of wasting, respectively. Over the 2 years of follow-up, potent combination ART use increased from 6% to 79%. Concurrently, a significant increase in mean body cell mass and a reduction in prevalence of wasting were seen, while total body weight, fat mass, and total body water did not change. Increasingly potent ART was associated with significant increases in body cell mass and reduction in wasting. Other significant correlates of increased body cell mass included higher CD4 count and decreased severity of HIV-related symptoms, fatigue and depression. The current study found that higher potency ART taken for relatively short term (2 years) was associated with an increase in body cell mass and a reduction in wasting and that these changes were associated with both medical (CD4, HIV symptoms) and behavioral (fatigue, depression) improvements. One caveat is this study did not distinguish among

  7. Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

    PubMed

    Necchi, Daniela; Pinto, Antonella; Tillhon, Micol; Dutto, Ilaria; Serafini, Melania Maria; Lanni, Cristina; Govoni, Stefano; Racchi, Marco; Prosperi, Ennio

    2015-10-01

    Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase β, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

  8. HIV-1 Tat Protein Increases Microglial Outward K+ Current and Resultant Neurotoxic Activity

    PubMed Central

    Liu, Jianuo; Xu, Peng; Collins, Cory; Liu, Han; Zhang, Jingdong; Keblesh, James P.; Xiong, Huangui

    2013-01-01

    Microglia plays a crucial role in the pathogenesis of HIV-1-associated neurocognitive disorders. Increasing evidence indicates the voltage-gated potassium (Kv) channels are involved in the regulation of microglia function, prompting us to hypothesize Kv channels may also be involved in microglia-mediated neurotoxic activity in HIV-1-infected brain. To test this hypothesis, we investigated the involvement of Kv channels in the response of microglia to HIV-1 Tat protein. Treatment of rat microglia with HIV-1 Tat protein (200 ng/ml) resulted in pro-inflammatory microglial activation, as indicated by increases in TNF-α, IL-1β, reactive oxygen species, and nitric oxide, which were accompanied by enhanced outward K+ current and Kv1.3 channel expression. Suppression of microglial Kv1.3 channel activity, either with Kv1.3 channel blockers Margatoxin, 5-(4-Phenoxybutoxy)psoralen, or broad-spectrum K+ channel blocker 4-Aminopyridine, or by knockdown of Kv1.3 expression via transfection of microglia with Kv1.3 siRNA, was found to abrogate the neurotoxic activity of microglia resulting from HIV-1 Tat exposure. Furthermore, HIV-1 Tat-induced neuronal apoptosis was attenuated with the application of supernatant collected from K+ channel blocker-treated microglia. Lastly, the intracellular signaling pathways associated with Kv1.3 were investigated and enhancement of microglial Kv1.3 was found to correspond with an increase in Erk1/2 mitogen-activated protein kinase activation. These data suggest targeting microglial Kv1.3 channels may be a potential new avenue of therapy for inflammation-mediated neurological disorders. PMID:23738010

  9. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

    PubMed Central

    2016-01-01

    ABSTRACT There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized. PMID:27624129

  10. Increased regulatory T cell counts in HIV-infected nonresponders to hepatitis B virus vaccine.

    PubMed

    del Pozo Balado, María del Mar; Leal, Manuel; Méndez Lagares, Gema; Mata, Rosario C; López-Cortés, Luis F; Viciana, Pompeyo; Pacheco, Yolanda M

    2010-08-15

    Hepatitis B virus (HBV) coinfection is a main cause of liver-related mortality in human immunodeficiency virus (HIV)-infected subjects. Unfortunately, HIV-infected subjects show a low rate of response to standard HBV vaccination (23%-56%), in contrast to rates >90% found in the general population, and the underlying causes (particularly cellular and molecular causes) are still unknown. We hypothesized that an increased frequency of regulatory T (T(reg)) cells could be involved in the low rate of seroconversion in HIV-infected subjects. Forty HIV-infected subjects were enrolled in the Assistance Vaccination Program against HBV of the Infectious Diseases Service from the Virgen del Rocío University Hospital, Seville, Spain. Freshly isolated peripheral blood mononuclear cells from baseline were immunophenotyped for T(reg) cells, CD4, and CD8 T cells in both naive and memory subpopulations and activation degree, as well as recent thymic emigrants. Baseline T(reg) cell frequency was found independently associated with the final nonresponse to HBV vaccine in HIV-infected subjects. Furthermore, a negative correlation between baseline frequency of T(reg) cells and antibody titers in the final response was found. These findings suggest an active role played by T(reg) cells on the immunization antigen-specific T and/or B cell responses with the final consequence of a B cell anti-HBs lower production.

  11. Four-Stage Audit Demonstrating Increased Uptake of HIV Testing in Acute Neurology Admissions Using Staged Practical Interventions

    PubMed Central

    Sokhi, Dilraj Singh; Oxenham, Chantal; Coates, Rebecca; Forbes, Mhairi; Gupta, Nadi K.; Blackburn, Daniel J.

    2015-01-01

    Background UK National Guidelines (UKNG) advise HIV testing in clinically indicated neurological presentations. We audited the impact of our practical strategies to increase uptake of HIV testing at a regional acute neurology admissions unit. Methods We audited HIV testing in 4 periods over 2 years: before we designed a UKNG-based “HIV testing in Neurology” protocol (“pre-protocol”); after dissemination of the protocol alone (“post-protocol”); post-protocol dissemination combined with both a tailored departmental admissions clerking proforma to prompt for HIV testing & consenting, and regular focussed tutorials to doctors on HIV testing in neurological patients (“post-proforma”); and finally one year after the post-proforma period (“+1 year”). We also looked at the total number of HIV tests sent from the unit during the two-year period. We assessed significance using Fisher’s exact test. Results 47.8% of all acute neurology non-stroke admissions were eligible for HIV testing during all the audit periods. Testing rates were as follows: pre-protocol 21.9%; post-protocol 36.6%; post-proforma 83.3%; and at +1 year 65.4% (p<0.05 for both post-protocol and +1 year when compared to pre-protocol). Documentation of consent for HIV testing improved from 25% to 67.6% with the HIV-tailored clerking proforma. The total number of HIV tests requested from the unit doubled in the post-proforma period compared to pre-protocol (p<0.05). Conclusion In conclusion: the combination of an HIV testing protocol, a tailored departmental clerking proforma and regular focussed teaching to doctors on indications for HIV testing led to a sustained increase in HIV testing uptake in our regional acute neurology admissions unit. PMID:26335351

  12. Enhanced non-inflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for HIV DNA vaccines.

    PubMed

    Qiao, Chenmeng; Liu, Jiandong; Yang, Jun; Li, Yan; Weng, Jie; Shao, Yiming; Zhang, Xin

    2016-04-01

    Human immunodeficiency virus (HIV) DNA vaccine can induce cellular and humoral immunity. A safe and effective HIV DNA vaccine is urgent need to prevent the spread of acquired immune deficiency syndrome (AIDS). The major drawback of DNA vaccines is the low immunogenicity, which is caused by the poor delivery to antigen presenting cells and insufficient antigen expression. Sparked by the capability of endosomal/lysosomal escape of the zwitterionic lipid distearoyl phosphoethanol-amine-polycarboxybetaine (DSPE-PCB), we attempted to develop a zwitterionic-based cationic liposome with enhanced immunogenicity of DNA vaccines. The mannosylated zwitterionic-based cationic liposome (man-ZCL) was constructed as a DNA vaccine adjuvant for HIV vaccination. Man-ZCL could complex with DNA antigens to form a tight structure and protect them from nuclei enzyme degradation. Benefited from the capability of the specific mannose receptor mediated antigen processing cells targeting and enhanced endosomal/lysosomal escape, the man-ZCL lipoplexes were supposed to promote antigen presentation and the immunogenicity of DNA vaccines. In vitro and in vivo results revealed that man-ZCL lipoplexes showed enhanced anti-HIV immune responses and lower toxicity compared with CpG/DNA and Lipo2k/DNA, and triggered a Th1/Th2 mixed immunity. An antigen-depot effect was observed in the administration site, and this resulted in enhanced retention of DNA antigens in draining lymph nodes. Most importantly, the man-ZCL could assist to activate T cells through a non-inflammasome pathway. These findings suggested that the man-ZCL could be potentially applied as a safe and efficient DNA adjuvant for HIV vaccines. PMID:26851653

  13. HIV infections and STI co-infections in men who have sex with men in Belgium: sustained increase in HIV diagnoses.

    PubMed

    Sasse, A; Defraye, A

    2009-01-01

    Belgium is currently experiencing an upward trend in the number of new HIV diagnoses characterised by a continuous increase in the number of cases among men who have sex with men (MSM). Based on surveillance data, in the past decade the yearly number of newly diagnosed HIV cases in MSM increased more than threefold, from 101 cases diagnosed in 1999 to 332 cases in 2008. During this period, the majority of new HIV infections in MSM were diagnosed among Belgian citizens (72%), followed by other European nationalities (13%). The increase in HIV diagnoses does not reflect an increase in HIV testing since the number of tests performed nationwide remained remarkably stable over time. The steady increase in the number of newly diagnosed HIV cases among MSM, and the high proportion of MSM among HIV-positive patients co-infected with other sexually transmitted infections (STI) (95.6% in 2008) indicate increases in unsafe sex practices in this group. Development of behavioural surveillance and more qualitative research on reasons for unsafe sex are needed in order to develop more effective prevention strategies.

  14. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

    PubMed

    Sarafianos, S G; Das, K; Tantillo, C; Clark, A D; Ding, J; Whitcomb, J M; Boyer, P L; Hughes, S H; Arnold, E

    2001-03-15

    We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

  15. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA

    PubMed Central

    Sarafianos, Stefan G.; Das, Kalyan; Tantillo, Chris; Clark, Arthur D.; Ding, Jianping; Whitcomb, Jeannette M.; Boyer, Paul L.; Hughes, Stephen H.; Arnold, Edward

    2001-01-01

    We have determined the 3.0 Å resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The ‘RNase H primer grip’, consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual ‘unzipping’ of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage. PMID:11250910

  16. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    SciTech Connect

    Ebina, Hirotaka Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  17. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

    PubMed Central

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F. J.; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT. PMID:26800261

  18. Efficiency of CCR5 coreceptor utilization by the HIV quasispecies increases over time, but is not associated with disease progression.

    PubMed

    Redd, Andrew D; Laeyendecker, Oliver; Kong, Xiangrong; Kiwanuka, Noah; Lutalo, Tom; Huang, Wei; Gray, Ronald H; Wawer, Maria J; Serwadda, David; Eshleman, Susan H; Quinn, Thomas C

    2012-03-01

    CCR5 is the primary coreceptor for HIV entry. Early after infection, the HIV viral population diversifies rapidly into a quasispecies. It is not known whether the initial efficiency of the viral quasispecies to utilize CCR5 is associated with HIV disease progression or if it changes in an infected individual over time. The CCR5 and CXCR4 utilization efficiencies (R5-UE and X4-UE) of the HIV quasispecies were examined using a pseudovirus, single-round infection assay for samples obtained from known seroconverters from Rakai district, Uganda (n=88). Initial and longitudinal R5-UE values were examined to assess the association of R5-UE with HIV disease progression using multivariate Cox proportional hazard models. Longitudinal samples were analyzed for 35 seroconverters who had samples available from multiple time points. There was no association between initial or longitudinal changes in R5-UE and the hazard of HIV disease progression (p=0.225 and p=0.942, respectively). In addition, R5-UE increased significantly over time after HIV seroconversion (p<0.001), regardless of HIV subtype or the emergence of CXCR4-tropic virus. These data demonstrate that the R5-UE of the viral quasispecies early in HIV infection is not associated with disease progression, and that R5-UE levels increase in HIV-infected individuals over time.

  19. Increasing HIV-related knowledge, communication, and testing intentions among Latinos: Protege tu Familia: Hazte la Prueba.

    PubMed

    Rios-Ellis, Britt; Espinoza, Lilia; Bird, Mara; Garcia, Melawhy; D'Anna, Laura Hoyt; Bellamy, Laura; Scolari, Rosana

    2010-08-01

    Latinos are less likely to be aware of their HIV seropositivity than African Americans and Whites. 'Protege tu Familia: Hazte la Prueba' is a culturally and linguistically-sensitive HIV/AIDS prevention and testing program targeting Latino families. Using community-based participatory research techniques, Spanish-speaking bicultural community health workers helped develop and then used an educational flip chart and materials to conduct outreach and HIV prevention education in diverse settings. The intervention was created to increase HIV/AIDS-related knowledge, to improve communication regarding sexual risk, and to augment intentions to use condoms and test for HIV. A secondary purpose was to decrease HIV-related stigma by improving knowledge about transmission and reducing homophobia. Participants demonstrated significant increases in HIV knowledge, intention to practice safer sex and communicate sexual risk to partner(s), and intention to test for HIV. Improvements were also found in self-reported comfort levels when interacting with and caring for the HIV positive, thus decreasing HIV/AIDS-related stigma.

  20. HIV-1 replication in human immune cells is independent of TAR DNA binding protein 43 (TDP-43) expression.

    PubMed

    Nehls, Julia; Koppensteiner, Herwig; Brack-Werner, Ruth; Floss, Thomas; Schindler, Michael

    2014-01-01

    The TAR DNA binding protein (TDP-43) was originally identified as a host cell factor binding to the HIV-1 LTR and thereby suppressing HIV-1 transcription and gene expression (Ou et al., J.Virol. 1995, 69(6):3584). TDP-43 is a global regulator of transcription, can influence RNA metabolism in many different ways and is ubiquitously expressed. Thus, TDP-43 could be a major factor restricting HIV-1 replication at the level of LTR transcription and gene expression. These facts prompted us to revisit the role of TDP-43 for HIV-1 replication. We utilized established HIV-1 cell culture systems as well as primary cell models and performed a comprehensive analysis of TDP-43 function and investigated its putative impact on HIV-1 gene expression. In HIV-1 infected cells TDP-43 was neither degraded nor sequestered from the nucleus. Furthermore, TDP-43 overexpression as well as siRNA mediated knockdown did not affect HIV-1 gene expression and virus production in T cells and macrophages. In summary, our experiments argue against a restricting role of TDP-43 during HIV-1 replication in immune cells.

  1. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    PubMed

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

  2. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    PubMed

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  3. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

    PubMed Central

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J.

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  4. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

    PubMed

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-02-02

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

  5. Increased sensitivity of HIV-1 p24 ELISA using a photochemical signal amplification system

    PubMed Central

    Bystryak, Simon; Santockyte, Rasa

    2016-01-01

    In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption (ICD and Non-ICD ELISA), by a factor of approximately 40-fold. ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. PMID:26090753

  6. Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders.

    PubMed

    Zucchini, Silvia; Pittaluga, Anna; Brocca-Cofano, Egidio; Summa, Maria; Fabris, Marina; De Michele, Rita; Bonaccorsi, Angela; Busatto, Graziella; Barbanti-Brodano, Giuseppe; Altavilla, Giuseppe; Verlengia, Gianluca; Cifelli, Pierangelo; Corallini, Alfredo; Caputo, Antonella; Simonato, Michele

    2013-07-01

    HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.

  7. The Amagugu Intervention: A Conceptual Framework for Increasing HIV Disclosure and Parent-Led Communication about Health among HIV-Infected Parents with HIV-Uninfected Primary School-Aged Children

    PubMed Central

    Rochat, Tamsen J.; Mitchell, Joanie; Stein, Alan; Mkwanazi, Ntombizodumo Brilliant; Bland, Ruth M.

    2016-01-01

    Advances in access to HIV prevention and treatment have reduced vertical transmission of HIV, with most children born to HIV-infected parents being HIV-uninfected themselves. A major challenge that HIV-infected parents face is disclosure of their HIV status to their predominantly HIV-uninfected children. Their children enter middle childhood and early adolescence facing many challenges associated with parental illness and hospitalization, often exacerbated by stigma and a lack of access to health education and support. Increasingly, evidence suggests that primary school-aged children have the developmental capacity to grasp concepts of health and illness, including HIV, and that in the absence of parent-led communication and education about these issues, HIV-exposed children may be at increased risk of psychological and social problems. The Amagugu intervention is a six-session home-based intervention, delivered by lay counselors, which aims to increase parenting capacity to disclose their HIV status and offer health education to their primary school-aged children. The intervention includes information and activities on disclosure, health care engagement, and custody planning. An uncontrolled pre–post-evaluation study with 281 families showed that the intervention was feasible, acceptable, and effective in increasing maternal disclosure. The aim of this paper is to describe the conceptual model of the Amagugu intervention, as developed post-evaluation, showing the proposed pathways of risk that Amagugu aims to disrupt through its intervention targets, mechanisms, and activities; and to present a summary of results from the large-scale evaluation study of Amagugu to demonstrate the acceptability and feasibility of the intervention model. This relatively low-intensity home-based intervention led to: increased HIV disclosure to children, improvements in mental health for mother and child, and improved health care engagement and custody planning for the child. The

  8. The Amagugu Intervention: A Conceptual Framework for Increasing HIV Disclosure and Parent-Led Communication about Health among HIV-Infected Parents with HIV-Uninfected Primary School-Aged Children

    PubMed Central

    Rochat, Tamsen J.; Mitchell, Joanie; Stein, Alan; Mkwanazi, Ntombizodumo Brilliant; Bland, Ruth M.

    2016-01-01

    Advances in access to HIV prevention and treatment have reduced vertical transmission of HIV, with most children born to HIV-infected parents being HIV-uninfected themselves. A major challenge that HIV-infected parents face is disclosure of their HIV status to their predominantly HIV-uninfected children. Their children enter middle childhood and early adolescence facing many challenges associated with parental illness and hospitalization, often exacerbated by stigma and a lack of access to health education and support. Increasingly, evidence suggests that primary school-aged children have the developmental capacity to grasp concepts of health and illness, including HIV, and that in the absence of parent-led communication and education about these issues, HIV-exposed children may be at increased risk of psychological and social problems. The Amagugu intervention is a six-session home-based intervention, delivered by lay counselors, which aims to increase parenting capacity to disclose their HIV status and offer health education to their primary school-aged children. The intervention includes information and activities on disclosure, health care engagement, and custody planning. An uncontrolled pre–post-evaluation study with 281 families showed that the intervention was feasible, acceptable, and effective in increasing maternal disclosure. The aim of this paper is to describe the conceptual model of the Amagugu intervention, as developed post-evaluation, showing the proposed pathways of risk that Amagugu aims to disrupt through its intervention targets, mechanisms, and activities; and to present a summary of results from the large-scale evaluation study of Amagugu to demonstrate the acceptability and feasibility of the intervention model. This relatively low-intensity home-based intervention led to: increased HIV disclosure to children, improvements in mental health for mother and child, and improved health care engagement and custody planning for the child. The

  9. The Amagugu Intervention: A Conceptual Framework for Increasing HIV Disclosure and Parent-Led Communication about Health among HIV-Infected Parents with HIV-Uninfected Primary School-Aged Children.

    PubMed

    Rochat, Tamsen J; Mitchell, Joanie; Stein, Alan; Mkwanazi, Ntombizodumo Brilliant; Bland, Ruth M

    2016-01-01

    Advances in access to HIV prevention and treatment have reduced vertical transmission of HIV, with most children born to HIV-infected parents being HIV-uninfected themselves. A major challenge that HIV-infected parents face is disclosure of their HIV status to their predominantly HIV-uninfected children. Their children enter middle childhood and early adolescence facing many challenges associated with parental illness and hospitalization, often exacerbated by stigma and a lack of access to health education and support. Increasingly, evidence suggests that primary school-aged children have the developmental capacity to grasp concepts of health and illness, including HIV, and that in the absence of parent-led communication and education about these issues, HIV-exposed children may be at increased risk of psychological and social problems. The Amagugu intervention is a six-session home-based intervention, delivered by lay counselors, which aims to increase parenting capacity to disclose their HIV status and offer health education to their primary school-aged children. The intervention includes information and activities on disclosure, health care engagement, and custody planning. An uncontrolled pre-post-evaluation study with 281 families showed that the intervention was feasible, acceptable, and effective in increasing maternal disclosure. The aim of this paper is to describe the conceptual model of the Amagugu intervention, as developed post-evaluation, showing the proposed pathways of risk that Amagugu aims to disrupt through its intervention targets, mechanisms, and activities; and to present a summary of results from the large-scale evaluation study of Amagugu to demonstrate the acceptability and feasibility of the intervention model. This relatively low-intensity home-based intervention led to: increased HIV disclosure to children, improvements in mental health for mother and child, and improved health care engagement and custody planning for the child. The

  10. Interaction of HIV-1 Gag protein components with single DNA molecules

    NASA Astrophysics Data System (ADS)

    Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

    2003-03-01

    The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

  11. DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

    PubMed

    Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

    2016-11-15

    Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. PMID:27295571

  12. Increased Cardiovascular Disease Risk In The HIV-Positive Population on ART: Potential Role of HIV-Nef and Tat

    PubMed Central

    Wang, Ting; Yi, Ru; Green, Linden Ann; Chelvanambi, Sarvesh; Seimetz, Michael; Clauss, Matthias

    2016-01-01

    With effective antiretroviral therapy (ART), many HIV-infected people die of diseases other than acquired immune deficiency syndrome (AIDS). In particular, coronary artery disease has emerged as one of most critical complications of HIV infection and a major cause of morbidity and mortality. Although reportedly antiretroviral combination therapy itself may accelerate atherosclerosis by enhancing dyslipidemia, most recent epidemiological studies support the notion that HIV infection itself contributes to cardiovascular disease. However, it is still a mystery how the virus can contribute to cardiovascular disease development even while suppressed by ARTs. This review discusses the current understanding of interactions between HIV infection and cardiovascular diseases in both clinical and experimental studies with special focus on those viral proteins which are still produced by HIV. This will help infectious disease/vascular biology experts to gain insights into the pathophysiological mechanisms of HIV associated cardiovascular disease and new trends to treat and prevent cardiovascular disease in the HIV infected population. PMID:26233281

  13. Increased utilisation of PEPFAR-supported laboratory services by non-HIV patents in Tanzania

    PubMed Central

    McNairy, Margaret L.; Gwynn, Charon; Rabkin, Miriam; Antelman, Gretchen; Wu, Yingfeng; Alemayehu, Bereket; Lim, Travis; Imtiaz, Rubina; Mosha, Fausta; Mwasekaga, Michael; Othman, Asha A.; Justman, Jessica

    2016-01-01

    Background It is unknown to what extent the non-HIV population utilises laboratories supported by the President's Emergency Plan for AIDS Relief (PEPFAR). Objectives We aimed to describe the number and proportion of laboratory tests performed in 2009 and 2011 for patients referred from HIV and non-HIV services (NHSs) in a convenience sample collected from 127 laboratories supported by PEPFAR in Tanzania. We then compared changes in the proportions of tests performed for patients referred from NHSs in 2009 vs 2011. Methods Haematology, chemistry, tuberculosis and syphilis test data were collected from available laboratory registers. Referral sources, including HIV services, NHSs, or lack of a documented referral source, were recorded. A generalised linear mixed model reported the odds that a test was from a NHS. Results A total of 94 132 tests from 94 laboratories in 2009 and 157 343 tests from 101 laboratories in 2011 were recorded. Half of all tests lacked a documented referral source. Tests from NHSs constituted 42% (66 084) of all tests in 2011, compared with 31% (29 181) in 2009. A test in 2011 was twice as likely to have been referred from a NHS as in 2009 (adjusted odds ratio: 2.0 [95% confidence interval: 2.0–2.1]). Conclusion Between 2009 and 2011, the number and proportion of tests from NHSs increased across all types of test. This finding may reflect increased documentation of NHS referrals or that the laboratory scale-up originally intended to service the HIV-positive population in Tanzania may be associated with a ‘spillover effect’ amongst the general population. PMID:26962475

  14. Increasing trends in primary NNRTI resistance among newly HIV-1-diagnosed individuals in Buenos Aires, Argentina

    PubMed Central

    Rodriguez-Rodrigues, Nahuel; Duran, Adriana; Bouzas, María Belen; Zapiola, Ines; Vila, Marcelo; Indyk, Debbie; Bissio, Emiliano; Salomon, Horacio; Dilernia, Dario A

    2013-01-01

    Objective Our objective was to estimate primary resistance in an urban setting in a developing country characterized by high antiretroviral (ARV) coverage over the diagnosed population and also by an important proportion of undiagnosed individuals, in order to determine whether any change in primary resistance occurred in the past five years. Design We carried out a multi-site resistance surveillance study according to WHO HIV resistance guidelines, using a weighted sampling technique based on annual HIV case reports per site. Methods Blood samples were collected from 197 drug-naive HIV-1-infected individuals diagnosed between March 2010 and August 2011 at 20 HIV voluntary counselling and testing centres in Buenos Aires. Clinical records of enrolled patients at the time of diagnosis were compiled. Viral load and CD4 counts were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbour-joining (NJ) trees and bootscanning analysis. Results We found that 12 (7.9%) of the 152 successfully sequenced samples harboured primary resistance mutations, of which K103N and G190A were the most prevalent. Non-nucleoside reverse transcriptase inhibitors (NNRTI) resistance mutations were largely the most prevalent (5.9%), accounting for 75% of all primary resistance and exhibiting a significant increase (p=0.0072) in prevalence during the past 10 years as compared to our previous study performed in 1997–2000 and in 2003–2005. Nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor primary resistance were low and similar to the one previously reported. Conclusions Levels of primary NNRTI resistance in Buenos Aires appear to be increasing in the context of a sustained ARV coverage and a high proportion of undiagnosed HIV-positive individuals. PMID:24093951

  15. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication.

    PubMed

    Berg, Randi K; Rahbek, Stine H; Kofod-Olsen, Emil; Holm, Christian K; Melchjorsen, Jesper; Jensen, David G; Hansen, Anne Louise; Jørgensen, Louise B; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S; Paludan, Søren R; Jakobsen, Martin R; Mogensen, Trine H

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.

  16. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Cancer.gov

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  17. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

    PubMed Central

    Richardson, C. D.; Ray, G. J.; Bray, N. L.; Corn, J. E.

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  18. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

    PubMed

    Richardson, C D; Ray, G J; Bray, N L; Corn, J E

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  19. Structural Studies of the HIV-1 Integrase Protein: Compound Screening and Characterization of a DNA-Binding Inhibitor

    PubMed Central

    Hassounah, Said; Mesplède, Thibault; Wainberg, Mark A.

    2015-01-01

    Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance. PMID:26046987

  20. Increased lung epithelial permeability in HIV-infected patients with isolated cytotoxic T-lymphocytic alveolitis

    SciTech Connect

    Meignan, M.; Guillon, J.M.; Denis, M.; Joly, P.; Rosso, J.; Carette, M.F.; Baud, L.; Parquin, F.; Plata, F.; Debre, P. )

    1990-05-01

    HIV-related lymphocytic alveolitis is common in HIV-seropositive patients without lung infection or tumor. In some of them a fraction of alveolar lymphocytes are HIV-specific cytotoxic T-lymphocytes (CTL) bearing the CD8 and D44 cell surface markers and capable of killing HIV-infected alveolar macrophages. In order to evaluate the in vivo effect of these CTL on lung function, we measured the pulmonary clearance of aerosolized 99mTc-diethylene triamine penta-acetate (DTPA-CI) on 24 occasions in 22 patients with lymphocytic alveolitis. DTPA-CI has been selected as a highly sensitive test to detect injury of the lung epithelium. In 13 of the patients, we found a high DTPA-CI of 4.56 +/- 2.54%.min-1 (mean +/- SD), suggesting an increase of the epithelial permeability. The lymphocytic alveolitis was then characterized by a high cellularity, a high proportion of lymphocytes (59 +/- 18%), mainly composed of CD8+D44+ T-lymphocytes (149 +/- 109 cells/mm3), which spontaneously exhibited a cytolytic activity against the autologous alveolar macrophages in a standard 51Cr release assay. In the remaining 11 patients, DTPA-CI was normal (less than 1.78%.min-1), lymphocytic alveolitis being characterized by a low number or an absence of CD8+D44+ alveolar lymphocytes (9 +/- 13 cells/mm3) with no significant cytolytic activity. In the whole group, a significant correlation (r = 0.74, p = 0.0004) was found between the DTPA-CI and the number of CD8+D44+ lymphocytes and their cytotoxic activity against alveolar macrophages. Altogether, these results suggest that an injury of the lung epithelium could result from a HIV-specific CTL-induced immunologic conflict.

  1. Neisseria gonorrhoeae-Induced Human Defensins 5 and 6 Increase HIV Infectivity: Role in Enhanced Transmission1

    PubMed Central

    Klotman, Mary E.; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A.; Lu, Wuyuan; Porter, Edith; Chang, Theresa L.

    2011-01-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4+ T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection. PMID:18424739

  2. Brief Intervention to Increase Emergency Department Uptake of Combined Rapid HIV and Hepatitis C Screening Among a Drug Misusing Population

    PubMed Central

    Merchant, Roland C.; Baird, Janette R.; Liu, Tao; Taylor, Lynn E.; Montague, Brian T.; Nirenberg, Ted D.

    2014-01-01

    Objectives In this study, Increasing Viral Testing in the Emergency Department (InVITED), the authors investigated if a brief intervention about human immunodeficiency virus (HIV) and hepatitis C virus (HCV) risk-taking behaviors and drug use and misuse in addition to a self-administered risk assessment, as compared to a self-administered risk assessment alone, increased uptake of combined screening for HIV and HCV, self-perception of HIV/HCV risk, and beliefs and opinions on HIV/HCV screening. Methods InVITED was a randomized, controlled trial conducted at two urban emergency departments (EDs) from February 2011 to March 2012. ED patients who self-reported drug use within the past three months were invited to enroll. Drug misuse severity and need for a brief or more intensive intervention was assessed using the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST). Participants were randomly assigned to one of two study arms: a self-administered HIV/HCV risk assessment alone (control arm), or the assessment plus a brief intervention about their drug misuse and screening for HIV/HCV (intervention arm). Beliefs on the value of combined HIV/HCV screening, self-perception of HIV/HCV risk, and opinions on HIV/HCV screening in the ED were measured in both study arms before the HIV/HCV risk assessment (pre), after the assessment in the control arm, and after the brief intervention in the intervention arm (post). Participants in both study arms were offered free combined rapid HIV/HCV screening. Uptake of screening was compared by study arm. Multivariable logistic regression models were used to evaluate factors related to uptake of screening. Results Of the 395 participants in the study, the median age was 28 years (IQR 23 to 38 years), 44.8% were female, 82.3% had ever been tested for HIV, and 67.3% had ever been tested for HCV. Uptake of combined rapid HIV/HCV screening was nearly identical by study arm (64.5% vs. 65.2%; Δ = −0.7%; 95% CI = −10.1% to 8

  3. 31P NMR conformational studies of non-palindromic DNA duplexes related to HIV-1 enhancer

    NASA Astrophysics Data System (ADS)

    Tisne, C.; Simenel, C.; Hantz, E.; Delepierre, M.

    1998-02-01

    Assignment of all 31P resonances of 16 base-pair DNA duplexes, 5' d(CTGGGGACTTTCCAGG)3' 5' d(CCTGGAAAGTCCCCAG)3', related to the DNA kappaB site of the HIV-1 LTR together with a mutated sequence 5' d(CTGCTCACTTTCCAGG)3' 5' d(CCTGGAAAGTGAGCAG)3' was determined by 2D heteronuclear inverse NMR spectroscopy (HSQC-TOCSY and heteronuclear COSY). ^3JH{3'}-P coupling constants for most of the phosphates of the mutated oligomer were obtained using heteronuclear selective COSY. L'attribution des résonances 31P du fragment d'ADN de seize paires de base 5' d(CTGGGGACTTTCCAGG)3' 5' d(CCTGGAAAGTCCCCAG)3' (16N) correspondant au site kappaB du LTR du HIV-1 et de la séquence mutée 5' d(CTGCTCACTTTCCAGG)3' 5' d(CCTGGAAAGTGAGCAG)3' (16M) a été déterminée à l'aide de méthodes 2D hétéronucléaires à détection indirecte (HSQC-TOCSY et COSY hétéronucléaires). Les constantes de couplage ^3JH{3'}-P ont été mesurées pour la plupart des phosphates de 16 M à l'aide d'expérience COSY hétéronucléaires sélectives.

  4. Use of a rapid and simple method to extract proviral DNA in the identification of HIV-1 by PCR.

    PubMed

    Tagliaferro, L; Corbelli, M; Maietta, G; Pellegrino, V; Pignatelli, P

    1995-07-01

    DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV-1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity.

  5. Induction of human immunodeficiency virus type-1-specific immunity with a novel gene transport unit (GTU)-MultiHIV DNA vaccine.

    PubMed

    Blazevic, Vesna; Männik, Andres; Malm, Maria; Sikut, Rein; Valtavaara, Minna; Toots, Urve; Ustav, Mart; Krohn, Kai

    2006-07-01

    A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria. PMID:16831091

  6. Structural factors that increase HIV/STI vulnerability among indigenous people in the Peruvian amazon.

    PubMed

    Orellana, E Roberto; Alva, Isaac E; Cárcamo, Cesar P; García, Patricia J

    2013-09-01

    We examined structural factors-social, political, economic, and environmental-that increase vulnerability to HIV among indigenous people in the Peruvian Amazon. Indigenous adults belonging to 12 different ethnic groups were purposively recruited in four Amazonian river ports and 16 indigenous villages. Qualitative data revealed a complex set of structural factors that give rise to environments of risk where health is constantly challenged. Ferryboats that cross Amazonian rivers are settings where unprotected sex-including transactional sex between passengers and boat crew and commercial sex work-often take place. Population mobility and mixing also occurs in settings like the river docks, mining sites, and other resource extraction camps, where heavy drinking and unprotected sex work are common. Multilevel, combination prevention strategies that integrate empirically based interventions with indigenous knowledge are urgently needed, not only to reduce vulnerability to HIV transmission, but also to eliminate the structural determinants of indigenous people's health.

  7. Structural Factors That Increase HIV/STI Vulnerability Among Indigenous People in the Peruvian Amazon

    PubMed Central

    Orellana, E. Roberto; Alva, Isaac E.; Cárcamo, Cesar P.; García, Patricia J.

    2015-01-01

    We examined structural factors—social, political, economic, and environmental—that increase vulnerability to HIV among indigenous people in the Peruvian Amazon. Indigenous adults belonging to 12 different ethnic groups were purposively recruited in four Amazonian river ports and 16 indigenous villages. Qualitative data revealed a complex set of structural factors that give rise to environments of risk where health is constantly challenged. Ferryboats that cross Amazonian rivers are settings where unprotected sex—including transactional sex between passengers and boat crew and commercial sex work—often take place. Population mobility and mixing also occurs in settings like the river docks, mining sites, and other resource extraction camps, where heavy drinking and unprotected sex work are common. Multilevel, combination prevention strategies that integrate empirically based interventions with indigenous knowledge are urgently needed, not only to reduce vulnerability to HIV transmission, but also to eliminate the structural determinants of indigenous people’s health. PMID:23925407

  8. Structural factors that increase HIV/STI vulnerability among indigenous people in the Peruvian amazon.

    PubMed

    Orellana, E Roberto; Alva, Isaac E; Cárcamo, Cesar P; García, Patricia J

    2013-09-01

    We examined structural factors-social, political, economic, and environmental-that increase vulnerability to HIV among indigenous people in the Peruvian Amazon. Indigenous adults belonging to 12 different ethnic groups were purposively recruited in four Amazonian river ports and 16 indigenous villages. Qualitative data revealed a complex set of structural factors that give rise to environments of risk where health is constantly challenged. Ferryboats that cross Amazonian rivers are settings where unprotected sex-including transactional sex between passengers and boat crew and commercial sex work-often take place. Population mobility and mixing also occurs in settings like the river docks, mining sites, and other resource extraction camps, where heavy drinking and unprotected sex work are common. Multilevel, combination prevention strategies that integrate empirically based interventions with indigenous knowledge are urgently needed, not only to reduce vulnerability to HIV transmission, but also to eliminate the structural determinants of indigenous people's health. PMID:23925407

  9. Ethnicity and Smoking-Associated DNA Methylation Changes at HIV Co-Receptor GPR15

    PubMed Central

    Dogan, Meeshanthini V.; Xiang, Jinhua; Beach, Steven R. H.; Cutrona, Carolyn; Gibbons, Frederick X.; Simons, Ronald L.; Brody, Gene H.; Stapleton, Jack T.; Philibert, Robert A.

    2015-01-01

    Smoking is associated with poorer health outcomes for both African and European Americans. In order to better understand whether ethnic-specific genetic variation may underlie some of these differences, we compared the smoking-associated genome-wide methylation signatures of African Americans with those of European Americans, and followed up this analysis with a focused examination of the most ethnically divergent locus, cg19859270, at the GPR15 gene. We examined the association of methylation at this locus to the rs2230344 SNP and GPR15 gene and protein expression. Consistent with prior analyses, AHRR residue cg05575921 was the most differentially methylated residue in both African Americans and European Americans. However, the second most differentially methylated locus in African Americans, cg19859270, was only modestly differentially methylated in European Americans. Interrogation of the methylation status of this CpG residue found in GPR15, a chemokine receptor involved in HIV pathogenesis, showed a significant interaction of ethnicity with smoking as well as a marginal effect of genotype at rs2230344, a neighboring non-synonymous SNP, but only among African Americans. Gene and protein expression analyses showed that demethylation at cg19859270 was associated with an increase in both mRNA and protein levels. Since GPR15 is involved in the early stages of viral replication for some HIV-1 and HIV-2 isolates, and the prevalence of HIV is increased in African Americans and smokers, these data support a possible role for GPR15 in the ethnically dependent differential prevalence of HIV. PMID:26441693

  10. Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats.

    PubMed

    Banerjee, Atrayee; Abdelmegeed, Mohamed A; Jang, Sehwan; Song, Byoung-Joon

    2015-01-01

    The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.

  11. Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats.

    PubMed

    Banerjee, Atrayee; Abdelmegeed, Mohamed A; Jang, Sehwan; Song, Byoung-Joon

    2015-01-01

    The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART. PMID:26484872

  12. Why Are the Benefits of Increased Resources Not Impacting the Risk of HIV Infection for High SES Women in Cameroon?

    PubMed Central

    Mumah, Joyce N.; Jackson-Smith, Douglas

    2014-01-01

    Background Despite evidence of a positive SES-HIV gradient in some SSA countries, researchers and policy-makers frequently assume that a range of protective interventions – increasing awareness of mechanisms of HIV transmission, techniques for prevention, greater access to health care facilities, and greater availability of condoms – will reduce the likelihood of contracting HIV, even among higher SES populations. We therefore explore the relationships between SES and these intervening behaviors to illuminate the complex factors that link SES and HIV among women in Cameroon. Methods We use bivariate and multivariate statistical analysis to examine patterns among the 5, 155 women aged 15–49 who participated in the 2004 CDHS. Results The results show a strong pattern where higher SES women have greater access to and use of health care facilities, higher levels of condom use, more HIV knowledge, and command higher power within their relationships, yet also have higher rates of HIV. These traditionally protective factors appear to be offset by riskier sexual behaviors on the part of women with increased resources, most notably longer years of premarital sexual experience, multiple partners in last 12 months, and sexual encounters outside of relationship. Multivariate analyses suggests net of the effect of other factors, women who command higher decision-making power, have greater access to health care, more negative attitudes toward wife beating, longer years of premarital sexual exposure, and partners with professional/white collar jobs (characteristics associated with rising SES) had higher odds of testing positive for HIV. Conclusion Results show that higher riskier sexual practices on part of high SES women offset benefits that may have accrued from their increased access to resources. The results suggest that traditional approaches to HIV prevention which rely on poverty reduction, improving access to health care, improving HIV knowledge, and boosting women

  13. Oral supplementation with whey proteins increases plasma glutathione levels of HIV-infected patients.

    PubMed

    Micke, P; Beeh, K M; Schlaak, J F; Buhl, R

    2001-02-01

    HIV infection is characterized by an enhanced oxidant burden and a systemic deficiency of the tripeptide glutathione (GSH), a major antioxidant. The semi-essential amino acid cysteine is the main source of the free sulfhydryl group of GSH and limits its synthesis. Therefore, different strategies to supplement cysteine supply have been suggested to increase glutathione levels in HIV-infected individuals. The aim of this study was to evaluate the effect of oral supplementation with two different cysteine-rich whey protein formulas on plasma GSH levels and parameters of oxidative stress and immune status in HIV-infected patients. In a prospective double blind clinical trial, 30 patients (25 male, 5 female; mean age (+/- SD) 42 +/- 9.8 years) with stable HIV infection (221 +/- 102 CD4 + lymphocytes L-1) were randomized to a supplemental diet with a daily dose of 45 g whey proteins of either Protectamin (Fresenius Kabi, Bad Hamburg, Germany) or Immunocal (Immunotec, Vandreuil, Canada) for two weeks. Plasma concentrations of total, reduced and oxidized GSH, superoxide anion (O2-) release by blood mononuclear cells, plasma levels of TNF-alpha and interleukins 2 and 12 were quantified with standard methods at baseline and after therapy. Pre-therapy, plasma GSH levels (Protectamin: 1.92 +/- 0.6 microM; Immunocal: 1.98 +/- 0.9 microM) were less than normal (2.64 +/- 0.7 microM, P = 0.03). Following two weeks of oral supplementation with whey proteins, plasma GSH levels increased in the Protectamin group by 44 +/- 56% (2.79 +/- 1.2 microM, P = 0.004) while the difference in the Immunocal group did not reach significance (+ 24.5 +/- 59%, 2.51 +/- 1.48 microM, P = 0.43). Spontaneous O2- release by blood mononuclear cells was stable (20.1 +/- 14.2 vs. 22.6 +/- 16.1 nmol h-1 10-6 cells, P = 0.52) whereas PMA-induced O2- release decreased in the Protectamin group (53.7 +/- 19 vs. 39.8 +/- 18 nmol h-1 10-6 cells, P = 0.04). Plasma concentrations of TNF-alpha and interleukins 2 and

  14. Increasing Access to Oral Health Care for People Living with HIV/AIDS in Rural Oregon

    PubMed Central

    Jones, Jill; Mofidi, Mahyar; Bednarsh, Helene; Gambrell, Alan; Tobias, Carol R.

    2012-01-01

    Access to oral health care for people living with HIV/AIDS is a severe problem. This article describes the design and impact of an Innovations in Oral Health Care Initiative program, funded through the Health Resources and Services Administration HIV/AIDS Bureau's Special Projects of National Significance (SPNS) program, that expanded oral health-care services for these individuals in rural Oregon. From April 2007 to August 2010, 473 patients received dental care (exceeding the target goal of 410 patients) and 153 dental hygiene students were trained to deliver oral health care to HIV-positive patients. The proportion of patients receiving oral health care increased from 10% to 65%, while the no-show rate declined from 40% to 10%. Key implementation components were leveraging SPNS funding and services to create an integrated delivery system, collaborations that resulted in improved service delivery systems, using dental hygiene students to deliver oral health care, enhanced care coordination through the services of a dental case manager, and program capacity to adjust to unanticipated needs. PMID:22547878

  15. Incident Major Depressive Episodes increase the severity and risk of apathy in HIV infection

    PubMed Central

    Kamat, Rujvi; Cattie, Jordan E.; Marcotte, Thomas D.; Woods, Steven Paul; Franklin, Donald R.; Corkran, Stephanie H.; Ellis, Ronald J.; Grant, Igor; Heaton, Robert K.

    2015-01-01

    Apathy and depression are inter-related yet separable and prevalent neuropsychiatric disturbances in persons infected with HIV. In the present study of 225 HIV+ persons, we investigated the role of an incident depressive episode in changes in apathy. Participants completed the apathy subscale of the Frontal Systems Behavior Scale during a detailed neuropsychiatric and neuromedical evaluation at visit 1 and again at approximately a 14 month follow-up. The Composite International Diagnostic Interview was used to obtain diagnoses of a new major depressive disorder. At their follow-up visit, participants were classified into four groups depending on their visit 1 elevation in apathy and new major depressive episode (MDE) status. Apathetic participants at baseline with a new MDE (n=23) were at risk for continued, clinically elevated apathy at follow-up, although severity of symptoms did not increase. Of the 144 participants without clinically elevated apathy at visit 1, those who developed a new MDE (n=16) had greater apathy symptomatology at follow-up than those without MDE. These findings suggest that HIV+ individuals, who do not as yet present with elevated apathy, may be at greater risk of elevated psychiatric distress should they experience a new/recurrent depressive episode. Thus, in the context of previous findings, it appears that although apathy and depression are separable constructs, they interact such that a new depressive episode is a risk factor for incident apathy. PMID:25679203

  16. Prevalence and risk factors for oral DNA tumor viruses in HIV-infected youth.

    PubMed

    Kahn, Jessica A; Rudy, Bret J; Xu, Jiahong; Kapogiannis, Bill; Secord, Elizabeth; Gillison, Maura

    2016-11-01

    Human papillomavirus (HPV), Epstein-Barr virus (EBV), and Kaposi sarcoma-associated herpes virus (KSHV) may promote oral cancers, especially among immunosuppressed individuals. The aims of this study were to examine whether demographic characteristics, medical history, sexual behaviors, substance use, CD4+ T-cell count, HIV viral load, and HPV vaccination were associated with HPV, EBV, and KSHV infection and viral load. Multivariable modeling using logistic or linear regression examined associations between independent variables and infection or viral load, respectively. Among 272 HIV-infected 12-24-year-old youth, 19.5% were positive for oral HPV, 88.2% for EBV, and 11.8% for KSHV. In multivariable models, recent marijuana use (OR 1.97, 95%CI 1.02-3.82) and lower CD4+ T-cell count (<350 vs. ≥350 cells/mm(3) : OR 1.92, 95%CI 1.003-3.69) were associated with HPV infection; lifetime tobacco use (estimated coefficient [EC] 1.55, standard error [SE] 0.53, P = 0.0052) with HPV viral load; recent tobacco use (OR 2.90, 95%CI 1.06-7.97), and higher HIV viral load (>400 vs. <400 copies/ml: OR 3.98, 95%CI 1.84-8.74) with EBV infection; Black versus White race (EC 1.18, SE 0.37, P = 0.0023), and lower CD4+ T-cell count (EC 0.70, SE 0.28, P = 0.017) with EBV viral load, male versus female gender (OR 10, 95%CI 1.32-100) with KSHV infection, and younger age at HIV diagnosis (1-14 vs. 18-20 years: EC 0.33, SE 0.16, P = 0.049; 15-17 vs. 18-20 years: EC 0.35, SE 0.13, P = 0.0099) with KSHV viral load. In conclusion, substance use and immunosuppression are associated with oral DNA tumor viruses in HIV-infected youth. J. Med. Virol. 88:1944-1952, 2016. © 2016 Wiley Periodicals, Inc. PMID:27096166

  17. Prevalence and risk factors for oral DNA tumor viruses in HIV-infected youth.

    PubMed

    Kahn, Jessica A; Rudy, Bret J; Xu, Jiahong; Kapogiannis, Bill; Secord, Elizabeth; Gillison, Maura

    2016-11-01

    Human papillomavirus (HPV), Epstein-Barr virus (EBV), and Kaposi sarcoma-associated herpes virus (KSHV) may promote oral cancers, especially among immunosuppressed individuals. The aims of this study were to examine whether demographic characteristics, medical history, sexual behaviors, substance use, CD4+ T-cell count, HIV viral load, and HPV vaccination were associated with HPV, EBV, and KSHV infection and viral load. Multivariable modeling using logistic or linear regression examined associations between independent variables and infection or viral load, respectively. Among 272 HIV-infected 12-24-year-old youth, 19.5% were positive for oral HPV, 88.2% for EBV, and 11.8% for KSHV. In multivariable models, recent marijuana use (OR 1.97, 95%CI 1.02-3.82) and lower CD4+ T-cell count (<350 vs. ≥350 cells/mm(3) : OR 1.92, 95%CI 1.003-3.69) were associated with HPV infection; lifetime tobacco use (estimated coefficient [EC] 1.55, standard error [SE] 0.53, P = 0.0052) with HPV viral load; recent tobacco use (OR 2.90, 95%CI 1.06-7.97), and higher HIV viral load (>400 vs. <400 copies/ml: OR 3.98, 95%CI 1.84-8.74) with EBV infection; Black versus White race (EC 1.18, SE 0.37, P = 0.0023), and lower CD4+ T-cell count (EC 0.70, SE 0.28, P = 0.017) with EBV viral load, male versus female gender (OR 10, 95%CI 1.32-100) with KSHV infection, and younger age at HIV diagnosis (1-14 vs. 18-20 years: EC 0.33, SE 0.16, P = 0.049; 15-17 vs. 18-20 years: EC 0.35, SE 0.13, P = 0.0099) with KSHV viral load. In conclusion, substance use and immunosuppression are associated with oral DNA tumor viruses in HIV-infected youth. J. Med. Virol. 88:1944-1952, 2016. © 2016 Wiley Periodicals, Inc.

  18. DNA Labeling Generates a Unique Amplification Probe for Sensitive Photoelectrochemical Immunoassay of HIV-1 p24 Antigen.

    PubMed

    Zhao, Wei-Wei; Han, Ying-Mei; Zhu, Yuan-Cheng; Zhang, Nan; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-06-01

    Photoelectrochemical (PEC) immunoassay is an attractive methodology as it allows for an elegant and sensitive protein assay. However, advanced PEC immunoassay remains challenging and the established amplifications rely almost exclusively on the labeling of various enzymes, which usually suffer the inferior stabilities. Here we report the development and validation of the DNA labeling that leads to a unique amplification probe for the sensitive PEC immunoassay of HIV-1 capsid protein, p24 antigen, an important biomarker of human immune deficiency virus (HIV). Following the sandwich immunobinding, the DNA tags could be released and the subsequent dipurinization of the oligonucleotide strands enables the easy oxidation of free nucleobases at a CdTe quantum dots (QDs) modified ITO transducer. Such DNA tags induced PEC amplification and readout permits the exquisite assay of HIV-1 p24 antigen with high sensitivity. As compared to the existing method of enzymatic labeling, the easy preparation and stability of these labels make them very suitable for PEC amplification. Another merit of this method is that it separates the immunobinding from the PEC transducer, which eliminates the commonly existing affection during the biorecognition processes. This work paves a new route for the PEC immunoassay of HIV-1 p24 antigen and provides a general format for the PEC biomolecular detection by means of the DNA labeling.

  19. Anti-human immunodeficiency virus 1 (HIV-1) activities of 3-deazaadenosine analogs: increased potency against 3'-azido-3'-deoxythymidine-resistant HIV-1 strains.

    PubMed Central

    Mayers, D L; Mikovits, J A; Joshi, B; Hewlett, I K; Estrada, J S; Wolfe, A D; Garcia, G E; Doctor, B P; Burke, D S; Gordon, R K

    1995-01-01

    3-Deazaadenosine (DZA), 3-deaza-(+/-)-aristeromycin (DZAri), and 3-deazaneplanocin A (DZNep) are powerful modulators of cellular processes. When tested against H9 cells infected acutely with two different strains of human immunodeficiency virus 1 (HIV-1) and in the chronically infected monocytoid cell lines U1 and THP-1, the 3-deazanucleosides caused a marked reduction in p24 antigen production. Similar reductions in p24 antigen were seen in phytohemagglutinin-stimulated peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Strikingly, in comparing the therapeutic indices between the paired pre- and post-3'-azido-3'-deoxythymidine (AZT) treatment HIV-1 isolates, DZNep and neplanocin A showed an increase of 3- to 18-fold in their potency against AZT-resistant HIV-1 isolates. In H9 cells treated with DZNep and DZAri, the formation of triphosphate nucleotides of DZNep and DZAri was observed. The mode of action of DZNep and DZAri appears complex, at least in part, at the level of infectivity as shown by decreases in syncytia formation in HIV-1-infected H9 cells and at the level of transcription as both drugs inhibited the expression of basal or tat-induced HIV-1 long terminal repeat chloramphenicol acetyltransferase activity in stably transfected cell lines. Since DZNep induced in H9 cells a rapid expression of nuclear binding factors that recognize the AP-1 transcription site, the anti-HIV-1 activity of the DZA analogs could partly be the induction of critical factors in the host cells. Thus, the 3-deazanucleoside drugs belong to an unusual class of anti-HIV-1 drugs, which may have therapeutic potential, in particular against AZT-resistant strains. Images Fig. 4 Fig. 5 PMID:7816820

  20. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

    PubMed

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

  1. Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap.

    PubMed

    Gray, Glenda E; Mayer, Kenneth H; Elizaga, Marnie L; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C; Sato, Alicia; Gu, Niya; Tomaras, Georgia D; Tucker, Timothy; Barnett, Susan W; Mkhize, Nonhlanhla N; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

    2016-06-01

    A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 10(9) PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4(+) T-cell and CD8(+) T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4(+) T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4(+) T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.).

  2. Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap.

    PubMed

    Gray, Glenda E; Mayer, Kenneth H; Elizaga, Marnie L; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C; Sato, Alicia; Gu, Niya; Tomaras, Georgia D; Tucker, Timothy; Barnett, Susan W; Mkhize, Nonhlanhla N; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

    2016-06-01

    A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 10(9) PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4(+) T-cell and CD8(+) T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4(+) T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4(+) T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.). PMID:27098021

  3. Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap

    PubMed Central

    Mayer, Kenneth H.; Elizaga, Marnie L.; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C.; Sato, Alicia; Gu, Niya; Tomaras, Georgia D.; Tucker, Timothy; Barnett, Susan W.; Mkhize, Nonhlanhla N.; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

    2016-01-01

    A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 109 PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4+ T-cell and CD8+ T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4+ T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4+ T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.) PMID:27098021

  4. The Affordable Care Act may increase the number of people getting tested for HIV by nearly 500,000 by 2017.

    PubMed

    Wagner, Zachary; Wu, Yanyu; Sood, Neeraj

    2014-03-01

    People are much less likely to engage in risky sexual behavior if they know that they are HIV-positive. Unfortunately, more than 18 percent of people living with HIV/AIDS in the United States are unaware of their HIV status, and about half of new HIV infections are transmitted from that "HIV unaware" population. For these reasons, HIV testing is at the forefront of HIV prevention strategies in the United States. The Affordable Care Act (ACA) may support these strategies, since gaining coverage increases the likelihood of being tested for HIV. We modeled the impact of the ACA on HIV testing, diagnoses, and awareness of being HIV-positive, assuming that only the eighteen states (and the District of Columbia) that had committed to expand Medicaid as of July 2013 did expand the program. We found that the ACA will result in an additional 466,153 people's being tested for HIV and 2,598 new diagnoses of HIV by 2017. Among people living with HIV/AIDS who gain insurance through the ACA, the share of the HIV unaware will decline by 22 percent. The impact on both HIV testing and new diagnoses would be nearly 30 percent larger if all fifty states expanded Medicaid. Policy makers should consider such epidemiological benefits when analyzing insurance expansion policies.

  5. Increasing polymer diffusivity by increasing the contour length: The surprising effect of YOYO-1 on DNA dynamics

    NASA Astrophysics Data System (ADS)

    Shin, Seunghwan; Dorfman, Kevin; Cheng, Xiang

    2015-03-01

    Double-stranded DNA (dsDNA) labeled with cyanine dyes such as YOYO-1 has been extensively used as a model to study equilibrium and dynamic properties of semiflexible polyelectrolytes. The ability to directly visualize the polymer dynamics is an attractive feature of these experiments, but positively charged cyanine dyes affect the physical properties of dsDNA, distorting the double helix and counterbalancing the intrinsic negative charge of the backbone. A variety of studies have been conducted to reveal the effect of the dye on the contour length and the persistence length of dsDNA. However, fewer efforts have been made to directly quantify the effect of dye on the diffusion behavior of dsDNA. In order to resolve this issue, we measured the in-plane diffusion coefficient of unconfined dsDNA using confocal microscopy. Although there is widespread consensus that intercalation increases the contour length of dsDNA, we find that increasing the dye:base pair ratio for YOYO-1 actually enhances the diffusion of dsDNA. This enhancement is more significant at lower ionic strengths, which implies that the increase in the diffusion coefficient by dye-DNA intercalation is mainly due to a reduction of excluded volume effect resulting from charge neutralization on the backbone.

  6. Using Social Media to Increase HIV Testing Among Gay and Bisexual Men, Other Men Who Have Sex With Men, and Transgender Persons: Outcomes From a Randomized Community Trial.

    PubMed

    Rhodes, Scott D; McCoy, Thomas P; Tanner, Amanda E; Stowers, Jason; Bachmann, Laura H; Nguyen, Annie L; Ross, Michael W

    2016-06-01

    We tested an intervention designed to increase human immunodeficiency virus (HIV) testing among men who have sex with men and transgender persons within existing and commonly used social media. At follow-up, intervention communities had significantly higher past 12-month HIV testing than the comparison communities. Findings suggest that promoting HIV testing via social media can increase testing. PMID:26980878

  7. Using Social Media to Increase HIV Testing Among Gay and Bisexual Men, Other Men Who Have Sex With Men, and Transgender Persons: Outcomes From a Randomized Community Trial.

    PubMed

    Rhodes, Scott D; McCoy, Thomas P; Tanner, Amanda E; Stowers, Jason; Bachmann, Laura H; Nguyen, Annie L; Ross, Michael W

    2016-06-01

    We tested an intervention designed to increase human immunodeficiency virus (HIV) testing among men who have sex with men and transgender persons within existing and commonly used social media. At follow-up, intervention communities had significantly higher past 12-month HIV testing than the comparison communities. Findings suggest that promoting HIV testing via social media can increase testing.

  8. APOBEC3H Haplotypes and HIV-1 Pro-Viral vif DNA Sequence Diversity in Early Untreated HIV-1 Infection

    PubMed Central

    Gourraud, PA; Karaouni, A; Woo, JM; Schmidt, T; Oksenberg, JR; Hecht, FM; Liegler, TJ; Barbour, JD

    2011-01-01

    We examined single nucleotide polymorphisms (SNP) in the APOBEC3 locus on chromosome 22, paired to population sequences of pro-viral HIV-1 vif of peripheral blood mononuclear cells (PBMC), from 96 recently HIV-1 infected treatment naïve adults. We found evidence for the existence of an APOBEC3H linkage disequilibrium (LD) block associated with variation in GA->AA, or APOBEC3F signature, sequence changes in pro-viral HIV-1 vif sequence (top significant 10 SNPs with a top-significant p=4.8×10−3). We identified a common 5 position risk haplotype distal to APOBEC3H (A3Hrh). These markers were in high LD (D′ = 1; r2=0.98) to a previously described A3H ‘RED’ haplotype containing a variant (E121) with enhanced susceptibility to HIV-1 Vif (Zhen et al 2009 [1]). This association is confirmed by a haplotype analysis: Homozygote carriers of the A3Hrh had lower GA->AA (A3F) sequence editing on pro-viral HIV-1 vif sequence (p = 0.01), and lower HIV-1 RNA levels over time during early, untreated HIV-1 infection, (p = 0.015 mixed effects model). This effect may be due to enhanced susceptibility of A3H forms to HIV-1 Vif mediated viral suppression of sequence editing activity, slowing viral diversification and escape from immune responses. PMID:21167246

  9. HIV-1 reverse transcriptase complex with DNA and nevirapine reveals nonnucleoside inhibition mechanism

    PubMed Central

    Das, Kalyan; Martinez, Sergio E.; Bauman, Joseph D.; Arnold, Eddy

    2012-01-01

    Combinations of nucleoside and nonnucleoside inhibitors (NNRTIs) of HIV-1 reverse transcriptase (RT) are widely used in anti-AIDS therapies. Five NNRTIs including nevirapine are clinical drugs; however, the molecular mechanism of inhibition by NNRTIs is not clear. We determined the crystal structures of RT–DNA–nevirapine, RT–DNA, and RT–DNA–AZT-triphosphate complexes at 2.85, 2.70, and 2.80 Å, respectively. The RT–DNA complex in the crystal could bind nevirapine or AZT-triphosphate; however, not both. Binding of nevirapine led to opening of the NNRTI-binding pocket. The pocket formation caused shifting of the 3’-end of DNA primer by ~5.5 Å away from its polymerase active site position. Nucleic acid interactions with fingers and palm subdomains were reduced, the dNTP-binding pocket was distorted, and the thumb opened up. The structures elucidate complementary roles of nucleoside and nonnucleoside inhibitors in inhibiting RT. PMID:22266819

  10. Virion-associated HIV-1 Vpr: variable amount in virus particles derived from cells upon virus infection or proviral DNA transfection.

    PubMed

    Singh, S P; Tungaturthi, P; Cartas, M; Tomkowicz, B; Rizvi, T A; Khan, S A; Kalyanaraman, V S; Srinivasan, A

    2001-04-25

    Human immunodeficiency virus type-1 (HIV-1) Vpr is a virion-associated protein implicated to have a role in AIDS pathogenesis. In regard to the amount of Vpr incorporated into virus particles, the published data vary widely. To address this, we quantitated Vpr in virus particles derived from diverse sources that are used to evaluate the biological effect of Vpr. Virus particles from infected cells showed only a small amount of Vpr. Interestingly, virus particles from cells cotransfected with HIV-1 proviral DNA lacking Vpr coding sequences (NLDeltaVpr) and a Vpr expression plasmid showed a drastic increase (29.4-fold) in the incorporation of Vpr. Furthermore, cotransfection involving NLDeltaVpr and different concentrations of Vpr expression plasmid resulted in virus particles containing Vpr in proportion to the Vpr expression plasmid used. The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr.

  11. Estimating the Impact of Earlier ART Initiation and Increased Testing Coverage on HIV Transmission among Men Who Have Sex with Men in Mexico using a Mathematical Model

    PubMed Central

    Caro-Vega, Yanink; del Rio, Carlos; Dias Lima, Viviane; Lopez-Cervantes, Malaquias; Crabtree-Ramirez, Brenda; Bautista-Arredondo, Sergio; Colchero, M. Arantxa; Sierra-Madero, Juan

    2015-01-01

    Objective To estimate the impact of late ART initiation on HIV transmission among men who have sex with men (MSM) in Mexico. Methods An HIV transmission model was built to estimate the number of infections transmitted by HIV-infected men who have sex with men (MSM-HIV+) MSM-HIV+ in the short and long term. Sexual risk behavior data were estimated from a nationwide study of MSM. CD4+ counts at ART initiation from a representative national cohort were used to estimate time since infection. Number of MSM-HIV+ on treatment and suppressed were estimated from surveillance and government reports. Status quo scenario (SQ), and scenarios of early ART initiation and increased HIV testing were modeled. Results We estimated 14239 new HIV infections per year from MSM-HIV+ in Mexico. In SQ, MSM take an average 7.4 years since infection to initiate treatment with a median CD4+ count of 148 cells/mm3(25th-75th percentiles 52–266). In SQ, 68% of MSM-HIV+ are not aware of their HIV status and transmit 78% of new infections. Increasing the CD4+ count at ART initiation to 350 cells/mm3 shortened the time since infection to 2.8 years. Increasing HIV testing to cover 80% of undiagnosed MSM resulted in a reduction of 70% in new infections in 20 years. Initiating ART at 500 cells/mm3 and increasing HIV testing the reduction would be of 75% in 20 years. Conclusion A substantial number of new HIV infections in Mexico are transmitted by undiagnosed and untreated MSM-HIV+. An aggressive increase in HIV testing coverage and initiating ART at a CD4 count of 500 cells/mm3 in this population would significantly benefit individuals and decrease the number of new HIV infections in Mexico. PMID:26302044

  12. Types of HIV/AIDS Antiretroviral Drugs

    MedlinePlus

    ... reverse transcriptase (RT) from converting single-stranded HIV RNA into double-stranded HIV DNA―a process called ... RT, interfering with its ability to convert HIV RNA into HIV DNA Integrase Inhibitors block the HIV ...

  13. Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers

    PubMed Central

    Guenaga, Javier; Dubrovskaya, Viktoriya; de Val, Natalia; Sharma, Shailendra K.; Carrette, Barbara; Ward, Andrew B.

    2015-01-01

    ABSTRACT Due to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimics, like the native flexibly linked (NFL) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055 Env sequences to form well-ordered, homogenous, and highly stable soluble trimers. The generation of these spike mimics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intraprotomer disulfide linkage in the prebridging sheet, I201C-A433C (CC), that locks the gp120 in the receptor nontriggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from the prototypic subtype A, B, and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimics that are resistant to CD4-induced changes. These advances will allow testing of the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small-animal models and primates. IMPORTANCE HIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimics, typified by NFL

  14. Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

    SciTech Connect

    Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia; Piubelli, Orlando; Alves de Brito, Cyro; Fusaro, Ana Elisa; Eurico de Alencar, Liciana Xavier; August, Thomas; Torres Azevedo Marques, Ernesto; Silva Duarte, Alberto Jose da; Sato, Maria Notomi

    2010-10-10

    Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

  15. Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition among Vaccinated Men

    PubMed Central

    Moodie, Zoe; Metch, Barbara; Bekker, Linda-Gail; Churchyard, Gavin; Nchabeleng, Maphoshane; Mlisana, Koleka; Laher, Fatima; Roux, Surita; Mngadi, Kathryn; Innes, Craig; Mathebula, Matsontso; Allen, Mary; Bentley, Carter; Gilbert, Peter B.; Robertson, Michael; Kublin, James; Corey, Lawrence; Gray, Glenda E.

    2015-01-01

    Background The Phase 2b double-blinded, randomized Phambili/HVTN 503 trial evaluated safety and efficacy of the MRK Ad5 gag/pol/nef subtype B HIV-1 preventive vaccine vs placebo in sexually active HIV-1 seronegative participants in South Africa. Enrollment and vaccinations stopped and participants were unblinded but continued follow-up when the Step study evaluating the same vaccine in the Americas, Caribbean, and Australia was unblinded for non-efficacy. Final Phambili analyses found more HIV-1 infections amongst vaccine than placebo recipients, impelling the HVTN 503-S recall study. Methods HVTN 503-S sought to enroll all 695 HIV-1 uninfected Phambili participants, provide HIV testing, risk reduction counseling, physical examination, risk behavior assessment and treatment assignment recall. After adding HVTN 503-S data, HIV-1 infection hazard ratios (HR vaccine vs. placebo) were estimated by Cox models. Results Of the 695 eligible, 465 (67%) enrolled with 230 from the vaccine group and 235 from the placebo group. 38% of the 184 Phambili dropouts were enrolled. Enrollment did not differ by treatment group, gender, or baseline HSV-2. With the additional 1286 person years of 503-S follow-up, the estimated HR over Phambili and HVTN 503-S follow-up was 1.52 (95% CI 1.08–2.15, p = 0.02, 82 vaccine/54 placebo infections). The HR was significant for men (HR = 2.75, 95% CI 1.49, 5.06, p = 0.001) but not for women (HR = 1.12, 95% CI 0.73, 1.72, p = 0.62). Conclusion The additional follow-up from HVTN 503-S supported the Phambili finding of increased HIV-1 acquisition among vaccinated men and strengthened the evidence of lack of vaccine effect among women. Trial Registration clinicaltrials.gov NCT00413725 SA National Health Research Database DOH-27-0207-1539 PMID:26368824

  16. Increased numbers of granzyme-B-expressing cytotoxic T-lymphocytes in the small intestine of HIV-infected patients.

    PubMed

    Snijders, F; Wever, P C; Danner, S A; Hack, C E; ten Kate, F J; ten Berge, I J

    1996-07-01

    The objective of this study was to determine whether granzyme B-expressing cells, which identify activated cytotoxic lymphocytes, are present in the small intestinal mucosa of human immunodeficiency virus (HIV)-infected patients with and without diarrhea. Therefore, duodenal biopsy specimens from 29 HIV-infected patients (11 with diarrhea and 18 without diarrhea) and 15 control patients were stained for the presence of granzyme B expressing cells. In HIV-infected patients, a significantly increased expression of granzyme B in the lamina propria was observed (p = 0.00001): In 22 of 29 patients, at least 5-10 cells per high-power field were counted. In contrast, in 13 of 15 control patients, granzyme B was not expressed or minimally so, and in two others a maximum of five granzyme-B-expressing cells could be detected per high-power field. No significant difference was found between the HIV-infected patients with and without diarrhea. Double staining revealed that the granzyme-B-expressing cells were mainly CD3 positive. These data show that activated cytotoxic T lymphocytes (CTLs) are present in the duodenal mucosa of HIV-infected patients. No relation between the number of CTLs and the presence of diarrhea was demonstrated. CTLs are known to be involved in the pathogenesis of HIV infection and in the production of tissue injury, but their functional role in intestinal HIV-related pathology has yet to be elucidated.

  17. Priming with a Simplified Intradermal HIV-1 DNA Vaccine Regimen followed by Boosting with Recombinant HIV-1 MVA Vaccine Is Safe and Immunogenic: A Phase IIa Randomized Clinical Trial

    PubMed Central

    Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L.; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

    2015-01-01

    Background Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. Methods In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two “simplified” regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. Results 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. Conclusions A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. Trial Registration World Health

  18. Increased DNA damage and oxidative stress among silver jewelry workers.

    PubMed

    Aktepe, Necmettin; Kocyigit, Abdurrahim; Yukselten, Yunus; Taskin, Abdullah; Keskin, Cumali; Celik, Hakim

    2015-04-01

    Silver has long been valued as a precious metal, and it is used to make ornaments, jewelry, high-value tableware, utensils, and currency coins. Human exposures to silver and silver compounds can occur oral, dermal, or by inhalation. In this study, we investigated genotoxic and oxidative effects of silver exposure among silver jewelry workers. DNA damage in peripheral mononuclear leukocytes was measured by using the comet assay. Serum total antioxidative status (TAS), total oxidative status (TOS), total thiol contents, and ceruloplasmin levels were measured by using colorimetric methods among silver jewelry workers. Moreover, oxidative stress index (OSI) was calculated. Results were compared with non-exposed healthy subjects. The mean values of mononuclear leukocyte DNA damage were significantly higher than control subjects (p < 0.001). Serum TOS, OSI, and ceruloplasmin levels were also found to be higher in silver particles exposed group than those of non-exposed group (p < 0.001, p < 0.001, p < 0.01, respectively). However, serum TAS levels and total thiol contents of silver exposed group were found significantly lower (p < 0.05, p < 0.001, respectively). Exposure to silver particles among silver jewelry workers caused oxidative stress and accumulation of severe DNA damage.

  19. Storytelling for Empowerment for Latino Teens: Increasing HIV Prevention Knowledge and Attitudes

    ERIC Educational Resources Information Center

    Nelson, Annabelle; Cordova, David; Walters, Andrew S.; Szecsy, Elsie

    2016-01-01

    Latino adolescents are disproportionately impacted by HIV, but researchers have documented few programs to prevent and reduce HIV risk. The Storytelling for Empowerment (SFE) "HIV StoryBook" was designed with an innovative ecodevelopment approach combining empowerment, family communication, and positive cultural identity. A mixed method…

  20. Increasing Parent Involvement in Youth HIV Prevention: A Randomized Caribbean Study

    ERIC Educational Resources Information Center

    Baptiste, Donna R.; Kapungu, Chisina; Miller, Steve; Crown, Laurel; Henry, David; Da Costa Martinez, Dona; Jo-Bennett, Karen

    2009-01-01

    This article presents preliminary findings of a randomized HIV prevention study in Trinidad and Tobago in the Caribbean. The study centers on a family HIV workshop aimed at strengthening parenting skills that are empirically linked to reducing adolescent HIV exposure and other sexual risks. These skills include parental monitoring; educating youth…

  1. Increases in Human Papillomavirus Detection During Early HIV Infection Among Women in Zimbabwe

    PubMed Central

    Nowak, Rebecca G.; Morrison, Charles S.; Gange, Stephen J.; Kwok, Cynthia; Oliver, Amy E.; Howard, Roslyn; Van Der Pol, Barbara; Salata, Robert A.; Padian, Nancy S.; Chipato, Tsungai; Munjoma, Marshall; Celentano, David D.

    2011-01-01

    Background. Individuals who acquire human immunodeficiency virus (HIV) may experience an immediate disruption of genital tract immunity, altering the ability to mount a local and effective immune response. This study examined the impact of early HIV infection on new detection of human papillomavirus (HPV). Methods. One hundred fifty-five Zimbabwean women with observation periods before and after HIV acquisition and 486 HIV-uninfected women were selected from a cohort study evaluating hormonal contraceptive use and risk of HIV acquisition. Study visits occurred at 3-month intervals. Cervical swab samples available from up to 6 months before, at, and up to 6 months after the visit when HIV was first detected were typed for 37 HPV genotypes or subtypes. Results. We observed ∼5-fold higher odds of multiple (≥2) new HPV detections only after HIV acquisition, relative to HIV-negative women after adjusting for sexual behavior and concurrent genital tract infections. We also observed ∼2.5-fold higher odds of single new HPV detections at visits before and after HIV acquisition, relative to HIV-uninfected women in multivariable models. Conclusions. These findings suggest that HIV infection has an immediate impact on genital tract immunity, as evidenced by the high risk of multiple new HPV detections immediately after HIV acquisition. PMID:21451006

  2. Nitrite inhalant use among young gay and bisexual men in Vancouver during a period of increasing HIV incidence

    PubMed Central

    Lampinen, Thomas M; Mattheis, Kelly; Chan, Keith; Hogg, Robert S

    2007-01-01

    Background Nitrite inhalants ("poppers") are peripheral vasodilators which, since the beginning of the epidemic, have been known to increase risk for acquiring HIV infection among men who have sex with men (MSM). However, few studies in recent years have characterized use. From 1999 to 2004, new HIV diagnoses among MSM in British Columbia increased 78%, prompting us to examine the prevalence and correlates of this modifiable HIV risk factor. Methods Self-administered questionnaires were completed between October 2002 and May 2004 as part of an open cohort study of HIV-seronegative young MSM. We measured nitrite inhalant use during the previous year and use during sexual encounters with casual partners specifically. Correlates of use were identified using odds ratios. Results Among 354 MSM surveyed, 31.6% reported any use during the previous year. Nitrite inhalant use during sexual encounters was reported by 22.9% of men and was strongly associated with having casual partners, with greater numbers of casual partners (including those with positive or unknown serostatus) and with anal intercourse with casual partners. Nitrite inhalant use was not associated with non-use of condoms with casual sexual partners per se. Conclusion Contemporary use of nitrite inhalants amongst young MSM is common and a strong indicator of anal intercourse with casual sexual partners. Since use appears to increase the probability of infection following exposure to HIV, efforts to reduce the use of nitrite inhalants among MSM should be a very high priority among HIV prevention strategies. PMID:17362516

  3. Using social and behavior change communication to increase HIV testing and condom use: the Malawi BRIDGE Project.

    PubMed

    Kaufman, Michelle R; Rimal, Rajiv N; Carrasco, Maria; Fajobi, OlaOluwa; Soko, Anthony; Limaye, Rupali; Mkandawire, Glory

    2014-01-01

    While overall HIV prevalence in Malawi has decreased, it is still high in the southern region of the country. Behavioral prevention activities are crucial to continue the reduction in HIV prevalence. Behavior change is influenced by many factors. Previous work indicates knowledge about HIV transmission, self-efficacy to protect oneself from exposure, and accurate risk perception of one's susceptibility all impact sexual behavior. The current study looks at the effects of a behavior change communication program in Malawi called the BRIDGE II Project on psychosocial and behavioral variables. The program sought to address barriers to individual action and confront societal norms related to sexual risk behavior through a mix of community-based activities and mass media messages delivered through local radio stations. Using cohort data (n = 594), we examined the effect of BRIDGE exposure on three variables that affect HIV behaviors: knowledge, self-efficacy, and risk perception, as well as two behavioral outcomes: HIV testing and condom use at last sex. Data were collected at baseline and for a midterm evaluation. Regression analyses showed exposure to BRIDGE was significantly associated with knowledge level (β = 0.20, p < .001) and self-efficacy (β = 0.35, p < .001) at midterm when controlling for baseline scores, but not risk perception. Psychosocial variables did not show a significant relationship to either behavioral outcome. However, program exposure was a significant predictor of both HIV testing in the past year (odds ratio [OR] = 1.40, p < .001) and condom use at last sex (OR = 1.26, p < .05). This study suggests such a communication intervention may play an important role in not only affecting HIV-related behaviors themselves, but also critical factors that affect HIV behaviors, including knowledge and self-efficacy. It is recommended that communication efforts around HIV risk reduction be increased. PMID:24735337

  4. Using social and behavior change communication to increase HIV testing and condom use: the Malawi BRIDGE Project.

    PubMed

    Kaufman, Michelle R; Rimal, Rajiv N; Carrasco, Maria; Fajobi, OlaOluwa; Soko, Anthony; Limaye, Rupali; Mkandawire, Glory

    2014-01-01

    While overall HIV prevalence in Malawi has decreased, it is still high in the southern region of the country. Behavioral prevention activities are crucial to continue the reduction in HIV prevalence. Behavior change is influenced by many factors. Previous work indicates knowledge about HIV transmission, self-efficacy to protect oneself from exposure, and accurate risk perception of one's susceptibility all impact sexual behavior. The current study looks at the effects of a behavior change communication program in Malawi called the BRIDGE II Project on psychosocial and behavioral variables. The program sought to address barriers to individual action and confront societal norms related to sexual risk behavior through a mix of community-based activities and mass media messages delivered through local radio stations. Using cohort data (n = 594), we examined the effect of BRIDGE exposure on three variables that affect HIV behaviors: knowledge, self-efficacy, and risk perception, as well as two behavioral outcomes: HIV testing and condom use at last sex. Data were collected at baseline and for a midterm evaluation. Regression analyses showed exposure to BRIDGE was significantly associated with knowledge level (β = 0.20, p < .001) and self-efficacy (β = 0.35, p < .001) at midterm when controlling for baseline scores, but not risk perception. Psychosocial variables did not show a significant relationship to either behavioral outcome. However, program exposure was a significant predictor of both HIV testing in the past year (odds ratio [OR] = 1.40, p < .001) and condom use at last sex (OR = 1.26, p < .05). This study suggests such a communication intervention may play an important role in not only affecting HIV-related behaviors themselves, but also critical factors that affect HIV behaviors, including knowledge and self-efficacy. It is recommended that communication efforts around HIV risk reduction be increased.

  5. Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

    PubMed Central

    Li, Nan; An, Lili; Hang, Haiying

    2015-01-01

    Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects. PMID:25915950

  6. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    PubMed

    Li, Nan; An, Lili; Hang, Haiying

    2015-01-01

    Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.

  7. Asymptomatic HIV-associated neurocognitive impairment increases risk for symptomatic decline

    PubMed Central

    Franklin, Donald R.; Deutsch, Reena; Woods, Steven P.; Vaida, Florin; Ellis, Ronald J.; Letendre, Scott L.; Marcotte, Thomas D.; Atkinson, J.H.; Collier, Ann C.; Marra, Christina M.; Clifford, David B.; Gelman, Benjamin B.; McArthur, Justin C.; Morgello, Susan; Simpson, David M.; McCutchan, John A.; Abramson, Ian; Gamst, Anthony; Fennema-Notestine, Christine; Smith, Davey M.; Heaton, Robert K.

    2014-01-01

    Objective: While HIV-associated neurocognitive disorders (HAND) remain prevalent despite combination antiretroviral therapy (CART), the clinical relevance of asymptomatic neurocognitive impairment (ANI), the most common HAND diagnosis, remains unclear. We investigated whether HIV-infected persons with ANI were more likely than those who were neurocognitively normal (NCN) to experience a decline in everyday functioning (symptomatic decline). Methods: A total of 347 human participants from the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort were NCN (n = 226) or had ANI (n = 121) at baseline. Neurocognitive assessments occurred approximately every 6 months, with median (interquartile range) follow-up of 45.2 (28.7–63.7) months. Symptomatic decline was based on self-report (SR) or objective, performance-based (PB) problems in everyday functioning. Proportional hazards modeling was used to generate risk ratios for progression to symptomatic HAND after adjusting for baseline and time-dependent covariates, including CD4+ T-lymphocyte count (CD4), virologic suppression, CART, and mood. Results: The ANI group had a shorter time to symptomatic HAND than the NCN after adjusting for baseline predictors: adjusted risk ratios for symptomatic HAND were 2.0 (confidence interval [CI] 1.1–3.6; p = 0.02) for SR, 5.8 (CI 3.2–10.7; p < 0.0001) for PB, and 3.2 (CI 2.0–5.0; p < 0.0001) for either SR or PB. Current CD4 and depression were significant time-dependent covariates, but antiretroviral regimen, virologic suppression, and substance abuse or dependence were not. Conclusions: This longitudinal study demonstrates that ANI conveys a 2-fold to 6-fold increase in risk for earlier development of symptomatic HAND, supporting the prognostic value of the ANI diagnosis in clinical settings. Identifying those at highest risk for symptomatic decline may offer an opportunity to modify treatment to delay progression. PMID:24814848

  8. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    SciTech Connect

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  9. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    SciTech Connect

    Li, Hui-Guang; Huang, Philip L.; Zhang, Dawei; Sun, Yongtao; Chen, Hao-Chia; Zhang, John; Huang, Paul L.; Kong, Xiang-Peng; Lee-Huang, Sylvia

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  10. Retinoic acid imprints a mucosal-like phenotype on dendritic cells with an increased ability to fuel HIV-1 infection.

    PubMed

    Guerra-Pérez, Natalia; Frank, Ines; Veglia, Filippo; Aravantinou, Meropi; Goode, Diana; Blanchard, James L; Gettie, Agegnehu; Robbiani, Melissa; Martinelli, Elena

    2015-03-01

    The tissue microenvironment shapes the characteristics and functions of dendritic cells (DCs), which are important players in HIV infection and dissemination. Notably, DCs in the gut have the daunting task of orchestrating the balance between immune response and tolerance. They produce retinoic acid (RA), which imprints a gut-homing phenotype and influences surrounding DCs. To investigate how the gut microenvironment impacts the ability of DCs to drive HIV infection, we conditioned human immature monocyte-derived DCs (moDCs) with RA (RA-DCs), before pulsing them with HIV and mixing them with autologous T cells. RA-DCs showed a semimature, mucosal-like phenotype and released higher amounts of TGF-β1 and CCL2. Using flow cytometry, Western blot, and microscopy, we determined that moDCs express the cell adhesion molecule mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and that RA increases its expression. MAdCAM-1 was also detected on a small population of DCs in rhesus macaque (Macaca mulata) mesenteric lymph node. RA-DCs formed more DC-T cell conjugates and promoted significantly higher HIV replication in DC-T cell mixtures compared with moDCs. This correlated with the increase in MAdCAM-1 expression. Blocking MAdCAM-1 partially inhibited the enhanced HIV replication. In summary, RA influences DC phenotype, increasing their ability to exacerbate HIV infection. We describe a previously unknown mechanism that may contribute to rapid HIV spread in the gut, a major site of HIV replication after mucosal exposure. PMID:25624458

  11. Increases in Recent HIV Testing Among Men Who Have Sex With Men Coincide With the Centers for Disease Control and Prevention's Expanded Testing Initiative

    PubMed Central

    Cooley, Laura A.; Wejnert, Cyprian; Rose, Charles E.; Paz-Bailey, Gabriela; Taussig, Jennifer; Gern, Robert; Hoyte, Tamika; Salazar, Laura; White, Jianglan; Todd, Jeff; Bautista, Greg; Flynn, Colin; Sifakis, Frangiscos; German, Danielle; Isenberg, Debbie; Driscoll, Maura; Hurwitz, Elizabeth; Doherty, Rose; Wittke, Chris; Prachand, Nikhil; Benbow, Nanette; Melville, Sharon; Pannala, Praveen; Yeager, Richard; Sayegh, Aaron; Dyer, Jim; Sheu, Shane; Novoa, Alicia; Thrun, Mark; Al-Tayyib, Alia; Wilmoth, Ralph; Higgins, Emily; Griffin, Vivian; Mokotoff, Eve; MacMaster, Karen; Wolverton, Marcia; Risser, Jan; Rehman, Hafeez; Padgett, Paige; Bingham, Trista; Sey, Ekow Kwa; LaLota, Marlene; Metsch, Lisa; Forrest, David; Beck, Dano; Cardenas, Gabriel; Nemeth, Chris; Anderson, Bridget J.; Watson, Carol-Ann; Smith, Lou; Robinson, William T.; Gruber, DeAnn; Barak, Narquis; Murrill, Chris; Neaigus, Alan; Jenness, Samuel; Hagan, Holly; Reilly, Kathleen H.; Wendel, Travis; Cross, Helene; Bolden, Barbara; D'Errico, Sally; Wogayehu, Afework; Godette, Henry; Brady, Kathleen A.; Kirkland, Althea; Sifferman, Andrea; Miguelino-Keasling, Vanessa; Velasco, Al; Tovar, Veronica; Raymond, H. Fisher; De León, Sandra Miranda; Rolón-Colón, Yadira; Marzan, Melissa; Courogen, Maria; Jaenicke, Tom; Thiede, Hanne; Burt, Richard; Jia, Yujiang; Opoku, Jenevieve; Sansone, Marie; West, Tiffany; Magnus, Manya; Kuo, Irene

    2015-01-01

    According to National HIV Behavioral Surveillance system data, human immunodeficiency virus (HIV) testing increased among gay, bisexual, and other men who have sex with men from 2008 to 2011 in cities funded by the Centers for Disease Control and Prevention's Expanded Testing Initiative, suggesting that focused HIV testing initiatives might have positive effects. PMID:25352589

  12. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR.

    PubMed

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2015-02-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

  13. Brief Training of HIV Medical Providers Increases Their Frequency of Delivering Prevention Counselling to Patients at Risk of Transmitting HIV to Others

    ERIC Educational Resources Information Center

    Patel, Shilpa N.; Marks, Gary; Gardner, Lytt; Golin, Carol E.; Shinde, Sanjyot; O'Daniels, Christine; Wilson, Tracey E.; Quinlivan, E. Byrd; Banderas, Julie W.

    2013-01-01

    Objective: The aim of this study was to examine whether brief training of human immunodeficiency virus (HIV) medical providers increased the frequency with which they routinely delivered prevention counselling to patients, and whether patient characteristics were associated with receipt of that counselling. Design: Longitudinal. Setting: Seven HIV…

  14. The Community Liaison Program: A Health Education Pilot Program to Increase Minority Awareness of HIV and Acceptance of HIV Vaccine Trials

    ERIC Educational Resources Information Center

    Kelley, R. T.; Hannans, A.; Kreps, G. L.; Johnson, K.

    2012-01-01

    This paper describes a 16-month health education pilot program based on diffusion of innovation and social network theories. The program was implemented by volunteer community liaisons for the purposes of increasing awareness of and support for HIV vaccine research in minority populations. This theoretically driven pilot program allowed the…

  15. Infant CD4 C868T polymorphism is associated with increased human immunodeficiency virus (HIV-1) acquisition.

    PubMed

    Choi, R Y; Farquhar, C; Juno, J; Mbori-Ngacha, D; Lohman-Payne, B; Vouriot, F; Wayne, S; Tuff, J; Bosire, R; John-Stewart, G; Fowke, K

    2010-06-01

    The C868T single nucleotide polymorphism (SNP) in the CD4 receptor encodes an amino acid change that could alter its structure and influence human immunodeficiency virus (HIV-1) infection risk. HIV-1-infected pregnant women in Nairobi were followed with their infants for 1 year postpartum. Among 131 infants, those with the 868T allele were more likely than wild-type infants to acquire HIV-1 overall [hazard ratio (HR) = 1.92, 95% confidence interval (CI) 1.05, 3.50, P = 0.03; adjusted HR = 2.03, 95% CI 1.03, 3.98, P = 0.04], after adjusting for maternal viral load. This SNP (an allele frequency of approximately 15% in our cohort) was associated with increased susceptibility to mother-to-child HIV-1 transmission, consistent with a previous study on this polymorphism among Nairobi sex workers.

  16. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa

    PubMed Central

    Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-01-01

    Abstract As more human immunodeficiency virus (HIV)–infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ2 test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ2 test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  17. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa.

    PubMed

    Manasa, Justen; Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-08-01

    As more human immunodeficiency virus (HIV)-infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ(2) test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ(2) test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  18. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

    PubMed

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis. PMID:27032385

  19. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

    PubMed

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis.

  20. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform

    PubMed Central

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis. PMID:27032385

  1. Increased Intrathecal Immune Activation in Virally Suppressed HIV-1 Infected Patients with Neurocognitive Impairment

    PubMed Central

    Edén, Arvid; Marcotte, Thomas D.; Heaton, Robert K.; Nilsson, Staffan; Zetterberg, Henrik; Fuchs, Dietmar; Franklin, Donald; Price, Richard W.; Grant, Igor; Letendre, Scott L.; Gisslén, Magnus

    2016-01-01

    Objective Although milder forms of HIV-associated neurocognitive disorder (HAND) remain prevalent, a correlation to neuronal injury has not been established in patients on antiretroviral therapy (ART). We examined the relationship between mild HAND and CSF neurofilament light protein (NFL), a biomarker of neuronal injury; and CSF neopterin, a biomarker of CNS immunoactivation, in virally suppressed patients on antiretroviral therapy (ART). Design and Methods We selected 99 subjects on suppressive ART followed longitudinally from the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) study. Based on standardized comprehensive neurocognitive performance (NP) testing, subjects were classified as neurocognitively normal (NCN; n = 29) or impaired (NCI; n = 70). The NCI group included subjects with asymptomatic (ANI; n = 37) or mild (MND; n = 33) HAND. CSF biomarkers were analyzed on two occasions. Results Geometric mean CSF neopterin was 25% higher in the NCI group (p = 0.04) and NFL and neopterin were significantly correlated within the NCI group (r = 0.30; p<0.001) but not in the NCN group (r = -0.13; p = 0.3). Additionally, a trend towards higher NFL was seen in the NCI group (p = 0.06). Conclusions Mild HAND was associated with increased intrathecal immune activation, and the correlation between neopterin and NFL found in NCI subjects indicates an association between neurocognitive impairment, CNS inflammation and neuronal damage. Together these findings suggest that NCI despite ART may represent an active pathological process within the CNS that needs further characterization in prospective studies. PMID:27295036

  2. Increased inflammation in sanctuary sites may explain viral blips in HIV infection

    DOE PAGESBeta

    Piovoso, Michael J.; Cardozo, E. Fabian; Zurakowski, Ryan

    2016-08-01

    Here, combined antiretroviral therapy (cART) suppress HIV-1 viral replication, such that viral load in plasma remains below the limit of detection in standard assays. However, intermittent episodes of transient viremia (blips) occur in a set of HIV-patients. Given that follicular hyperplasia occurs during lymphoid inflammation as a normal response to infection, it is hypothesised that when the diameter of the lymph node follicle (LNF) increases and crosses a critical size, a viral blip occurs due to cryptic viremia. To study this hypothesis, a theoretical analysis of a mathematical model is performed to find the conditions for virus suppression in allmore » compartments and different scenarios of LNF size changes are simulated. According to the analysis, blips with duration of around 30 days arise when the diameter rise rate is between 0.02 and 0.03 days–1. Moreover, the final diameter of the site is directly related to the steady states of the virus load after the occurrence of a blip. When the value of R0 is around 2.1, to have a steady-state below the limit of detection after the viral blip, the maximum final diameters should be greater than 0.7 mm so that there is a relative loss of connection between compartments.« less

  3. Multimeric soluble CD40 ligand (sCD40L) efficiently enhances HIV specific cellular immune responses during DNA prime and boost with attenuated poxvirus vectors MVA and NYVAC expressing HIV antigens.

    PubMed

    Gómez, Carmen E; Nájera, José L; Sánchez, Raquel; Jiménez, Victoria; Esteban, Mariano

    2009-05-21

    The attenuated poxvirus vectors MVA and NYVAC are now in clinical trials against HIV/AIDS. Due to the vectors restricted replication capacity in human cells, approaches to enhance their immunogenicity are highly desirable. Here, we have analyzed the ability of a soluble form of hexameric CD40L (sCD40L) to stimulate specific immune responses to HIV antigens when inoculated in mice during priming with DNA and in the booster with MVA or NYVAC, expressing the vectors HIV-1 Env, Gag, Pol and Nef antigens from clade B. Our findings revealed that sCD40L in DNA/poxvirus combination enhanced the magnitude about 2-fold (DNA-B/MVA-B) and 4-fold (DNA-B/NYVAC-B), as well as the breath of the HIV antigen specific cellular immune responses. sCD40L was necessary in both prime and boost inoculations triggering a potent polarization of the Th response towards a Th1 type. In DNA-B/NYVAC-B regime the addition of sCD40L significantly enhanced the humoral immune response against HIV gp160, but not in DNA-B/MVA-B combination. These findings provided evidence for the immunostimulatory benefit of sCD40L when DNA and the poxvirus vectors MVA and NYVAC are used as immunogens.

  4. Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

    PubMed

    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 (p<0.05). At month 30, 33 out of 67 patients (49.2%) showed at least one sign of lipodystrophy. The mean plasma mitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (p<0.001). The receiver operating curve analysis demonstrated that using plasma mitochondrial DNA level (with cut-off value <5.09 log10 copies/ml) as a molecular marker allowed identification of patients with lipodystrophy with a sensitivity of 64.2% and a specificity of 73.0%. Our data suggest that mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk.

  5. Declining Inconsistent Condom Use but Increasing HIV and Syphilis Prevalence Among Older Male Clients of Female Sex Workers

    PubMed Central

    Chen, Yi; Abraham Bussell, Scottie; Shen, Zhiyong; Tang, Zhenzhu; Lan, Guanghua; Zhu, Qiuying; Liu, Wei; Tang, Shuai; Li, Rongjian; Huang, Wenbo; Huang, Yuman; Liang, Fuxiong; Wang, Lu; Shao, Yiming; Ruan, Yuhua

    2016-01-01

    Abstract Clients of female sex workers (CFSWs) are a bridge population for the spread of HIV and syphilis to low or average risk heterosexuals. Most studies have examined the point prevalence of these infections in CFSWs. Limited evidence suggests that older age CFSWs are at a higher risk of acquiring sexually transmitted diseases compared with younger clients. Thus, we sought to describe long-term trends in HIV, syphilis, and hepatitis C (HCV) to better understand how these infections differ by sex worker classification and client age. We also examined trends in HIV, syphilis, and HCV among categories of female sex workers (FSWs). We conducted serial cross-sectional studies from 2010 to 2015 in Guangxi autonomous region, China. We collected demographic and behavior variables. FSWs and their clients were tested for HIV, syphilis, and HCV antibodies. Positive HIV and syphilis serologies were confirmed by Western blot and rapid plasma regain, respectively. Clients were categorized as middle age (40–49 years) and older clients (≥50 years). FSWs were categorized as high-tier, middle-tier, or low-tier based on the payment amount charged for sex and their work venue. Chi-square test for trends was used for testing changes in prevalence over time. By 2015, low-tier FSWs (LTFSWs) accounted for almost half of all FSWs; and they had the highest HIV prevalence at 1.4%. HIV prevalence declined significantly for FSWs (high-tier FSW, P = 0.003; middle-tier FSWs; P = 0.021; LTFSWs, P < 0.001). Syphilis infections significantly declined for FSWs (P < 0.001) but only to 7.3% for LTFSWs. HCV and intravenous drug use were uncommon in FSWs. HIV prevalence increased for older age clients (1.3%–2.0%, P = 0.159) while syphilis prevalence remained stable. HCV infections were halved among older clients in 3 years (1.7%–0.8%, P < 0.001). Condom use during the last sexual encounter increased for FSWs and CFSWs. Few clients reported sex with men or intravenous

  6. Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture.

    PubMed

    Islas-Lugo, Fabiola; Vega-Estrada, Jesús; Alvis, Christian Ariel; Ortega-López, Jaime; Del Carmen Montes-Horcasitas, María

    2016-09-10

    Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions. PMID:27374404

  7. Outcomes of a Behavioral Intervention to Increase Condom Use and Reduce HIV Risk Among Urban African American Young Adults.

    PubMed

    Henry Akintobi, Tabia; Trotter, Jennie; Zellner, Tiffany; Lenoir, Shelia; Evans, Donoria; Rollins, Latrice; Miller, Assia

    2016-09-01

    African Americans comprise nearly half of people in the United States living with the human immunodeficiency virus (HIV) but compose one tenth of the population. Infection rate among young African American adults is 11 times that of Whites. The Color It Real Program was a seven-session, weekly administered, age-specific, and culturally tailored intervention designed to provide HIV education and address behavioral motivations (risk awareness, decisional balance exercises, partner negotiation, and attitudes) associated with HIV risk among African Americans ages 18 to 24 years in Atlanta, Georgia. Effectiveness was assessed through a quasi-experimental study design that consisted of intervention (n = 88) and control (n = 52) groups completing a 45-item survey. When controlling for gender and education, repeated measures analysis of variance revealed that the intervention group had significant increases in HIV transmission knowledge (F = 4.84, p = .0305), condom use, and intentions to use condoms (F = 4.38, p = .0385). Risky sexual behavior means did not significantly differ between groups (F = 1.44, p = .2331). Results indicate the value of culturally tailored educational strategies toward improved HIV knowledge and adoption of risk reduction strategies. Future studies investigating the differential impact of programs by gender and sexual orientation are also critical. Continued innovation and tailoring of risk reduction strategies for minority young adults will contribute to reducing HIV incidence and prevalence over the life course. PMID:27216874

  8. Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir.

    PubMed

    Tuske, Steve; Sarafianos, Stefan G; Clark, Arthur D; Ding, Jianping; Naeger, Lisa K; White, Kirsten L; Miller, Michael D; Gibbs, Craig S; Boyer, Paul L; Clark, Patrick; Wang, Gang; Gaffney, Barbara L; Jones, Roger A; Jerina, Donald M; Hughes, Stephen H; Arnold, Eddy

    2004-05-01

    Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.

  9. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    SciTech Connect

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  10. [THE ORGANIZATIONAL TECHNOLOGIES OF INCREASING OF EFFECTIVENESS OF DISPENSARY MONITORING OF HIV-INFECTED PERSONS].

    PubMed

    Moskvitcheva, M G; Yu, Kitmanova L

    2015-01-01

    The organizational technologies of increasing effectiveness ofdispensarization monitoring of HIV-infected persons are to targeted to development in patients commitment to get medical care. The cohort monitoring of registered patients receiving anti-retrovirus therapy permitted to evaluate effectiveness of organizational model of multi-professional team developing commitment ofpatients to anti-retrovirus therapy in conditions of center ofprevention and struggle with AIDS and infectious diseases. The criteria ofeffectiveness offunctioning ofmulti-professional team are developed and implemented The list of criteria include percentage of patients in cohort with optimal commitment (not lower than 95% of applied dosage of anti-retrovirus pharmaceuticals at 12th, 24th, 36th, 48th and 60th month), percentage ofpatients with achieved effect of anti-retrovirus therapy, percentage of patients proceeding anti-retrovirus therapy. The multi-professional team implemented motivational techniques of behavior alteration and patient-oriented care. The main strategy of development of of commitment to anti-retrovirus therapy under HIV-infection is determined as management of resources and risks capable decreasing commitment to dispensarization monitoring. The analysis of problems permitted to structure them in risks of commitment failure: medical (13.7%), medical biological under using psychoactive substances (43.1%), psychological (27.7%), social (15.5%). This listing determined the profile of specialists of multi-professional team. The ranking of risks lead out to the first ranking place medical risks, including diagnosed tuberculosis, combination of secondary and concomitant diseases inpatient, number of intaking pills more than 7 per day. The second ranking place took medical biological risks in users of psychoactive substances. Up to 60th month the anti-retrovirus therapy was proceeded by 61.5% of users of psychoactive substances with optimal commitment in 60%. The implementation

  11. [THE ORGANIZATIONAL TECHNOLOGIES OF INCREASING OF EFFECTIVENESS OF DISPENSARY MONITORING OF HIV-INFECTED PERSONS].

    PubMed

    Moskvitcheva, M G; Yu, Kitmanova L

    2015-01-01

    The organizational technologies of increasing effectiveness ofdispensarization monitoring of HIV-infected persons are to targeted to development in patients commitment to get medical care. The cohort monitoring of registered patients receiving anti-retrovirus therapy permitted to evaluate effectiveness of organizational model of multi-professional team developing commitment ofpatients to anti-retrovirus therapy in conditions of center ofprevention and struggle with AIDS and infectious diseases. The criteria ofeffectiveness offunctioning ofmulti-professional team are developed and implemented The list of criteria include percentage of patients in cohort with optimal commitment (not lower than 95% of applied dosage of anti-retrovirus pharmaceuticals at 12th, 24th, 36th, 48th and 60th month), percentage ofpatients with achieved effect of anti-retrovirus therapy, percentage of patients proceeding anti-retrovirus therapy. The multi-professional team implemented motivational techniques of behavior alteration and patient-oriented care. The main strategy of development of of commitment to anti-retrovirus therapy under HIV-infection is determined as management of resources and risks capable decreasing commitment to dispensarization monitoring. The analysis of problems permitted to structure them in risks of commitment failure: medical (13.7%), medical biological under using psychoactive substances (43.1%), psychological (27.7%), social (15.5%). This listing determined the profile of specialists of multi-professional team. The ranking of risks lead out to the first ranking place medical risks, including diagnosed tuberculosis, combination of secondary and concomitant diseases inpatient, number of intaking pills more than 7 per day. The second ranking place took medical biological risks in users of psychoactive substances. Up to 60th month the anti-retrovirus therapy was proceeded by 61.5% of users of psychoactive substances with optimal commitment in 60%. The implementation

  12. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    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

  13. Identifying Opportunities to Increase HIV Testing among Mexican Migrants: A Call to Step Up Efforts in Health Care and Detention Settings

    PubMed Central

    Martínez-Donate, Ana P.; Rangel, Maria Gudelia; Rhoads, Natalie; Zhang, Xiao; Hovell, Melbourne; Magis-Rodriguez, Carlos; González-Fagoaga, Eduardo

    2015-01-01

    HIV testing and counseling is a critical component of HIV prevention efforts and core element of current “treatment as prevention” strategies. Mobility, low education and income, and limited access to health care put Latino migrants at higher risk for HIV and represent barriers for adequate levels of HIV testing in this population. We examined correlates of, and missed opportunities to increase, HIV testing for circular Mexican migrants in the U.S. We used data from a probability-based survey of returning Mexican migrants (N=1161) conducted in the border city of Tijuana, Mexico. We estimated last 12-months rates of HIV testing and the percentage of migrants who received other health care services or were detained in an immigration center, jail, or prison for 30 or more days in the U.S., but were not tested for HIV. Twenty-two percent of migrants received HIV testing in the last 12 months. In general, utilization of other health care services or detention for 30 or more days in the U.S. was a significant predictor of last 12-months HIV testing. Despite this association, we found evidence of missed opportunities to promote testing in healthcare and/or correctional or immigration detention centers. About 27.6% of migrants received other health care and/or were detained at least 30 days but not tested for HIV. Health care systems, jails and detention centers play an important role in increasing access to HIV testing among circular migrants, but there is room for improvement. Policies to offer opt-out, confidential HIV testing and counseling to Mexican migrants in these settings on a routine and ethical manner need to be designed and pilot tested. These policies could increase knowledge of HIV status, facilitate engagement in HIV treatment among a highly mobile population, and contribute to decrease incidence of HIV in the host and receiving communities. PMID:25860261

  14. Identifying opportunities to increase HIV testing among mexican migrants: a call to step up efforts in health care and detention settings.

    PubMed

    Martínez-Donate, Ana P; Rangel, Maria Gudelia; Rhoads, Natalie; Zhang, Xiao; Hovell, Melbourne; Magis-Rodriguez, Carlos; González-Fagoaga, Eduardo

    2015-01-01

    HIV testing and counseling is a critical component of HIV prevention efforts and core element of current "treatment as prevention" strategies. Mobility, low education and income, and limited access to health care put Latino migrants at higher risk for HIV and represent barriers for adequate levels of HIV testing in this population. We examined correlates of, and missed opportunities to increase, HIV testing for circular Mexican migrants in the U.S. We used data from a probability-based survey of returning Mexican migrants (N=1161) conducted in the border city of Tijuana, Mexico. We estimated last 12-months rates of HIV testing and the percentage of migrants who received other health care services or were detained in an immigration center, jail, or prison for 30 or more days in the U.S., but were not tested for HIV. Twenty-two percent of migrants received HIV testing in the last 12 months. In general, utilization of other health care services or detention for 30 or more days in the U.S. was a significant predictor of last 12-months HIV testing. Despite this association, we found evidence of missed opportunities to promote testing in healthcare and/or correctional or immigration detention centers. About 27.6% of migrants received other health care and/or were detained at least 30 days but not tested for HIV. Health care systems, jails and detention centers play an important role in increasing access to HIV testing among circular migrants, but there is room for improvement. Policies to offer opt-out, confidential HIV testing and counseling to Mexican migrants in these settings on a routine and ethical manner need to be designed and pilot tested. These policies could increase knowledge of HIV status, facilitate engagement in HIV treatment among a highly mobile population, and contribute to decrease incidence of HIV in the host and receiving communities.

  15. Computer-Based Video to Increase HIV Testing Among Emergency Department Patients Who Decline

    PubMed Central

    Aronson, Ian David; Marsch, Lisa A.; Rajan, Sonali; Koken, Juline; Bania, Theodore C.

    2014-01-01

    The Centers for Disease Control and Prevention (CDC) recommend routine HIV screening in clinical settings, including emergency departments (EDs), because earlier diagnosis enables treatment before symptoms develop and delivery of interventions to reduce continued transmission. However, patients frequently decline testing. This study delivered a 16-minute video-based intervention to 160 patients who declined HIV tests in a high volume, urban ED. One third of participants (N=53) accepted an HIV test post-intervention. Interviews with a subset of participants (n=40) show that before the video, many were unaware HIV testing could be conducted without drawing blood, or that results could be delivered in 20 minutes. PMID:25064047

  16. Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia.

    PubMed

    Seu, Lillian; Mwape, Innocent; Guffey, M Bradford

    2014-07-01

    The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes.

  17. Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia.

    PubMed

    Seu, Lillian; Mwape, Innocent; Guffey, M Bradford

    2014-07-01

    The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes. PMID:24667303

  18. Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia

    PubMed Central

    Seu, Lillian; Mwape, Innocent; Guffey, M. Bradford

    2014-01-01

    The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5′ and 3′ region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5′ and 3′ proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes. PMID:24667303

  19. Widespread rape does not directly appear to increase the overall HIV prevalence in conflict-affected countries: so now what?

    PubMed Central

    Anema, Aranka; Joffres, Michel R; Mills, Edward; Spiegel, Paul B

    2008-01-01

    Background Sub-Saharan Africa (SSA) is severely affected by HIV/AIDS and conflict. Sexual violence as a weapon of war has been associated with concerns about heightened HIV incidence among women. Widespread rape by combatants has been documented in Burundi, Sierra Leone, Rwanda, Democratic Republic of Congo, Liberia, Sudan and Uganda. To examine the assertion that widespread rape may not directly increase HIV prevalence at the population level, we built a model to determine the potential impact of varying scenarios of widespread rape on HIV prevalence in the above seven African countries. Discussion Our findings show that even in the most extreme situations, where 15% of the female population was raped, where HIV prevalence among assailants was 8 times the country population prevalence, and where the HIV transmission rate was highest at 4 times the average high rate, widespread rape increased the absolute HIV prevalence of these countries by only 0.023%. These projections support the finding that widespread rape in conflict-affected countries in SSA has not incurred a major direct population-level change in HIV prevalence. However, this must not be interpreted to say that widespread rape does not pose serious problems to women's acquisition of HIV on an individual basis or in specific settings. Furthermore, direct and indirect consequences of sexual violence, such as physical and psychosocial trauma, unwanted pregnancies, and stigma and discrimination cannot be understated. Summary The conclusions of this article do not significantly change current practices in the field from an operational perspective. Proper care and treatment must be provided to every survivor of rape regardless of the epidemiological effects of HIV transmission at the population level. Sexual violence must be treated as a protection issue and not solely a reproductive health and psychosocial issue. It is worth publishing data and conclusions that could be misconstrued and may not make much of a

  20. Oxidative mitochondrial DNA damage in peripheral blood mononuclear cells is associated with reduced volumes of hippocampus and subcortical gray matter in chronically HIV-infected patients

    PubMed Central

    Kallianpur, Kalpana J.; Gerschenson, Mariana; Mitchell, Brooks I.; LiButti, Daniel E.; Umaki, Tracie M.; Ndhlovu, Lishomwa C.; Nakamoto, Beau K.; Chow, Dominic C.; Shikuma, Cecilia M.

    2016-01-01

    Cross-sectional relationships were examined between regional brain volumes and mitochondrial DNA (mtDNA) 8-hydroxy-2-deoxyguanosine (8-oxo-dG) in peripheral blood mononuclear cells (PBMCs) of 47 HIV patients [mean age 51 years; 81% with HIV RNA ≤50 copies/mL] on combination antiretroviral therapy. The gene-specific DNA damage and repair assay measured mtDNA 8-oxo-dG break frequency. Magnetic resonance imaging was performed at 3 T. Higher mtDNA 8-oxo-dG was associated with lateral ventricular enlargement and with decreased volumes of hippocampus, pallidum, and total subcortical gray matter, suggesting the involvement of systemic mitochondrial-specific oxidative stress in chronic HIV-related structural brain changes and cognitive difficulties. Clarification of the mechanism may provide potential therapeutic targets. PMID:26923169

  1. Comparing the impact of increasing condom use or HIV pre-exposure prophylaxis (PrEP) use among female sex workers.

    PubMed

    Mukandavire, Zindoga; Mitchell, Kate M; Vickerman, Peter

    2016-03-01

    In many settings, interventions targeting female sex workers (FSWs) could significantly reduce the overall transmission of HIV. To understand the role HIV pre-exposure prophylaxis (PrEP) could play in controlling HIV transmission amongst FSWs, it is important to understand how its impact compares with scaling-up condom use-one of the proven HIV prevention strategies for FSWs. It is important to remember that condoms also have other benefits such as reducing the incidence of sexually transmitted infections and preventing pregnancy. A dynamic deterministic model of HIV transmission amongst FSWs, their clients and other male partners (termed 'pimps') was used to compare the protection provided by PrEP for HIV-negative FSWs with FSWs increasing their condom use with clients and/or pimps. For different HIV prevalence scenarios, levels of pimp interaction, and baseline condom use, we estimated the coverage of PrEP that gives the same reduction in endemic FSW HIV prevalence or HIV infections averted as different increases in condom use. To achieve the same impact on FSW HIV prevalence as increasing condom use by 1%, the coverage of PrEP has to increase by >2%. The relative impact of PrEP increases for scenarios where pimps contribute to HIV transmission, but not greatly, and decreases with higher baseline condom use. In terms of HIV infections averted over 10 years, the relative impact of PrEP compared to condoms was reduced, with a >3% increase in PrEP coverage achieving the same impact as a 1% increase in condom use. Condom promotion interventions should remain the mainstay HIV prevention strategy for FSWs, with PrEP only being implemented once condom interventions have been maximised or to fill prevention gaps where condoms cannot be used.

  2. Socioeconomic Disconnection as a Risk Factor for Increased HIV Infection in Young Men Who Have Sex with Men

    PubMed Central

    Gayles, Travis A.; Kuhns, Lisa M.; Kwon, Soyang; Mustanski, Brian

    2016-01-01

    Abstract Purpose: HIV disproportionately affects young men who have sex with men (YMSM), particularly black YMSM. Increasingly, researchers are turning to social, economic, and structural factors to explain these disproportionate rates. In this study, we explore the relationship between socioeconomic disconnection and HIV status and factors related to HIV infection, including drug use, condomless anal sex, and binge drinking. We operationalize socioeconomic disconnection in this young population as lack of engagement in educational and employment opportunities. Methods: Baseline data were analyzed from a longitudinal cohort study of YMSM aged 16–20 years recruited from the Chicago area (N = 450). Bivariate analyses of the association of socioeconomic disconnection and HIV-positive status, drug and alcohol use, and condomless anal sex were assessed using chi-square tests. The relationship of socioeconomic disconnection and HIV-positive status was then examined in multivariate logistic regression models, controlling for age and race/ethnicity and significant behavioral factors. Results: Among study participants, 112 (25%) were not in school, 310 (69%) were not currently working, and 81 (18%) were neither in school nor working. Black MSM were more likely to be socioeconomically disconnected (neither in school nor working; n = 56, 23.3%). The results revealed that disconnected YMSM were more likely to binge drink (AOR = 2.34; 95% CI = 1.16, 4.74) and be HIV positive (AOR = 2.24; 95% CI = 1.04, 4.83). Subpopulation analysis for black participants revealed similar associations (AOR of binge drinking = 2.92; 95% CI = 1.07, 8.01; AOR of HIV positive = 2.38; 95% CI = 1.03, 5.51). Controlling for substance use, the association between disconnection and HIV-positive status remained significant (AOR = 2.37; 95% CI = 1.08, 5.20). Conclusion: Socioeconomic disconnection is significantly and positively associated with HIV status

  3. Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming.

    PubMed

    Picard, Martin; Zhang, Jiangwen; Hancock, Saege; Derbeneva, Olga; Golhar, Ryan; Golik, Pawel; O'Hearn, Sean; Levy, Shawn; Potluri, Prasanth; Lvova, Maria; Davila, Antonio; Lin, Chun Shi; Perin, Juan Carlos; Rappaport, Eric F; Hakonarson, Hakon; Trounce, Ian A; Procaccio, Vincent; Wallace, Douglas C

    2014-09-23

    Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling. PMID:25192935

  4. Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse.

    PubMed

    Dillon, Lloye M; Williams, Siôn L; Hida, Aline; Peacock, Jacqueline D; Prolla, Tomas A; Lincoln, Joy; Moraes, Carlos T

    2012-05-15

    Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.

  5. Development of a Multilevel Intervention to Increase HIV Clinical Trial Participation among Rural Minorities

    ERIC Educational Resources Information Center

    Corbie-Smith, Giselle; Odeneye, Ebun; Banks, Bahby; Shandor Miles, Margaret; Roman Isler, Malika

    2013-01-01

    Minorities are disproportionately affected by HIV/AIDS in the rural Southeast; therefore, it is important to develop targeted, culturally appropriate interventions to support rural minority participation in HIV/AIDS research. Using intervention mapping, we developed a comprehensive multilevel intervention for service providers (SPs) and people…

  6. Increased Thermal Stability of Chromatin Containing 5-Bromodeoxyuridine-Substituted DNA

    PubMed Central

    David, John; Gordon, Joel S.; Rutter, William J.

    1974-01-01

    The replacement of thymidine by 5-bromodeoxyuridine in DNA leads to a greatly enhanced stability of chromatin from hepatoma tissue culture or embryonic rat pancreas, as measured by thermal chromatography on hydroxylapatite. The increased stability is directly correlated with the degree of bromodeoxyuridine substitution. On the other hand, the incorporation of bromodeoxyuridine into DNA results in a modest stabilization of purified DNA. Substitution of nucleotide also alters slightly the hyperchromicity profile generated during the thermal denaturation of purified DNA and chromatin. The observed changes can best be explained by an altered interaction between the bromodeoxyuridine-DNA and other chromatin components, presumably proteins. These results suggests that the selective effects of bromodeoxyuridine on cytodifferentiation may be due to an increased affinity of regulatory proteins for bromodeoxyuridine-DNA. PMID:4368811

  7. Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

    PubMed Central

    Coley, Jacqueline S.; Calderon, Tina M.; Gaskill, Peter J.; Eugenin, Eliseo A.; Berman, Joan W.

    2015-01-01

    Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain

  8. Increased Hepatitis E Virus Seroprevalence Correlates with Lower CD4+ Cell Counts in HIV-Infected Persons in Argentina

    PubMed Central

    Debes, José D.; Martínez Wassaf, Maribel; Pisano, María Belén; Isa, María Beatriz; Lotto, Martin; Marianelli, Leonardo G.; Frassone, Natalia; Ballari, Estefania; Bohjanen, Paul R.; Hansen, Bettina E.; Ré, Viviana

    2016-01-01

    Hepatitis E virus (HEV) is a single-stranded RNA virus that can cause hepatitis in an epidemic fashion. HEV usually causes asymptomatic or limited acute infections in immunocompetent individuals, whereas in immunosuppressed individuals such as transplant recipients, HEV can cause chronic infections. The risks and outcomes of HEV co-infection in patients infected with human immunodeficiency virus (HIV) are poorly characterized. We used a third generation immunoassay to measure serum IgG antibodies specific for HEV in 204 HIV-infected individuals from Argentina and a control group of 433 HIV-negative individuals. We found 15 of 204 (7.3%, 95%CI 3.74–10.96%) individuals in the HIV-positive group to have positive HEV IgG levels suggestive of previous infection, compared to 19 of 433 (4.4%, 95% CI 2.5–6.3%) individuals in the HIV-negative control group (p = 0.12). Among HIV-positive individuals, those with HEV seropositivity had lower CD4 counts compared to those that were HEV seronegative (average CD4 count of 234 vs 422 mm3, p = 0.01), indicating that patients with lower CD4 counts were more likely to be HEV IgG positive. Moreover, HEV seropositivity in patients with CD4 counts <200 mm3 was 16%, compared to 4.5% in those with CD4 counts >200 mm3 (p = 0.012). We found a positive PCR result for HEV in one individual. Our study found that increased seroprevalence of HEV IgG correlated with lower CD4 counts in HIV-infected patients in Argentina. PMID:27467394

  9. Increased Hepatitis E Virus Seroprevalence Correlates with Lower CD4+ Cell Counts in HIV-Infected Persons in Argentina.

    PubMed

    Debes, José D; Martínez Wassaf, Maribel; Pisano, María Belén; Isa, María Beatriz; Lotto, Martin; Marianelli, Leonardo G; Frassone, Natalia; Ballari, Estefania; Bohjanen, Paul R; Hansen, Bettina E; Ré, Viviana

    2016-01-01

    Hepatitis E virus (HEV) is a single-stranded RNA virus that can cause hepatitis in an epidemic fashion. HEV usually causes asymptomatic or limited acute infections in immunocompetent individuals, whereas in immunosuppressed individuals such as transplant recipients, HEV can cause chronic infections. The risks and outcomes of HEV co-infection in patients infected with human immunodeficiency virus (HIV) are poorly characterized. We used a third generation immunoassay to measure serum IgG antibodies specific for HEV in 204 HIV-infected individuals from Argentina and a control group of 433 HIV-negative individuals. We found 15 of 204 (7.3%, 95%CI 3.74-10.96%) individuals in the HIV-positive group to have positive HEV IgG levels suggestive of previous infection, compared to 19 of 433 (4.4%, 95% CI 2.5-6.3%) individuals in the HIV-negative control group (p = 0.12). Among HIV-positive individuals, those with HEV seropositivity had lower CD4 counts compared to those that were HEV seronegative (average CD4 count of 234 vs 422 mm3, p = 0.01), indicating that patients with lower CD4 counts were more likely to be HEV IgG positive. Moreover, HEV seropositivity in patients with CD4 counts <200 mm3 was 16%, compared to 4.5% in those with CD4 counts >200 mm3 (p = 0.012). We found a positive PCR result for HEV in one individual. Our study found that increased seroprevalence of HEV IgG correlated with lower CD4 counts in HIV-infected patients in Argentina. PMID:27467394

  10. Prognostic Evaluation of DNA Index in HIV-HPV Co-Infected Women Cervical Samples Attending in Reference Centers for HIV-AIDS in Recife

    PubMed Central

    Martins, Albert Eduardo Silva; Lucena-Silva, Norma; Garcia, Renan Gomes; Welkovic, Stefan; Barbosa, Aureliana; Menezes, Maria Luiza Bezerra; Tenório, Terezinha; Maruza, Magda; Ximenes, Ricardo A. A.

    2014-01-01

    Introduction Persistence of cervical infection caused by human papillomavirus (HPV) types with high oncogenic risk may lead to cervical intraepithelial neoplasia (CIN). The aim of the present study was to evaluate whether, in HIV-positive women, the presence of aneuploidy in cervical cell samples is associated with presence and evolution of CIN. Methods The present study had two stages. In the first stage, comprising a cross-sectional study, the association between the presence of aneuploidy seen via flow cytometry and sociodemographic characteristics, habits and characteristics relating to HPV and HIV infection was analyzed. In the second stage, comprising a cohort study, it was investigated whether aneuploidy was predictive of CIN evolution. Results No association was observed between the presence of aneuploidy and HPV infection, or between its presence and alterations seen in oncotic cytological analysis. On the other hand, aneuploidy was associated with the presence of CIN (p = 0.030) in histological analysis and with nonuse of antiretroviral therapy (p = 0.001). Most of the HIV-positive women (234/272) presented normal CD4+ T lymphocyte counts (greater than 350 cells/mm3) and showed a greater aneuploidy regression rate (77.5%) than a progression rate (23.9%) over a follow-up of up to two years. Conclusion Although there was an association between the presence of cervical tissue lesions and the DNA index, the latter was not predictive of progression of the cervical lesion. This suggests that progression of the cervical lesion to cancer in HIV-positive women may also be changed through improvement of the immunological state enabled by using antiretroviral therapy. PMID:25144309

  11. A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation.

    PubMed

    delBarco-Trillo, Javier; García-Álvarez, Olga; Soler, Ana Josefa; Tourmente, Maximiliano; Garde, José Julián; Roldan, Eduardo R S

    2016-03-16

    Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.

  12. Ultrasound-assessed perirenal fat is related to increased ophthalmic artery resistance index in HIV-1 patients

    PubMed Central

    2010-01-01

    Background The introduction of highly active antiretroviral therapy (HAART) has dramatically changed the prognosis of human immunodeficiency virus (HIV) infection, with a significant decline in morbidity and mortality. Changes in body fat distribution are a common finding in individuals with HIV infection being treated with antiretrovirals, and this condition (collectively termed lipodystrophy syndrome) is associated with depletion of subcutaneous fat, increased triglycerides and insulin resistance. Obesity, particularly visceral obesity, is associated with increased risk of cardiovascular disease. Therefore, estimating visceral fat distribution is important in identifying subjects at high risk for cardiovascular disease. The aim of our study was to evaluate whether perirenal fat thickness (PRFT), a parameter of central obesity, is related to ophthalmic artery resistance index (OARI), an index of occlusive carotid artery disease in HIV-1 infected patients. Methods We enrolled 88 consecutive HIV-1-infected patients receiving highly active antiretroviral therapy for more than 12 months, in a prospective cohort study. Echographically measured PRFT and OARI, as well as serum metabolic parameters, were evaluated. PRFT and OARI were measured by 3.75 MHz convex and 7.5 MHz linear probe, respectively. Results The means of PRFT and OARI in HIV-1-infected patients with visceral obesity was considerably higher than in patients without it (p < 0.0001 and p < 0.001, respectively). Using the average OARI as the dependent variable, total serum cholesterol level, HDL, triglycerides, glycemia, sex, blood pressure, age and PRFT were independent factors associated with OARI. A PRFT of 6.1 mm was the most discriminatory value for predicting an OARI > 0.74 (sensitivity 78.9%, specificity 82.8%). Conclusions Our data indicate that ultrasound assessment of PRFT may have potential as a marker of increased endothelial damage with specific involvement of the ocular vascular region in HIV-1

  13. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys

    PubMed Central

    Castro, María del R.; Suarez, Edu; Kraiselburd, Edmundo; Isidro, Angel; Paz, José; Ferder, León; Ayala-Torres, Sylvette

    2013-01-01

    While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging. PMID:22027539

  14. HIV Infection and Antiretroviral Therapy Have Divergent Effects on Mitochondria in Adipose Tissue

    PubMed Central

    Morse, Caryn G.; Voss, Joachim G.; Rakocevic, Goran; McLaughlin, Mary; Vinton, Carol L.; Huber, Charles; Hu, Xiaojun; Yang, Jun; Huang, Da Wei; Logun, Carolea; Danner, Robert L.; Rangel, Zoila G.; Munson, Peter J.; Orenstein, Jan M.; Rushing, Elisabeth J.; Lempicki, Richard A.; Dalakas, Marinos C.; Kovacs, Joseph A.

    2012-01-01

    Background. Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. Methods. Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)–based ART, and HIV-negative controls. Results. The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+/HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. Conclusions. HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART. PMID:22476717

  15. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

    PubMed

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Livneh, Zvi; Paz-Elizur, Tamar

    2014-12-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT.

  16. Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter.

    PubMed

    el Kharroubi, A; Verdin, E

    1994-08-01

    We have examined by in vitro footprinting a region located downstream of the human immunodeficiency virus, type 1 (HIV-1) promoter found to be hypersensitive to DNase I digestion in vivo. Recognition sites for several constitutive or inducible DNA binding factors were identified. Three AP-1 binding sites and an AP-3-like motif were situated within the R-U5 region of the long terminal repeat. A novel purine-rich motif (5'-GAAAGC-GAAAGDD-3' (D represents G, A, or T residues)), which interacts with a nuclear factor designated downstream binding factor 1 (DBF1), and two juxtaposed Sp-1 binding sites were located in the untranslated sequence immediately downstream of the 5'-long terminal repeat. Genomic footprinting of these sequence elements in the HIV-1 chronically infected cell lines revealed that the DBF1 and Sp-1 sites are occupied in vivo. Furthermore, transient transfection assays showed that point mutations in the DBF1 binding site decreased significantly the HIV-1 basal promoter activity. Taken together, these results suggest that the DBF1 play a role in the HIV-1 transcription regulation. PMID:8051074

  17. Analysis of the Prevalence of HTLV-1 Proviral DNA in Cervical Smears and Carcinomas from HIV Positive and Negative Kenyan Women.

    PubMed

    He, Xiaotong; Maranga, Innocent O; Oliver, Anthony W; Gichangi, Peter; Hampson, Lynne; Hampson, Ian N

    2016-01-01

    The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV-ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck(®)) and cytology. This showed 22/113 (19.5%) of LBC's from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV-ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07-26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV-ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear. PMID:27608036

  18. How does sex trafficking increase the risk of HIV Infection? An observational study from Southern India.

    PubMed

    Wirth, Kathleen E; Tchetgen Tchetgen, Eric J; Silverman, Jay G; Murray, Megan B

    2013-02-01

    Studies have documented the substantial risk of human immunodeficiency virus (HIV) infection endured by sex-trafficked women, but it remains unclear how exposure to trafficking puts its victims at risk. We assessed whether the association between sex trafficking and HIV could be explained by self-reported forced prostitution or young age at entry into prostitution using cross-sectional data collected from 1,814 adult female sex workers in Karnataka, India, between August 2005 and August 2006. Marginal structural logistic regression was used to estimate adjusted odds ratios for HIV infection. Overall, 372 (21%) women met 1 or both criteria used to define sex trafficking: 278 (16%) began sex work before age 18 years, and 107 (5%) reported being forcibly prostituted. Thirteen (0.7%) met both criteria. Forcibly prostituted women were more likely to be HIV-infected than were women who joined the industry voluntarily, independent of age at entering prostitution (odds ratio = 2.30, 95% confidence interval: 1.08, 4.90). Conversely, after adjustment for forced prostitution and other confounders, no association between age at entry into prostitution and HIV was observed. The association between forced prostitution and HIV infection became stronger in the presence of sexual violence (odds ratio = 11.13, 95% confidence interval: 2.41, 51.40). These findings indicate that forced prostitution coupled with sexual violence probably explains the association between sex trafficking and HIV. PMID:23324332

  19. Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

    PubMed

    Leung, Michael Y K; Cohen, Fredric S

    2011-04-20

    T-20/Fuzeon/Enfuvirtide (ENF), a peptide inhibitor of HIV-1 infection, targets the grooves created by heptad repeat 2 (HR2) of Env's coiled-coil, but mutants resistant to ENF emerge. In this study, ENF-resistant mutants--V38A, N43D, N43D/S138A, Q40H/L45M--were combined with modified inhibitory peptides to identify what we believe to be novel ways to improve peptide efficacy. V38A did not substantially reduce infectivity, but was relatively resistant to inhibitory peptides. N43D was more resistant to inhibitory peptides than wild-type, but infectivity was reduced. The additional mutation S138A (N43D/S138A) increased infectivity and further reduced peptide inhibitory potency. It is concluded that S138A increased binding of HR2/ENF into grooves and that S138A compensated for electrostatic repulsion between N43D and HR2. The six-helix bundle structure indicated that E148A should increase hydrophobic interactions between the coiled-coil and peptide. Importantly, the modifications S138A and E148A in the same peptide retained potency against ENF-escape mutants. The double mutant's increase in potency was greater than the increases from the sum of S138A and E148A individually, showing that these two altered residues synergistically contributed to peptide binding. Isothermal titration calorimetry established that hydrophobic substitutions at positions S138 and E148 improved potency of inhibitory peptides against escape mutants by increasing enthalpic release of energy upon peptide binding.

  20. S100A9 Tetramers, Which are Ligands of CD85j, Increase the Ability of MVAHIV-Primed NK Cells to Control HIV Infection

    PubMed Central

    Moreno-Nieves, Uriel Y.; Didier, Céline; Lévy, Yves; Barré-Sinoussi, Françoise; Scott-Algara, Daniel

    2015-01-01

    Natural killer (NK) cells are the major antiviral effector population of the innate immune system. We previously found that S100A9 is a novel ligand of the receptor CD85j and that S100A9 tetramers enhance the anti-HIV activity of NK cells. Also, we found that dendritic cells (DCs) infected by the HIV vaccine candidate, MVAHIV, prime NK cells to specifically control HIV infection in autologous CD4+ T cells. In this study, we analyzed whether stimulation of NK cells by S100A9 tetramers prior to the priming by MVAHIV-infected DCs modulates the subsequent anti-HIV activity of NK cells. We found that S100A9 tetramers activate NK cells and that DCs enhance the anti-HIV activity of NK cells. Interestingly, we observed that stimulation of NK cells by S100A9 tetramers, prior to the priming, significantly increased the subsequent anti-HIV activity of NK cells and that the enhanced anti-HIV activity was observed following different conditions of priming, including the MVAHIV-priming. As S100A9 tetramers alone directly increase the anti-HIV activity of NK cells and as this increased anti-HIV activity is also observed following the interaction of NK cells with MVAHIV-infected DCs, we propose S100A9 tetramers as potential adjuvants to stimulate the anti-HIV activity of NK cells. PMID:26441983

  1. Collaborative study for the calibration of HCV RNA, HBV DNA and HIV RNA reference preparations against the relative international standards.

    PubMed

    Pisani, Giulio; Marino, Francesco; Cristiano, Karen; Bisso, Guillermo Mario; Mele, Caludio; Luciani, Francesca; Wirz, Maria; Gentili, Giuliano

    2007-01-01

    We organised a collaborative study to calibrate three new ISS reference preparations (ISS: Istituto Superiore di Sanità), one for HCV RNA, one for HIV RNA and one for HBV DNA, to be used for nucleic acid amplification techniques (NAT) in blood testing. Serial dilution of the ISS reference preparations and the respective international standards were tested in different days by each participating laboratory using two commercial NAT assays. Data were collected by the ISS for statistical analysis. Based on the mean potency of the HCV RNA and HIV RNA preparations, calculated from the results provided by the 12 participating laboratories, a definitive concentrations of 5700 IU/mL and 4000 IU/mL, respectively, were assigned to the reference materials. On the contrary, it was not possible to obtain a consensus titre for the HBV DNA reference material. These new Italian reference preparations (HCV RNA ISS/1005 and HIV RNA ISS/1005) calibrated against the respective international standards are available free of charge to any laboratory upon request.

  2. Fractionated neem leaf extract is safe and increases CD4+ cell levels in HIV/AIDS patients.

    PubMed

    Mbah, A U; Udeinya, I J; Shu, E N; Chijioke, C P; Nubila, T; Udeinya, F; Muobuike, Angela; Mmuobieri, Ancila; Obioma, Maria S

    2007-01-01

    The safety and effect of an acetone-water neem leaf extract (IRAB) on CD4 cells was investigated in 60 HIV/AIDS patients as part of an ongoing study to determine the influence of neem on immunity and viral load in HIV/AIDS. Patients were confirmed as HIV I or II positive, as having CD4 cell count, less than 300 cells/microL, and as antiretrovirally naïve. They were given oral IRAB (1.0 g daily for 12 weeks). Clinical and laboratory tests were carried out at baseline and at 4 weekly intervals. Thus, the patients served as their own controls. Sixty patients completed treatment. Fifty (83.33%) were completely compliant with respect to laboratory tests. Increase in mean CD4 cells, 266 cells/microL (159%), for the 50 patients was significant (P < 0.001) between baseline and week 12. Erythrocyte sedimentation rate (64 mm/hr at baseline) was 16 mm/hr at week 12, whereas total number of incidences of HIV/AIDS-related pathologies decreased from 120 at baseline to 5. Mean bodyweight, hemoglobin concentration, and lymphocyte differential count increased significantly by 12% (P < 0.05), 24% (P < 0.0001), and 20% (P < 0.0001), respectively. There were no adverse effects and no abnormalities in kidney and liver function parameters. The results support the safety of IRAB in HIV/AIDS, and its significant influence on CD4 cells may be useful in the formulation of multidrug combination therapies for HIV/AIDS. However, its antiretroviral activity is being evaluated in our laboratory.

  3. Fractionated neem leaf extract is safe and increases CD4+ cell levels in HIV/AIDS patients.

    PubMed

    Mbah, A U; Udeinya, I J; Shu, E N; Chijioke, C P; Nubila, T; Udeinya, F; Muobuike, Angela; Mmuobieri, Ancila; Obioma, Maria S

    2007-01-01

    The safety and effect of an acetone-water neem leaf extract (IRAB) on CD4 cells was investigated in 60 HIV/AIDS patients as part of an ongoing study to determine the influence of neem on immunity and viral load in HIV/AIDS. Patients were confirmed as HIV I or II positive, as having CD4 cell count, less than 300 cells/microL, and as antiretrovirally naïve. They were given oral IRAB (1.0 g daily for 12 weeks). Clinical and laboratory tests were carried out at baseline and at 4 weekly intervals. Thus, the patients served as their own controls. Sixty patients completed treatment. Fifty (83.33%) were completely compliant with respect to laboratory tests. Increase in mean CD4 cells, 266 cells/microL (159%), for the 50 patients was significant (P < 0.001) between baseline and week 12. Erythrocyte sedimentation rate (64 mm/hr at baseline) was 16 mm/hr at week 12, whereas total number of incidences of HIV/AIDS-related pathologies decreased from 120 at baseline to 5. Mean bodyweight, hemoglobin concentration, and lymphocyte differential count increased significantly by 12% (P < 0.05), 24% (P < 0.0001), and 20% (P < 0.0001), respectively. There were no adverse effects and no abnormalities in kidney and liver function parameters. The results support the safety of IRAB in HIV/AIDS, and its significant influence on CD4 cells may be useful in the formulation of multidrug combination therapies for HIV/AIDS. However, its antiretroviral activity is being evaluated in our laboratory. PMID:17667213

  4. 2'-deoxy-5-azacytidine increases binding of cisplatin to DNA by a mechanism independent of DNA hypomethylation.

    PubMed Central

    Ellerhorst, J. A.; Frost, P.; Abbruzzese, J. L.; Newman, R. A.; Chernajovsky, Y.

    1993-01-01

    The chemotherapeutic agents 2'-deoxy-5-azacytidine (DAC) and cisplatin (cDDP) have been shown in vitro to be synergystic in their cytotoxicity toward human tumour cells. We have investigated possible molecular mechanisms underlying this synergy using the plasmid pSVE3 in vitro and after transfection into CMT3 cells. Increased binding of cDDP to DAC-substituted DNA generated in vivo was confirmed by flameless atomic absorption spectrophotometry (FAAS). The plasmid used in these experiments was unmethylated suggesting that DAC was effective in enhancing cDDP binding to DNA without acting as a hypomethylating agent, but by directly changing the topology of DNA. The role of DNA methylation in cDDP binding was studied using methylated and unmethylated plasmid incubated in vitro with cDDP. Restriction analyses and FAAS measurement of bound platinum indicated that methylated DNA bound more cDDP than unmethylated DNA. In addition, in vivo studies confirmed the in vitro observations since replication of methylated plasmid was inhibited to a greater extent than unmethylated plasmid. Images Figure 2 Figure 3 Figure 4 PMID:7679279

  5. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

    PubMed

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-04-02

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

  6. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs

    PubMed Central

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-01-01

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors. PMID:25837486

  7. Mononuclear phagocyte accumulation in visceral tissue in HIV encephalitis: evidence for increased monocyte/macrophage trafficking and altered differentiation.

    PubMed

    Fischer, Tracy; Wyatt, Christina M; D'Agati, Vivette D; Croul, Sidney; McCourt, Laura; Morgello, Susan; Rappaport, Jay

    2014-01-01

    The invasion of circulating monocytes/macrophages (MΦ)s from the peripheral blood into the central nervous system (CNS) appears to play an important role in the pathogenesis of HIV dementia (HIV-D), the most severe form of HIV-associated neurocognitive disorders (HAND), often confirmed histologically as HIV encephalitis (HIVE). In order to determine if trafficking of monocytes/MΦs is exclusive to the CNS or if it also occurs in organs outside of the brain, we have focused our investigation on visceral tissues of patients with HIVE. Liver, lymph node, spleen, and kidney autopsy tissues from the same HIVE cases investigated in earlier studies were examined by immunohistochemistry for the presence of CD14, CD16, CD68, Ki-67, and HIV-1 p24 expression. Here, we report a statistically significant increase in accumulation of MΦs in kidney, spleen, and lymph node tissues in specimens from patients with HIVE. In liver, we did not observe a significant increase in parenchymal macrophage accumulation, although perivascular macrophage accumulation was consistently observed with nodular lesions in 4 of 5 HIVE cases. We also observed an absence of CD14 expression on splenic MΦs in HIVE cases, which may implicate the spleen as a potential source of increased plasma soluble CD14 in HIV infection. HIV-1 p24 expression was observed in liver, lymph node and spleen but not kidney. Interestingly, renal pathology suggestive of chronic tubulointerstitial nephritis (possibly due to chronic pyelonephritis), including tubulointerstitial scarring, chronic interstitial inflammation and focal global glomerulosclerosis, without evidence of HIV-associated nephropathy (HIVAN), was seen in four of eight HIVE cases. Focal segmental and global glomerulosclerosis with tubular dilatation and prominent interstitial inflammation, consistent with HIVAN, was observed in two of the eight cases. Abundant cells expressing monocyte/MΦ cell surface markers, CD14 and CD68, were also CD16(+) and found

  8. Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1 Transmission: A Prospective Cohort Analysis among African Couples

    PubMed Central

    Cohen, Craig R.; Lingappa, Jairam R.; Baeten, Jared M.; Ngayo, Musa O.; Spiegel, Carol A.; Hong, Ting; Donnell, Deborah; Celum, Connie; Kapiga, Saidi; Delany, Sinead; Bukusi, Elizabeth A.

    2012-01-01

    Background Bacterial vaginosis (BV), a disruption of the normal vaginal flora, has been associated with a 60% increased risk of HIV-1 acquisition in women and higher concentration of HIV-1 RNA in the genital tract of HIV-1–infected women. However, whether BV, which is present in up to half of African HIV-1–infected women, is associated with an increase in HIV-1 transmission to male partners has not been assessed in previous studies. Methods and Findings We assessed the association between BV on female-to-male HIV-1 transmission risk in a prospective study of 2,236 HIV-1–seropositive women and their HIV-1 uninfected male partners from seven African countries from a randomized placebo-controlled trial that enrolled heterosexual African adults who were seropositive for both HIV-1 and herpes simplex virus (HSV)-2, and their HIV-1–seronegative partners. Participants were followed for up to 24 months; every three months, vaginal swabs were obtained from female partners for Gram stain and male partners were tested for HIV-1. BV and normal vaginal flora were defined as a Nugent score of 7–10 and 0–3, respectively. To reduce misclassification, HIV-1 sequence analysis of viruses from seroconverters and their partners was performed to determine linkage of HIV-1 transmissions. Overall, 50 incident HIV-1 infections occurred in men in which the HIV-1–infected female partner had an evaluable vaginal Gram stain. HIV-1 incidence in men whose HIV-1–infected female partners had BV was 2.91 versus 0.76 per 100 person-years in men whose female partners had normal vaginal flora (hazard ratio 3.62, 95% CI 1.74–7.52). After controlling for sociodemographic factors, sexual behavior, male circumcision, sexually transmitted infections, pregnancy, and plasma HIV-1 RNA levels in female partners, BV was associated with a greater than 3-fold increased risk of female-to-male HIV-1 transmission (adjusted hazard ratio 3.17, 95% CI 1.37–7.33). Conclusions This study identified an

  9. Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).

    PubMed Central

    Patterson, B K; Jiyamapa, D; Mayrand, E; Hoff, B; Abramson, R; Garcia, P M

    1996-01-01

    The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals. PMID:8836201

  10. Increased breadth and depth of cytotoxic T lymphocytes responses against HIV-1-B Nef by inclusion of epitope variant sequences.

    PubMed

    Rolland, Morgane; Frahm, Nicole; Nickle, David C; Jojic, Nebojsa; Deng, Wenjie; Allen, Todd M; Brander, Christian; Heckerman, David E; Mullins, James I

    2011-03-28

    Different vaccine approaches cope with HIV-1 diversity, ranging from centralized(1-4) to variability-encompassing(5-7) antigens. For all these strategies, a concern remains: how does HIV-1 diversity impact epitope recognition by the immune system? We studied the relationship between HIV-1 diversity and CD8(+) T Lymphocytes (CTL) targeting of HIV-1 subtype B Nef using 944 peptides (10-mers overlapping by nine amino acids (AA)) that corresponded to consensus peptides and their most common variants in the HIV-1-B virus population. IFN-γ ELISpot assays were performed using freshly isolated PBMC from 26 HIV-1-infected persons. Three hundred and fifty peptides elicited a response in at least one individual. Individuals targeted a median of 7 discrete regions. Overall, 33% of responses were directed against viral variants but not elicited against consensus-based test peptides. However, there was no significant relationship between the frequency of a 10-mer in the viral population and either its frequency of recognition (Spearman's correlation coefficient ρ = 0.24) or the magnitude of the responses (ρ = 0.16). We found that peptides with a single mutation compared to the consensus were likely to be recognized (especially if the change was conservative) and to elicit responses of similar magnitude as the consensus peptide. Our results indicate that cross-reactivity between rare and frequent variants is likely to play a role in the expansion of CTL responses, and that maximizing antigenic diversity in a vaccine may increase the breadth and depth of CTL responses. However, since there are few obvious preferred pathways to virologic escape, the diversity that may be required to block all potential escape pathways may be too large for a realistic vaccine to accommodate. Furthermore, since peptides were not recognized based on their frequency in the population, it remains unclear by which mechanisms variability-inclusive antigens (i.e., constructs enriched with frequent

  11. Increased mitochondrial DNA deletions in substantia nigra dopamine neurons of the aged rat.

    PubMed

    Parkinson, Gemma M; Dayas, Christopher V; Smith, Doug W

    2014-01-01

    The dopaminergic neurons of the substantia nigra (SN), which constitute the origin of the nigrostriatal system, are vulnerable to age-related degenerative processes. For example, in humans there is a relatively small age-related loss of neurons but a marked decline of the dopaminergic phenotype associated with impaired voluntary motor control. However, the mechanisms responsible for the dysfunction and degeneration of SN dopamine neurons remain poorly understood. One potential contributor is mitochondrial dysfunction, resulting from an increased abundance of mitochondrial DNA (mtDNA) mutations such as deletions. Human studies have identified relatively high levels of mtDNA deletions in these cells in both aging and Parkinson's disease (>35%), with a higher abundance of deletions (>60%) in individual neurons with mitochondrial dysfunction. However, it is unknown whether similar mtDNA mutations occur in other species such as the rat. In the present study, we quantified mtDNA deletion abundance in laser microdissected SN dopaminergic neurons from young and old F344 rats. Our results indicate that mtDNA deletions accumulated with age, with approximately 20% more mtDNA deletions in SN dopaminergic neurons from old compared to young animals. Thus, while rat SN dopaminergic neurons do accumulate mtDNA deletions with aging, this does not reflect the deletion burden in humans, and other mechanisms may be operating to compensate for age-related mtDNA damage in the rat SN dopaminergic neurons. PMID:25612740

  12. Competitive assembly to increase the performance of the DNA/carbon-nanomaterial-based sensing platform.

    PubMed

    Tang, Qiao; Zhang, Qier; Jiang, Ying; Li, Jishan; Zheng, Jing; Li, Yinhui; Yang, Ronghua; Tan, Weihong

    2014-08-27

    Increasing the rate of target binding on the surface and enhancing the fluorescence signal restoration efficiency are critical to the desirable biomedical application of carbon nanomaterials, for example, single-walled carbon nanotubes (SWNTs). We describe here a strategy to increase the target binding rate and enhance the fluorescence signal restoration efficiency on the DNA-functionalized SWNT surface using a short complementary DNA (scDNA) strand. The scDNA causes up to a 2.5-fold increase in association rate and 4-fold increase in fluorescence signal restoration by its competitive assembly on the nanostructure's surface and inducing a conformational change that extends the DNA away from the surface, making it more available to bind target nucleic acids. The scDNA-induced enhancement of binding kinetics and fluorescence signal restoration efficiency is a general phenomenon that occurred with all sequences and surfaces investigated. Through this competitive assembly strategy of scDNA, performance improvement of the carbon-nanomaterial-based biosensing platform for both in vitro detection and live cell imaging can be reached.

  13. Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

    PubMed Central

    Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

    2016-01-01

    ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's

  14. D-Dimer Levels before HIV Seroconversion Remain Elevated Even after Viral Suppression and Are Associated with an Increased Risk of Non-AIDS Events

    PubMed Central

    Freiberg, Matthew S.; Bebu, Ionut; Tracy, Russell; So-Armah, Kaku; Okulicz, Jason; Ganesan, Anuradha; Armstrong, Adam; O’Bryan, Thomas; Rimland, David; Justice, Amy C.; Agan, Brian K.

    2016-01-01

    The mechanism underlying the excess risk of non-AIDS diseases among HIV infected people is unclear. HIV associated inflammation/hypercoagulability likely plays a role. While antiretroviral therapy (ART) may return this process to pre-HIV levels, this has not been directly demonstrated. We analyzed data/specimens on 249 HIV+ participants from the US Military HIV Natural History Study, a prospective, multicenter observational cohort of >5600 active duty military personnel and beneficiaries living with HIV. We used stored blood specimens to measure D-dimer and Interleukin-6 (IL-6) at three time points: pre-HIV seroconversion, ≥6 months post-HIV seroconversion but prior to ART initiation, and ≥6 months post-ART with documented HIV viral suppression on two successive evaluations. We evaluated the changes in biomarker levels between time points, and the association between these biomarker changes and future non-AIDS events. During a median follow-up of 3.7 years, there were 28 incident non-AIDS diseases. At ART initiation, the median CD4 count was 361cells/mm3; median duration of documented HIV infection 392 days; median time on ART was 354 days. Adjusted mean percent increase in D-dimer levels from pre-seroconversion to post-ART was 75.1% (95% confidence interval 24.6–148.0, p = 0.002). This increase in D-dimer was associated with a significant 22% increase risk of future non-AIDS events (p = 0.03). Changes in IL-6 levels across time points were small and not associated with future non-AIDS events. In conclusion, ART initiation and HIV viral suppression does not eliminate HIV associated elevation in D-dimer levels. This residual pathology is associated with an increased risk of future non-AIDS diseases. PMID:27088215

  15. D-Dimer Levels before HIV Seroconversion Remain Elevated Even after Viral Suppression and Are Associated with an Increased Risk of Non-AIDS Events.

    PubMed

    Freiberg, Matthew S; Bebu, Ionut; Tracy, Russell; So-Armah, Kaku; Okulicz, Jason; Ganesan, Anuradha; Armstrong, Adam; O'Bryan, Thomas; Rimland, David; Justice, Amy C; Agan, Brian K

    2016-01-01

    The mechanism underlying the excess risk of non-AIDS diseases among HIV infected people is unclear. HIV associated inflammation/hypercoagulability likely plays a role. While antiretroviral therapy (ART) may return this process to pre-HIV levels, this has not been directly demonstrated. We analyzed data/specimens on 249 HIV+ participants from the US Military HIV Natural History Study, a prospective, multicenter observational cohort of >5600 active duty military personnel and beneficiaries living with HIV. We used stored blood specimens to measure D-dimer and Interleukin-6 (IL-6) at three time points: pre-HIV seroconversion, ≥6 months post-HIV seroconversion but prior to ART initiation, and ≥6 months post-ART with documented HIV viral suppression on two successive evaluations. We evaluated the changes in biomarker levels between time points, and the association between these biomarker changes and future non-AIDS events. During a median follow-up of 3.7 years, there were 28 incident non-AIDS diseases. At ART initiation, the median CD4 count was 361cells/mm3; median duration of documented HIV infection 392 days; median time on ART was 354 days. Adjusted mean percent increase in D-dimer levels from pre-seroconversion to post-ART was 75.1% (95% confidence interval 24.6-148.0, p = 0.002). This increase in D-dimer was associated with a significant 22% increase risk of future non-AIDS events (p = 0.03). Changes in IL-6 levels across time points were small and not associated with future non-AIDS events. In conclusion, ART initiation and HIV viral suppression does not eliminate HIV associated elevation in D-dimer levels. This residual pathology is associated with an increased risk of future non-AIDS diseases.

  16. D-Dimer Levels before HIV Seroconversion Remain Elevated Even after Viral Suppression and Are Associated with an Increased Risk of Non-AIDS Events.

    PubMed

    Freiberg, Matthew S; Bebu, Ionut; Tracy, Russell; So-Armah, Kaku; Okulicz, Jason; Ganesan, Anuradha; Armstrong, Adam; O'Bryan, Thomas; Rimland, David; Justice, Amy C; Agan, Brian K

    2016-01-01

    The mechanism underlying the excess risk of non-AIDS diseases among HIV infected people is unclear. HIV associated inflammation/hypercoagulability likely plays a role. While antiretroviral therapy (ART) may return this process to pre-HIV levels, this has not been directly demonstrated. We analyzed data/specimens on 249 HIV+ participants from the US Military HIV Natural History Study, a prospective, multicenter observational cohort of >5600 active duty military personnel and beneficiaries living with HIV. We used stored blood specimens to measure D-dimer and Interleukin-6 (IL-6) at three time points: pre-HIV seroconversion, ≥6 months post-HIV seroconversion but prior to ART initiation, and ≥6 months post-ART with documented HIV viral suppression on two successive evaluations. We evaluated the changes in biomarker levels between time points, and the association between these biomarker changes and future non-AIDS events. During a median follow-up of 3.7 years, there were 28 incident non-AIDS diseases. At ART initiation, the median CD4 count was 361cells/mm3; median duration of documented HIV infection 392 days; median time on ART was 354 days. Adjusted mean percent increase in D-dimer levels from pre-seroconversion to post-ART was 75.1% (95% confidence interval 24.6-148.0, p = 0.002). This increase in D-dimer was associated with a significant 22% increase risk of future non-AIDS events (p = 0.03). Changes in IL-6 levels across time points were small and not associated with future non-AIDS events. In conclusion, ART initiation and HIV viral suppression does not eliminate HIV associated elevation in D-dimer levels. This residual pathology is associated with an increased risk of future non-AIDS diseases. PMID:27088215

  17. Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

    PubMed Central

    Calloway, Cassandra

    2016-01-01

    BACKGROUND. Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS. Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS. Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20–40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION. Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. FUNDING. This project was supported by Children’s Hospital & Research Center Oakland Institutional Research Award and by the National Center for Advancing Translational Sciences, NIH, through UCSF-CTSI grant UL1 TR000004. PMID:27583305

  18. Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

    PubMed Central

    Lal, Ashutosh; Gomez, Esteban; Calloway, Cassandra

    2016-01-01

    BACKGROUND Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20–40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. PMID:27583305

  19. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice

    PubMed Central

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

    2016-01-01

    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction. PMID:26305669

  20. Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; De Spiegelaere, Ward

    2014-01-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  1. Educational Intervention Increased Referrals to Allopathic Care by Traditional Healers in Three High HIV-Prevalence Rural Districts in Mozambique

    PubMed Central

    Audet, Carolyn M.; Salato, José; Blevins, Meridith; Amsalem, David; Vermund, Sten H.; Gaspar, Felisbela

    2013-01-01

    Introduction Delayed uptake of clinical services impedes favorable clinical outcomes in Mozambique. Care is delayed among patients who initiate care with traditional healers; patients with conditions like human immunodeficiency virus (HIV) or tuberculosis are rarely referred to the health system in a timely fashion. Methods We conducted a pre-post educational intervention with traditional healers, assessing healer referral rates and HIV knowledge in three rural districts in Zambézia Province. Results The median monthly referral rate prior to the intervention was 0.25 patients (interquartile range [IQR]: 0–0.54) compared with a post-intervention rate of 0.34 patients (IQR: 0–0.71), a 35% increase (p = 0.046). A median HIV knowledge score of 67% (IQR: 59–78) was noted 4-months pre-intervention and a median score of 81% (IQR: 74–89) was recorded 2½ months post-intervention (p<0.001). One hundred and eleven healers referred 127 adults, 36 pregnant women, and 188 children to health facilities. Referred patients were most likely to be diagnosed with bronchopneumonia (20% adults; 13% children) and/or malaria (15% adults; 37% children). Of 315 non-pregnant persons referred, 3.5% were tested for HIV and 2.5% were tested for tuberculosis. Discussion We engaged traditional healers with some success; referral rates were low, but increased post-intervention. Once seen in the clinics, patients were rarely tested for HIV or tuberculosis, though symptoms suggested screening was indicated. We found increased referral rates through an inexpensive intervention with traditional healers, a viable, cost-effective method of directing patients to health facilities. However, quality improvement within the clinics is necessary before a substantial impact can be expected. PMID:23936407

  2. Enhancing benefits or increasing harms: community responses for HIV among men who have sex with men, transgender women, female sex workers, and people who inject drugs.

    PubMed

    Baral, Stefan; Holland, Claire E; Shannon, Kate; Logie, Carmen; Semugoma, Paul; Sithole, Bhekie; Papworth, Erin; Drame, Fatou; Beyrer, Chris

    2014-08-15

    Studies completed over the past 15 years have consistently demonstrated the importance of community-level determinants in potentiating or mitigating risks for the acquisition and transmission of HIV. Structural determinants are especially important in mediating HIV risk among key populations, including men who have sex with men, people who inject drugs, sex workers of all genders, and transgender women. The objective of this systematic review was to synthesize the evidence characterizing the community-level determinants that potentiate or mitigate HIV-related outcomes for key populations. The results of the review suggest that although health communication programs represent community-level strategies that have demonstrated the effectiveness in increasing the uptake of HIV testing and decreasing the experienced stigma among people living with HIV, there are limited studies focused on key populations in low- and middle-income settings. Moreover, interpretation from the 22 studies that met inclusion and exclusion criteria reinforce the importance of the continued measurement of community-level determinants of HIV risks and of the innovation in tools to effectively address these risks as components of the next generation of the HIV response. Consequently, the next generation of effective HIV prevention science research must improve our understanding of the multiple levels of HIV risk factors, while programming for key populations must address each of these risk levels. Failure to do so will cost lives, harm communities, and undermine the gains of the HIV response.

  3. Strong HIV-specific CD4+ and CD8+ T-lymphocyte proliferative responses in healthy individuals immunized with an HIV-1 DNA vaccine and boosted with recombinant modified vaccinia virus ankara expressing HIV-1 genes.

    PubMed

    Aboud, Said; Nilsson, Charlotta; Karlén, Katarina; Marovich, Mary; Wahren, Britta; Sandström, Eric; Gaines, Hans; Biberfeld, Gunnel; Godoy-Ramirez, Karina

    2010-07-01

    We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag, env, rev, and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag, env, and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [(3)H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed (n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [(3)H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4(+) T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8(+) T-cell responses. There was strong correlation between the proliferative responses measured by the [(3)H]thymidine uptake assay and CD4(+) T-cell FASCIA-WB (r = 0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4(+) T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8(+) T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [(3)H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [(3)H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

  4. Increased leukocyte mitochondrial DNA copy number is associated with oral premalignant lesions: an epidemiology study

    PubMed Central

    He, Yonggang; Gong, Yilei; Gu, Jian; Lee, J.Jack; Lippman, Scott M.; Wu, Xifeng

    2014-01-01

    Although changes in the mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) have been linked to increased susceptibility to several cancers, the relationship between the mtDNA copy number in PBLs and the risk of cancer precursors has not been investigated. In this study, we measured the relative mtDNA copy number in PBLs of 143 patients with histologically confirmed oral premalignant lesions (OPLs) and of 357 healthy controls that were frequency-matched to patients according to age, sex and race. OPL patients had a significantly higher mtDNA copy number than the controls (1.36±0.74 versus 1.11±0.32; P < 0.001). In analyses stratified by sex, race, alcohol consumption and smoking status, the mtDNA copy number was higher in the OPL patients than in the controls in all the strata. Using the median mtDNA copy number in the control group as a cutoff, we found that individuals with a high mtDNA copy number had significantly higher risk of having OPLs than individuals with a low mtDNA copy number (adjusted odds ratio, 1.93; 95% confidence interval, 1.23–3.05, P = 0.004). Analysis of the joint effect of alcohol consumption and smoking revealed even greater risk for OPLs. Our results suggest that high mtDNA copy number in PBLs is significantly associated with having OPLs. To our knowledge, this is the first epidemiologic study to show that the mtDNA copy number may indicate the risk of cancer precursors. PMID:24743515

  5. Lysine directed cross-linking of viral DNA-RNA:DNA hybrid substrate to the isolated RNase H domain of HIV-1 reverse transcriptase.

    PubMed

    Guaitiao, Juan P; Zúñiga, Roberto A; Roth, Monica J; Leon, Oscar

    2004-02-10

    An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA-RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn(2+). The Mn(2+) titration of cross-linking paralleled the Mn(2+) requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H-nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.

  6. A randomized controlled trial of a culturally congruent intervention to increase condom use and HIV testing among heterosexually active immigrant Latino men.

    PubMed

    Rhodes, Scott D; McCoy, Thomas P; Vissman, Aaron T; DiClemente, Ralph J; Duck, Stacy; Hergenrather, Kenneth C; Foley, Kristie Long; Alonzo, Jorge; Bloom, Fred R; Eng, Eugenia

    2011-11-01

    This randomized controlled trial tested the efficacy of an HIV prevention intervention to increase condom use and HIV testing among Spanish-speaking, heterosexually active immigrant Latino men. A community-based participatory research partnership developed the intervention and selected the study design. Following baseline data collection, 142 immigrant Latino men were randomized to the HIV prevention intervention or the cancer education intervention. Three-month follow-up data were collected from 139 participants, for a 98% retention rate. Mean age of participants was 31.6 years and 60% reported being from Mexico. Adjusting for baseline behaviors, relative to their peers in the cancer education comparison, participants in the HIV prevention intervention were more likely to report consistent condom use and receiving an HIV test. Community-based interventions for immigrant Latino men that are built on state of the art prevention science and developed in partnership with community members can greatly enhance preventive behaviors and may reduce HIV infection.

  7. Increasing quality of life and reducing HIV burden: the PATH+ intervention.

    PubMed

    Blank, Michael B; Hennessy, Michael; Eisenberg, Marlene M

    2014-04-01

    The heightened risk of persons with serious mental illness (SMI) to contract and transmit human immunodeficiency virus (HIV) is a public health problem. Our objective was test the effectiveness of a community-based advanced practice nurse intervention to promote adherence to HIV and psychiatric treatment regimens call Preventing AIDS Through Health for Positives (PATH+). We enrolled 238 HIV-positive subjects with SMI who were in treatment at community HIV provider agencies from 2004 to 2009. Participants in the intervention group were assigned an advanced practice nurse who provided community-based care management at a minimum of one visit/week and coordinated their medical and mental healthcare for 12 months. A parallel process latent growth curve model using three data points for biomarkers (baseline, 12 and 24 months) and five data points for health related quality of life (baseline, 3, 6, 12, and 24 months) showed moderate to excellent fit for modeling changes in CD4, viral load, and mental and physical SF-12 subscales. Results suggest that positive effects for PATH+ persisted at 24 months; 12 months after the intervention ended. This project demonstrates the effectiveness of a nurse-led, community-based, individually tailored adherence intervention. We demonstrated improved outcomes in individuals with HIV/SMI and regarding health-related quality of life and reductions in disease burden. PMID:24000053

  8. Does hormonal contraceptive use increase women’s risk of HIV acquisition? A meta-analysis of observational studies

    PubMed Central

    Ralph, Lauren J.; McCoy, Sandra I.; Shiu, Karen; Padian, Nancy S.

    2015-01-01

    Background Epidemiologic research has yielded inconsistent evidence on whether use of hormonal contraception (HC) increases women’s risk of HIV acquisition. A robust meta-analysis of existing data can yield a valid summary estimate to inform guidelines, models and future studies. Methods We updated a recent systematic review to identify studies examining the relationship between various HC methods and women’s risk of HIV. We assessed statistical heterogeneity, and, when appropriate, combined point estimates using random effects models. We explored heterogeneity through subgroup and stratified analyses according to study populations and design features. Findings We identified 26 studies, 12 of which met inclusion criteria. There was evidence of a modest increase in HIV risk in the ten studies examining depot-medroxyprogesterone acetate (DMPA) [pooled relative risk (RR) =1.40, 95% CI: 1.16, 1.69]. This risk was lower in the eight studies conducted with women in the general population [pooled RR=1.31, 95% CI: 1.10, 1.57]. There was substantial between study heterogeneity in secondary analyses of trials (n=7, I2=51.1%). Although individual study estimates suggested an elevated risk, substantial heterogeneity between the two studies conducted with high risk women (I2=54%) precluded pooling estimates. There was no evidence of an elevated HIV risk in the ten studies examining oral contraceptive pills (OCPs) [pooled RR = 1.00, 95% CI: 0.86, 1.16] or the five studies examining norethisterone enanthate (Net-En) ([pooled RR=1.10; 95% CI: 0.88, 1.37]. Interpretation The risks of HIV found here would not merit complete withdrawal of DMPA, OCPs, or Net-En from the contraceptive method mix in most settings for women in the general population. PMID:25578825

  9. Discrepancies in diagnosis of incident HIV infection between antibody- and DNA-based tests in a phase III prevention trial in southern Africa.

    PubMed

    Montgomery, E T; van der Pol, B; van der Straten, A; Ramjee, G; de Bruyn, G; Chipato, T; Blanchard, K; Padian, N S

    2012-09-01

    Dried blood spots (DBS) are widely used to test for HIV in a variety of research and service delivery settings; however, uniform guidelines regarding collection, storage and DNA extraction processes have neither been developed nor evaluated. Previously published reports suggested DBS may be stored at room temperature for up to 60 days, and intensive stability tests have shown that DBS can withstand high temperatures, humidity and freeze-thawing. During the implementation of a large randomized controlled trial (RCT) in southern Africa, with HIV acquisition as the primary endpoint, we observed 65 instances when DBS samples collected from the same day as a positive HIV antibody test yielded negative DNA polymerase chain reaction (PCR) results. The source of this discrepancy may have been due to inadequate specimen volume, filter paper or DNA extraction procedures, but were most likely due to storage conditions that have been reported as acceptable in other settings. PMID:23033520

  10. Increased sensitivity to cocaine self-administration in HIV-1 transgenic rats is associated with changes in striatal dopamine transporter binding

    PubMed Central

    McIntosh, Scot; Sexton, Tammy; Pattison, Lindsey P.; Childers, Steven R.; Hemby, Scott E.

    2015-01-01

    Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [125I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([125I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the

  11. Increased Sensitivity to Cocaine Self-Administration in HIV-1 Transgenic Rats is Associated with Changes in Striatal Dopamine Transporter Binding.

    PubMed

    McIntosh, Scot; Sexton, Tammy; Pattison, Lindsey P; Childers, Steven R; Hemby, Scott E

    2015-09-01

    Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [(125)I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125)I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the

  12. Increased Drug Use and STI Risk with Injection Drug Use Among HIV-Seronegative Heterosexual Methamphetamine Users†

    PubMed Central

    Cheng, W. Susan; Garfein, Richard S.; Semple, Shirley J.; Strathdee, Steffanie A.; Zians, James K.; Patterson, Thomas L.

    2010-01-01

    Methamphetamine (MA) use has been found to be associated with increased risk of HIV and sexually transmitted infections (STI) among men having sex with men, but it is unknown whether those who inject MA are at greater risk for these infections than those who administer MA by other routes. Furthermore, comparable data from heterosexual MA users are lacking. We investigated whether the HIV and STI risks of male and female heterosexual MA users who inject MA differ from those of comparable users who do not inject. Between 2001 and 2005, we interviewed 452 HIV-negative men and women aged 18 and older who had recently used MA and engaged in unprotected sex. Their mean age was 36.6 years; 68% were male; ethnicity was 49.4% Caucasian, 26.8% African-American, and 12.8% Hispanic. Logistic regression identified factors associated with injecting MA. Compared to non-IDU, IDU were more likely to: be Caucasian; be homeless; have used MA for a longer period and used more grams of MA in the last 30 days; have a history of felony conviction; and report a recent STI. HIV and STI prevention interventions should be tailored according to MA users’ method of administration. PMID:20464802

  13. Folate supplementation increases genomic DNA methylation in the liver of elder rats.

    PubMed

    Choi, Sang-Woon; Friso, Simonetta; Keyes, Mary K; Mason, Joel B

    2005-01-01

    The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.

  14. Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

    PubMed Central

    Palmer, S; Hamblin, A S

    1993-01-01

    In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease. PMID:8103716

  15. Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides.

    PubMed

    Rollenhagen, C; Lathrop, M J; Macura, S L; Doncel, G F; Asin, S N

    2014-09-01

    Herpes Simplex virus Type-2 (HSV-2) increases the risk of HIV-1 acquisition, yet the mechanism for this viral pathogen to regulate the susceptibility of the cervicovaginal mucosa to HIV-1 is virtually unknown. Using ex vivo human ectocervical tissue models, we report greater levels of HIV-1 reverse transcription, DNA integration, RNA expression, and virions release in HIV-1/HSV-2 co-infected tissues compared with HIV-1 only infected tissues (P<0.05). Enhanced HIV-1 replication was associated with increased CD4, CCR5, and CD38 transcription (P<0.05) and increased number of CD4(+)/CCR5(+)/CD38(+) T cells in HIV-1/HSV-2 co-infected tissues compared with tissues infected with HIV-1 alone. Tenofovir (TFV) 1% gel, the leading microbicide candidate, demonstrated only partial protection against HIV-1, when applied vaginally before and after sexual intercourse. It is possible that mucosal inflammation, in particular that induced by HSV-2 infection, may have decreased TFV efficacy. HSV-2 upregulated the number of HIV-1-infected cells and elevated the concentration of TFV needed to decrease HIV-1 infection. Similarly, only high concentrations of TFV inhibited HSV-2 replication in HIV-1/HSV-2-infected tissues. Thus, HSV-2 co-infection and mucosal immune cell activation should be taken into consideration when designing preventative strategies for sexual transmission of HIV-1.

  16. Mechanisms of HIV Entry into the CNS: Increased Sensitivity of HIV Infected CD14+CD16+ Monocytes to CCL2 and Key Roles of CCR2, JAM-A, and ALCAM in Diapedesis

    PubMed Central

    Williams, Dionna W.; Calderon, Tina M.; Lopez, Lillie; Carvallo-Torres, Loreto; Gaskill, Peter J.; Eugenin, Eliseo A.; Morgello, Susan; Berman, Joan W.

    2013-01-01

    As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14+CD16+ monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14+CD16+ monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14+CD16+ monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14+ cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14+CD16+ monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14+CD16+ monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14+CD16+ monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis. PMID:23922698

  17. A sandwich-hybridization assay for simultaneous determination of HIV and tuberculosis DNA targets based on signal amplification by quantum dots-PowerVision™ polymer coding nanotracers.

    PubMed

    Yan, Zhongdan; Gan, Ning; Zhang, Huairong; Wang, De; Qiao, Li; Cao, Yuting; Li, Tianhua; Hu, Futao

    2015-09-15

    A novel sandwich-hybridization assay for simultaneous electrochemical detection of multiple DNA targets related to human immune deficiency virus (HIV) and tuberculosis (TB) was developed based on the different quantum dots-PowerVision(TM) polymer nanotracers. The polymer nanotracers were respectively fabricated by immobilizing SH-labeled oligonucleotides (s-HIV or s-TB), which can partially hybrid with virus DNA (HIV or TB), on gold nanoparticles (Au NPs) and then modified with PowerVision(TM) (PV) polymer-encapsulated quantum dots (CdS or PbS) as signal tags. PV is a dendrimer enzyme linked polymer, which can immobilize abundant QDs to amplify the stripping voltammetry signals from the metal ions (Pb or Cd). The capture probes were prepared through the immobilization of SH-labeled oligonucleotides, which can complementary with HIV and TB DNA, on the magnetic Fe3O4@Au (GMPs) beads. After sandwich-hybridization, the polymer nanotracers together with HIV and TB DNA targets were simultaneously introduced onto the surface of GMPs. Then the two encoding metal ions (Cd(2+) and Pb(2+)) were used to differentiate two viruses DNA due to the different subsequent anodic stripping voltammetric peaks at -0.84 V (Cd) and -0.61 V (Pb). Because of the excellent signal amplification of the polymer nanotracers and the great specificity of DNA targets, this assay could detect targets DNA as low as 0.2 femtomolar and exhibited excellent selectivity with the dynamitic range from 0.5 fM to 500 pM. Those results demonstrated that this electrochemical coding assay has great potential in applications for screening more viruses DNA while changing the probes.

  18. Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus

    PubMed Central

    Hao, Xiao-Dan; Chen, Zhao-Li; Qu, Ming-Li; Zhao, Xiao-Wen; Li, Su-Xia; Chen, Peng

    2016-01-01

    Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC. PMID:27783701

  19. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

    PubMed Central

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  20. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    PubMed

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  1. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    PubMed

    Muthumani, Kar; Wise, Megan C; Broderick, Kate E; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y; Weiner, David B

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  2. HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

    PubMed Central

    Muthumani, Kar; Wise, Megan C.; Broderick, Kate E.; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B.; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y.; Weiner, David B.

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  3. Label-free photoelectrochemical detection of double-stranded HIV DNA by means of a metallointercalator-functionalized electrogenerated polymer.

    PubMed

    Haddache, Fatima; Le Goff, Alan; Reuillard, Bertrand; Gorgy, Karine; Gondran, Chantal; Spinelli, Nicolas; Defrancq, Eric; Cosnier, Serge

    2014-11-17

    The design of photoactive functionalized electrodes for the sensitive transduction of double-stranded DNA hybridization is reported. Multifunctional complex [Ru(bpy-pyrrole)2 (dppn)](2+) (bpy-pyrrole=4-methyl-4'-butylpyrrole-2,2'-bipyridine, dppn=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) exhibiting photosensitive, DNA-intercalating, and electropolymerizable properties was synthesized and characterized. The pyrrole groups undergo oxidative electropolymerization on planar electrodes forming a metallopolymer layer on the electrode. Thanks to the photoelectrochemical and intercalating properties of the immobilized Ru(II) complex, the binding of a double-stranded HIV DNA target was photoelectrochemically detected on planar electrodes. Photocurrent generation through visible irradiation was correlated to the interaction between double-stranded DNA and the metallointercalator polymer. These interactions were well fitted by using a Langmuir isotherm, which allowed a dissociation constant of 2×10(6)  L mol(-1) to be estimated. The low detection limit of 1 fmol L(-1) and sensitivity of 0.01 units per decade demonstrate excellent suitability of these modified electrodes for detection of duplex DNA.

  4. Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.

    PubMed Central

    Jenkins, T M; Esposito, D; Engelman, A; Craigie, R

    1997-01-01

    Analysis of the crystal structure of HIV-1 integrase reveals a cluster of lysine residues near the active site. Using site-directed mutagenesis and photo-crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3' processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication-defective at the step of integration. Photo-crosslinking, using 5-iododeoxyuracil-substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction. PMID:9362498

  5. Concurrent Sexual Partnerships Among Young Heterosexual Adults at Increased HIV Risk: Types and Characteristics

    PubMed Central

    Warren, Jocelyn T.; Harvey, S. Marie; Washburn, Isaac Joel; Sanchez, Diana Maria; Schoenbach, Victor J.; Agnew, Christopher R.

    2015-01-01

    Background The impact of concurrency on STI transmission depends upon coital frequency, condom use, duration of relationship overlap, and number of partners. Previous research has identified distinct concurrency types; however, little is known about their risk characteristics. Methods Men (n=261) and women (n=275) ages 18-30 years at increased risk of acquiring HIV were recruited from community locations in Los Angeles. Participants completed four in-person interviews over 12 months. Partnership data were used to characterize the prevalence of four types of concurrency: transitional (two overlapping relationships in which the first relationship ended before the second); single day (a second relationship of one day's duration during the course of another relationship); contained (a second relationship longer than 1 day began and ended during the course of another); and multiple (three or more overlapping relationships). Multilevel random intercept models were used to estimate mean coital frequency, proportion of condom-protected acts, total duration of overlap, and lifetime sex partners. Results At baseline, 47% of male and 32% of female participants reported any type of concurrency in the previous 4 months, and 26% of men and 10% of women reported multipleconcurrencies. Condom use ranged from 56% - 64%, with the highest use in transitional concurrency (61% for men, 68% for women) and the lowest in contained (52% for men, 54% for women). Coital frequency, total overlap, and lifetime sex partners also varied by concurrency type. Conclusions Inconsistent condom use and repeated opportunities for exposure characterize common types of concurrency among high-risk young adults. PMID:25763670

  6. A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA

    PubMed Central

    Lyonnais, Sébastien; Gorelick, Robert J.; Heniche-Boukhalfa, Fatima; Bouaziz, Serge; Parissi, Vincent; Mouscadet, Jean-François; Restle, Tobias; Gatell, Jose Maria; Le Cam, Eric; Mirambeau, Gilles

    2012-01-01

    Summary HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing preintegration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely Integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin. PMID:23017337

  7. Increased mitochondrial DNA content in saliva associated with head and neck cancer.

    PubMed

    Jiang, Wei-Wen; Masayesva, Brett; Zahurak, Marianna; Carvalho, Andre Lopes; Rosenbaum, Eli; Mambo, Elizabeth; Zhou, Shaoyu; Minhas, Khalid; Benoit, Nicole; Westra, William H; Alberg, Anthony; Sidransky, David; Koch, Wayne; Califano, Joseph

    2005-04-01

    Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

  8. Gender equality and education: Increasing the uptake of HIV testing among married women in Kenya, Zambia and Zimbabwe.

    PubMed

    Singh, Kavita; Luseno, Winnie; Haney, Erica

    2013-01-01

    Gender equality and education are being promoted as strategies to combat the HIV epidemic in Africa, but few studies have looked at the role of gender equality and education in the uptake of a vital service - HIV testing. This study looks at the associations between education (a key input needed for gender equality) and key gender equality measures (financial decision making and attitudes toward violence) with ever tested for HIV and tested for HIV in the past year. The study focused on currently married women ages between15-24 and 25-34 in three countries - Kenya, Zambia, and Zimbabwe. The data came from the Demographic and Health Surveys. Logistic regression was used to study the role of gender equality and education on the HIV testing outcomes after controlling for both social and biological factors. Results indicated that education had a consistent positive relationship with testing for both age groups, and the associations were always significant for young women aged 15-24 years (p<0.01). The belief that gender-based violence is unacceptable was positively associated with testing for women aged 25-34 in all the three countries, although the associations were only significant in Kenya (among women reporting ever being tested: OR 1.58, p<0.00; among women reporting being tested in the past year: OR 1.34, p<0.05) and Zambia (among women reporting ever being tested: OR 1.24, p<0.10; among women reporting being tested in the past year: OR 1.29, p<0.05). High financial decision making was associated with testing for women aged 25-34 in Zimbabwe only (among women reporting ever being tested: OR 1.66, p<0.01). Overall, the findings indicate that the education and the promotion of gender equality are important strategies for increasing uptake of a vital HIV service, and thus are important tools for protecting girls and young women against HIV. PMID:23438082

  9. Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination

    PubMed Central

    Fredriksen, Agnete Brunsvik; Løset, Geir Åge; Vikse, Elisabeth; Fugger, Lars

    2016-01-01

    It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II–targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II–targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans. PMID:27671110

  10. [Syphilis epidemic and an increase of the number of HIV infections among homosexual men attending the Amsterdam venereal disease clinic].

    PubMed

    Fennema, J S A; Cairo, I; Spaargaren, J; Dukers, N H T M; Coutinho, R A

    2002-03-30

    The registered number of cases of early infectious syphilis and of (ano)genital gonorrhoea among the attendees of the outpatient clinic for sexually transmitted diseases of the Amsterdam municipal health service shows a strong increase for both diagnoses in the period 1990-2001, notably in the last few years. Nearly all of this increase is accounted for by homosexual men. Syphilis increased mostly among men aged 35 years and over, gonorrhoea mostly among younger men. The population of older men also showed a distinct increase since 1997 in HIV incidence.

  11. Structures of HIV-1 reverse transcriptase with pre- and post-translocation AZTMP-terminated DNA.

    PubMed

    Sarafianos, Stefan G; Clark, Arthur D; Das, Kalyan; Tuske, Steve; Birktoft, Jens J; Ilankumaran, Palanichamy; Ramesha, Andagar R; Sayer, Jane M; Jerina, Donald M; Boyer, Paul L; Hughes, Stephen H; Arnold, Eddy

    2002-12-01

    AZT (3'-azido-3'-deoxythymidine) resistance involves the enhanced excision of AZTMP from the end of the primer strand by HIV-1 reverse transcriptase. This reaction can occur when an AZTMP-terminated primer is bound at the nucleotide-binding site (pre-translocation complex N) but not at the 'priming' site (post-translocation complex P). We determined the crystal structures of N and P complexes at 3.0 and 3.1 A resolution. These structures provide insight into the structural basis of AZTMP excision and the mechanism of translocation. Docking of a dNTP in the P complex structure suggests steric crowding in forming a stable ternary complex that should increase the relative amount of the N complex, which is the substrate for excision. Structural differences between complexes N and P suggest that the conserved YMDD loop is involved in translocation, acting as a springboard that helps to propel the primer terminus from the N to the P site after dNMP incorporation.

  12. Increasing Antiretroviral Adherence for HIV-Positive African Americans (Project Rise): A Treatment Education Intervention Protocol

    PubMed Central

    Bogart, Laura M; Mutchler, Matt G; McDavitt, Bryce; Mutepfa, Kieta D; Risley, Brian

    2016-01-01

    Background HIV-positive African Americans have been shown to have lower adherence to antiretroviral therapy (ART) than those of other races/ethnicities, yet adherence interventions have rarely been tailored to the needs of this population. Objective We developed and will evaluate a treatment education adherence intervention (called Rise) that was culturally adapted to address the needs of African Americans living with HIV. Methods This randomized controlled trial will examine the effects of the Rise intervention on ART adherence and HIV viral load. African Americans on ART who report adherence problems will be recruited from the community and randomly assigned to receive the intervention or usual care for 6 months. The intervention consists of 6-10 individual counseling sessions, with more sessions provided to those who demonstrate lower adherence. Primary outcomes include adherence as monitored continuously with Medication Event Monitoring Systems (MEMS) caps, and viral load data received from the participant’s medical provider. Survey assessments will be administered at baseline and month 6. Results The trial is ongoing. Conclusions If effective, the Rise intervention will provide community-based organizations with an intervention tailored to address the needs of African Americans for promoting optimal ART adherence and HIV clinical outcomes. Trial Registration Clinicaltrials.gov NCT01350544; https://clinicaltrials.gov/ct2/show/NCT01350544 (Archived by WebCite at http://www.webcitation.org/6fjqqnmn0). PMID:27025399

  13. Enhancement of anti-proliferative activities of Metformin, when combined with Celecoxib, without increasing DNA damage.

    PubMed

    Ullah, Asad; Ashraf, Muhammad; Javeed, Aqeel; Anjum, Aftab Ahmad; Attiq, Ali; Ali, Sarwat

    2016-07-01

    Pathophysiological changes in diabetes like hyperglycemia, oxidative stress, insulin resistance and compensatory hyperinsulinemia predispose cells to malignant transformation and damage DNA repair mechanism. This study was designed to explore the potential synergistic toxic effects of anti-diabetic drug (Metformin), and an analgesic drug (Celecoxib) at cellular level. MTT assay run on Vero cell line revealed that the combinations of Metformin and Celecoxib augment the anti-proliferative effects, whereas Single cell gel electrophoresis spotlighted that Metformin produce non-significant DNA damage with the threshold concentration of 400μg/ml in peripheral blood mononuclear cells (lymphocytes and monocytes), while Celecoxib produced significant (P<0.05) DNA damage (class III comets) above the concentration of 75μg/ml, however the DNA damage or DNA tail protrusions by combinations of both drugs were less than what was observed with Celecoxib alone. Metformin or Celecoxib did not appear mutagenic against any mutant strains (TA 100 and TA 98) but their combination exhibited slight mutagenicity at much higher concentration. The results obtained at concentrations higher than the therapeutic level of drugs and reflect that Metformin in combination with Celecoxib synergistically inhibits the cell proliferation in a concentration dependent pattern. Since, this increase in cytotoxicity did not confer an increase in DNA damage; this combination could be adopted to inhibit the growth of malignant cell without producing any genotoxic or mutagenic effects at cellular level. PMID:27327526

  14. DNA evidence for strong genetic stability and increasing heritability of intelligence from age 7 to 12.

    PubMed

    Trzaskowski, M; Yang, J; Visscher, P M; Plomin, R

    2014-03-01

    Two genetic findings from twin research have far-reaching implications for understanding individual differences in the development of brain function as indexed by general cognitive ability (g, aka intelligence): (1) The same genes affect g throughout development, even though (2) heritability increases. It is now possible to test these hypotheses using DNA alone. From 1.7 million DNA markers and g scores at ages 7 and 12 on 2875 children, the DNA genetic correlation from age 7 to 12 was 0.73, highly similar to the genetic correlation of 0.75 estimated from 6702 pairs of twins from the same sample. DNA-estimated heritabilities increased from 0.26 at age 7 to 0.45 at age 12; twin-estimated heritabilities also increased from 0.35 to 0.48. These DNA results confirm the results of twin studies indicating strong genetic stability but increasing heritability for g, despite mean changes in brain structure and function from childhood to adolescence.

  15. Preparation of DNA-adsorbed TiO2 particles--augmentation of performance for environmental purification by increasing DNA adsorption by external pH regulation.

    PubMed

    Amano, Takeharu; Toyooka, Tatsushi; Ibuki, Yuko

    2010-01-01

    We have previously developed a novel photocatalyst, DNA-attached titanium dioxide (DNA-TiO(2)), useful for the recovery and decomposition of chemicals [Suzuki et al. Environ. Sci. Technol. 42, 8076, 2008]. Chemicals accumulated in DNA near the surface of TiO(2) and were degraded under UV light. The efficiency of their removal was dependent on the amount of DNA adsorbed on TiO(2), indicating the attachment of larger amounts of DNA to result in higher efficiency. In this study, we succeeded in improving the performance of DNA-TiO(2) by increasing the amount of DNA adsorbed by regulating the external pH. The adsorption of DNA by TiO(2) dramatically increased at pH2, to about fourfold that at other pH values (pH4-10). Repeating the process of DNA addition increased the adsorption further. The attached DNA was stable on the surface of TiO(2) at pH2-10 and 4-56 degrees C, the same as DNA-TiO(2) prepared at pH7. As the DNA-TiO(2) prepared at pH2 retained much DNA on its surface, chemicals (methylene blue, ethidium bromide, etc.) which could intercalate or react with DNA were effectively removed from solutions. The photocatalytic degradation was slow at first, but the final degradation rate was higher than for non-adsorbed TiO(2) and DNA-TiO(2) prepared at pH7. These results indicated that preparation of DNA-TiO(2) at pH2 has advantages in that much DNA can be attached and large amounts of chemicals can be concentrated in the DNA, resulting in extensive decomposition under UV light.

  16. Increasing HIV Incidence among Men Who Have Sex with Men in Jiangsu Province, China: Results from Five Consecutive Surveys, 2011–2015

    PubMed Central

    Hu, Haiyang; Liu, Xiaoyan; Zhang, Zhi; Xu, Xiaoqin; Shi, Lingen; Fu, Gengfeng; Huan, Xiping; Zhou, Ying

    2016-01-01

    Epidemics of HIV among men who have sex with men (MSM) are major public health concerns in most parts of China. This study examined the trends in HIV incidence and associated factors among MSM in Jiangsu Province. Five consecutive cross-sectional surveys were conducted among MSM from 2011 to 2015 in eight cities throughout Jiangsu Province. Participants were recruited from MSM venues or via the internet. Demographic and behavioral data were collected through HIV bio-behavioral surveys. Blood specimens were collected to test for HIV and syphilis. HIV incidence was estimated by the IgG-capture BED-EIA (BED) method and a chi-square trend test was used to compare differences over the years. Multivariate logistic regression analysis was used to identify factors associated with recent infection. A total of 2433, 2678, 2591, 2610 and 2541 participants were enrolled in 2011, 2012, 2013, 2014 and 2015, respectively. HIV incidence increased from 5.10% in 2011 to 6.62% in 2015 (p = 0.025). MSM who had an education level of junior high school or less (aOR = 1.472, p = 0.018), engaged in condomless anal sex in the past 6 months (aOR = 2.389, p < 0.001), did not have an HIV test in the past 12 months (aOR = 3.215, p < 0.001), and were currently infected with syphilis (aOR = 2.025, p = 0.001) were likely to be recently infected with HIV. HIV incidence is increasing among MSM in Jiangsu Province, China. Condom usage and HIV testing promotion should be prioritized when attempting to reduce HIV transmission among MSM in China. PMID:27509513

  17. Increasing HIV Incidence among Men Who Have Sex with Men in Jiangsu Province, China: Results from Five Consecutive Surveys, 2011-2015.

    PubMed

    Hu, Haiyang; Liu, Xiaoyan; Zhang, Zhi; Xu, Xiaoqin; Shi, Lingen; Fu, Gengfeng; Huan, Xiping; Zhou, Ying

    2016-01-01

    Epidemics of HIV among men who have sex with men (MSM) are major public health concerns in most parts of China. This study examined the trends in HIV incidence and associated factors among MSM in Jiangsu Province. Five consecutive cross-sectional surveys were conducted among MSM from 2011 to 2015 in eight cities throughout Jiangsu Province. Participants were recruited from MSM venues or via the internet. Demographic and behavioral data were collected through HIV bio-behavioral surveys. Blood specimens were collected to test for HIV and syphilis. HIV incidence was estimated by the IgG-capture BED-EIA (BED) method and a chi-square trend test was used to compare differences over the years. Multivariate logistic regression analysis was used to identify factors associated with recent infection. A total of 2433, 2678, 2591, 2610 and 2541 participants were enrolled in 2011, 2012, 2013, 2014 and 2015, respectively. HIV incidence increased from 5.10% in 2011 to 6.62% in 2015 (p = 0.025). MSM who had an education level of junior high school or less (aOR = 1.472, p = 0.018), engaged in condomless anal sex in the past 6 months (aOR = 2.389, p < 0.001), did not have an HIV test in the past 12 months (aOR = 3.215, p < 0.001), and were currently infected with syphilis (aOR = 2.025, p = 0.001) were likely to be recently infected with HIV. HIV incidence is increasing among MSM in Jiangsu Province, China. Condom usage and HIV testing promotion should be prioritized when attempting to reduce HIV transmission among MSM in China. PMID:27509513

  18. Sexual mixing patterns and partner characteristics of black MSM in Massachusetts at increased risk for HIV infection and transmission.

    PubMed

    Mimiaga, Matthew J; Reisner, Sari L; Cranston, Kevin; Isenberg, Deborah; Bright, Donna; Daffin, Gary; Bland, Sean; Driscoll, Maura A; Vanderwarker, Rodney; Vega, Benny; Mayer, Kenneth H

    2009-07-01

    Black men who have sex with men (MSM) are at increased risk for HIV infection in the United States compared to other MSM. The aim of this study was to investigate Black MSM's sexual mixing patterns and partner characteristics in relation to sexual risk taking, as a possible explanation for this observed increase in HIV incidence. Between January and July 2008, 197 Black MSM were recruited via modified respondent-driven sampling and completed optional pretest and post-test HIV serological testing, counseling, and a demographic, behavioral, and psychosocial assessment battery. Bivariate and multivariable logistic regression procedures were used to examine predictors of risky sex across partner types. Overall, 18% of the sample was HIV-infected; 50% reported unprotected intercourse with men, 30% with women, and 5% with transgender partners. Fifty-three percent identified as bisexual or straight, although all reported oral or anal sex with another man in the prior 12 months. Significant predictors of engaging in at least one episode of: (1) serodiscordant unprotected anal sex (UAS) with a male partner in the past 12 months: individuals at risk for social isolation (AOR = 4.23; p = 0.03), those with unstable housing (AOR = 4.19; p = 0.03), and those who used poppers at least weekly during sex (AOR = 5.90; p = 0.05); (2) UAS and/or unprotected vaginal intercourse with a female partner in the past 12 months: those with unstable housing (AOR = 4.85; p = 0.04), those who used cocaine at least weekly during sex (AOR = 16.78; p = 0.006), being HIV-infected (AOR = 0.07; p = 0.02), and feeling social norms favor condom use (AOR = 0.60; p = 0.05); (3) UAS with the participants' most recent nonmain male sex partner: use of alcohol and drugs during last sex by participant (AOR = 4.04; p = 0.01), having sex with a Hispanic/Latino male (AOR = 2.71; p = 0.04) or a Black male (AOR = 0.50; p = 0.05) compared to a White male, and lower education (AOR = 1.31; p = 0.02). Findings suggest

  19. Age-related increases in human lymphocyte DNA damage: is there a role of aerobic fitness?

    PubMed

    Soares, Jorge Pinto; Mota, Maria Paula; Duarte, José Alberto; Collins, Andrew; Gaivão, Isabel

    2013-12-01

    Oxidative stress has been advanced as one of the major causes of damage to DNA and other macromolecules. Although physical exercise may also increase oxidative stress, an important role has been recognized for regular exercise in improving the overall functionality of the body, as indicated by an increase in maximal aerobic uptake ((V)O2max), and in resistance to cell damage. The aims of this study were 1) to evaluate the association between DNA damage in human lymphocytes and age and 2) to evaluate the association between DNA damage in human lymphocytes and ((V)O2max. The sample was composed of 36 healthy and nonsmoking males, aged from 20 to 84 years. ((V)O2max was evaluated through the Bruce protocol with direct measurement of oxygen consumption. The comet assay was used to evaluate the DNA damage, strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. We found a positive correlation of age with DNA strand breaks but not with FPG-sensitive sites. ((V)O2max was significantly inversely related with DNA strand breaks, but this relation disappeared when adjusted for age. A significantly positive relation between ((V)O2max and FPG-sensitive sites was verified. In conclusion, our results showed that younger subjects have lower DNA strand breaks and higher (V)O2max compared with older subjects and FPG-sensitive sites are positively related with ((V)O2max, probably as transient damage due to the acute effects of daily physical activity. PMID:24446564

  20. Breaking the Glass Ceiling: Increasing the Meaningful Involvement of Women Living With HIV/AIDS (MIWA) in the Design and Delivery of HIV/AIDS Services.

    PubMed

    Carter, Allison; Greene, Saara; Nicholson, Valerie; O'Brien, Nadia; Sanchez, Margarite; de Pokomandy, Alexandra; Loutfy, Mona; Kaida, Angela

    2015-01-01

    The meaningful involvement of women living with HIV/AIDS (MIWA) is a key feature of women-centred HIV care, yet little is known about transforming MIWA from principle to practice. Drawing on focus group data from the Canadian HIV Women's Sexual and Reproductive Health Cohort Study (CHIWOS), we explored HIV-positive women's meaningful involvement in the design and delivery of HIV/AIDS services in British Columbia, Canada. In this article, we highlight the benefits and tensions that emerge as women traverse multiple roles as service users and service providers within their care communities, and the impact this has on their access to care and overall health.

  1. PEPFAR's past and future efforts to cut costs, improve efficiency, and increase the impact of global HIV programs.

    PubMed

    Holmes, Charles B; Blandford, John M; Sangrujee, Nalinee; Stewart, Scott R; DuBois, Amy; Smith, Tyler R; Martin, Julia C; Gavaghan, Ann; Ryan, Caroline A; Goosby, Eric P

    2012-07-01

    Amid the global economic crisis, the President's Emergency Plan for AIDS Relief (PEPFAR) and other organizations have been pressed to do more with constrained resources to meet unmet needs in the worldwide HIV/AIDS pandemic. PEPFAR has approached this challenge through the development of an Impact and Efficiency Acceleration Plan, which includes improving the collection and use of economic and financial data, increasing the efficiency of HIV/AIDS program implementation, and collaborating with governments and multilateral organizations to maximize the impact of the resources provided by the United States. For example, by linking financial data with program outputs, PEPFAR was able to help its implementing partners in Mozambique reduce mean unit expenditures for people receiving antiretroviral treatment by 45 percent, from $265 to $145 per person, between 2009 and 2011. This article describes the plan's elements, provides examples of progress and challenges to its implementation, and assesses the prospects for further improvements in efficiency and impact. PMID:22778345

  2. Brief Report: Gonorrhea and Chlamydia Testing Increasing but Still Lagging in HIV Clinics in the United States.

    PubMed

    Berry, Stephen A; Ghanem, Khalil G; Mathews, William Christopher; Korthuis, Philip Todd; Yehia, Baligh R; Agwu, Allison L; Lehmann, Christoph U; Moore, Richard D; Allen, Sara L; Gebo, Kelly A

    2015-11-01

    Screening persons living with HIV for gonorrhea and chlamydia has been recommended since 2003. We compared annual gonorrhea/chlamydia testing to syphilis and lipid testing among 19,368 adults (41% men who have sex with men, 30% heterosexual men, and 29% women) engaged in HIV care. In 2004, 22%, 62%, and 70% of all patients were tested for gonorrhea/chlamydia, syphilis, and lipid levels, respectively. Despite increasing steadily [odds ratio per year (95% confidence interval): 1.14 (1.13 to 1.15)], gonorrhea/chlamydia testing in 2010 remained lower than syphilis and lipid testing (39%, 77%, 76%, respectively). Interventions to improve gonorrhea/chlamydia screening are needed. A more targeted screening approach may be warranted.

  3. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

    PubMed

    Darfeuille, Fabien; Hansen, Jens Bo; Orum, Henrik; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2004-01-01

    One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets. PMID:15181175

  4. Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

    PubMed

    Li, Tsai-Kun; Barbieri, Christopher M; Lin, Hsin-Chin; Rabson, Arnold B; Yang, Gengcheng; Fan, Yupeng; Gaffney, Barbara L; Jones, Roger A; Pilch, Daniel S

    2004-08-01

    RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

  5. Increase in Phi X174 DNA radiation sensitivity due to electric fields

    SciTech Connect

    McCormack, Percival D.; Swenberg, Charles E.

    1985-01-01

    The object of this research was to establish whether or not orientation of DNA in electric fields would result in a significant increase in its sensitivity to damage by ionizing radiation. The application of an external electric field simultaneously with gamma irradiation to an aqueous suspension of Phi X 174 (in the RFI form) is shown to increase significantly the number of strand breaks. Tritiated DNA allowed the number of single-strand breaks to be estimated from changes in the scintillation of electrophoretic gel band associated with the fastest mobility moiety. At 400 V ( approx. 2400 V/cm) the corrected increase (corrected for phoresis of DNA on the stainless steel plates) in the G-value yield is 38%. The increase in damage with field strength appears to follow the increase in reduced dichroism. Dichroism results correspond at 400 V to approximately 10% of the maximum orientation. These results support the conjecture that this significant increase in DNA-radiation interaction with an electric field is due to field-induced conformation changes in the molecule. Keywords: Polyelectrolytes, Polynucleotides, Polypeptides, Birefringence, Dipole, and Moments.

  6. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    NASA Astrophysics Data System (ADS)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  7. The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

    PubMed Central

    Janocko, Laura; Althouse, Andrew D.; Brand, Rhonda M.; Cranston, Ross D.

    2015-01-01

    Abstract The ex vivo mucosal explant model is frequently used to test the efficacy of microbicides that have the potential for preventing HIV-1 transmission. The conventional assessment of product efficacy has been the extent of HIV-1 p24 suppression in supernatant fluids sampled up to day 14 after HIV-1 challenge ex vivo. The purpose of this study was to determine if measurement of HIV-1 nucleic acids by real-time PCR and HIV-1 integration by Alu-gag PCR provides advantages with regard to monitoring HIV-1 infection in explants. Rectal biopsies from HIV-1-negative individuals were challenged with 1 × 105 virions/ml of HIV-1BaL or HIV-1CH077 ex vivo. HIV-1 RNA and HIV-1 p24 in supernatant fluids and HIV-1 nucleic acids and integrated provirus in individual biopsies were measured at days 1–14 after infection. HIV-1 RNA and proviral DNA were measured by quantitative real-time PCR (qRT-PCR) while integrated virus was detected by Alu-gag PCR. Real-time PCR assays detecting HIV-1 DNA and RNA performed similarly provided that the infecting virus sequences were a good match with the sequences of the assay primers and probes. Increased HIV-1 nucleic acid levels and DNA integration were measurable on days 11 and 14 after infection. The magnitude of explant infection was similar after challenge with HIV-1BaL and HIV-1CH077, although the trajectory of infection was delayed in the HIV-1CH077-infected biopsies. In the majority of experiments, qRT-PCR did not appreciably shorten the time necessary to detect evidence of HIV-1 infection. PMID:26214703

  8. A genomic DNA segment from Petunia hybrida leads to increased transformation frequencies and simple integration patterns.

    PubMed Central

    Meyer, P; Kartzke, S; Niedenhof, I; Heidmann, I; Bussmann, K; Saedler, H

    1988-01-01

    A 2-kilobase (kb) genomic fragment was selected from Petunia hybrida that increased transformation efficiencies by at least a factor of 20 after direct DNA transfer to petunia and tobacco protoplasts when supercoiled plasmid DNA was used. Because of this effect this fragment was named transformation booster sequence (TBS). Increased transformation frequencies were observed for plasmids that contained either the 2-kb fragment in dimeric or monomeric form or an internal 1.1-kb fragment of TBS. Analysis of transformants revealed that preferentially one copy of foreign DNA is integrated. Thus, TBS improves the poor transformation frequencies of direct gene transfer using circular plasmids, while it conserves the simple integration pattern that is important for practical applications. Possible mechanisms of TBS action are discussed. Images PMID:3186747

  9. In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

    PubMed

    Logan, Angela; Shabalina, Irina G; Prime, Tracy A; Rogatti, Sebastian; Kalinovich, Anastasia V; Hartley, Richard C; Budd, Ralph C; Cannon, Barbara; Murphy, Michael P

    2014-08-01

    In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria-targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro-apoptotic and pro-inflammatory redox signaling pathways.

  10. Analysis of the Prevalence of HTLV-1 Proviral DNA in Cervical Smears and Carcinomas from HIV Positive and Negative Kenyan Women

    PubMed Central

    He, Xiaotong; Maranga, Innocent O.; Oliver, Anthony W.; Gichangi, Peter; Hampson, Lynne; Hampson, Ian N.

    2016-01-01

    The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV−ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck®) and cytology. This showed 22/113 (19.5%) of LBC’s from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV−ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07–26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV−ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear. PMID:27608036

  11. Different mutagenic potential of HIV-1 restriction factors APOBEC3G and APOBEC3F is determined by distinct single-stranded DNA scanning mechanisms.

    PubMed

    Ara, Anjuman; Love, Robin P; Chelico, Linda

    2014-03-01

    The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹² motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F ¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F ¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif

  12. Different Mutagenic Potential of HIV-1 Restriction Factors APOBEC3G and APOBEC3F Is Determined by Distinct Single-Stranded DNA Scanning Mechanisms

    PubMed Central

    Ara, Anjuman; Love, Robin P.; Chelico, Linda

    2014-01-01

    The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (−)HIV-1 DNA. Upon replication of the (−)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an 190NPM192 motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F 191Pro to 191Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F 190NGM192 could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5

  13. Development of a Multilevel Intervention to Increase HIV Clinical Trial Participation among Rural Minorities

    PubMed Central

    Corbie-Smith, Giselle; Odeneye, Ebun; Banks, Bahby; Miles, Margaret Shandor; Isler, Malika Roman

    2013-01-01

    Minorities are disproportionately affected by HIV/AIDS in the rural Southeast; therefore, it is important to develop targeted, culturally appropriate interventions to support rural minority participation in HIV/AIDS research. Using Intervention Mapping, we developed a comprehensive multilevel intervention for service providers (SPs) and people living with HIV/AIDS (PLWHA). We collected data from both groups through 11 focus groups and 35 individual interviews. Resultant data were used to develop matrices of behavioral outcomes, performance objectives and learning objectives. Each performance objective was mapped with changeable, theory-based determinants to inform components of the intervention. Behavioral outcomes for the intervention included: (a) Eligible PLWHA will enroll in clinical trials; and (b) SPs will refer eligible PLWHA to clinical trials. The ensuing intervention consists of four SPs and six PLWHA educational sessions. Its contents, methods and strategies were grounded in the theory of reasoned action, social cognitive theory, and the concept of social support. All materials were pretested and refined for content appropriateness and effectiveness. PMID:22991051

  14. HIV Treatment Produces Economic Returns Through Increased Work And Education, And Warrants Continued US Support

    PubMed Central

    Thirumurthy, Harsha; Galárraga, Omar; Larson, Bruce; Rosen, Sydney

    2013-01-01

    Federal expenditures are under scrutiny in the United States, and the merits of continuing and expanding the President’s Emergency Plan for AIDS Relief (PEPFAR) to support access to antiretroviral therapy have become a topic of debate. A growing body of research on the economic benefits of treatment with antiretroviral therapy has important implications for these discussions. For example, research conducted since the inception of PEPFAR shows that HIV-infected adults who receive antiretroviral therapy often begin or resume productive work, and that children living in households with infected adults who are on treatment are more likely to attend school than those in households with untreated adults. These benefits should be considered when weighing the overall benefits of providing antiretroviral therapy against its costs, particularly in the context of discussions about the future of PEPFAR. A modest case can also be made in favor of having private companies in HIV-affected countries provide antiretroviral therapy to their employees and dependents, thus sharing some of the burden of funding HIV treatment. PMID:22778336

  15. Increased regression and decreased incidence of human papillomavirus-related cervical lesions among HIV-infected women on HAART

    PubMed Central

    Adler, David H.; Kakinami, Lisa; Modisenyane, Tebogo; Tshabangu, Nkeko; Mohapi, Lerato; De Bruyn, Guy; Martinson, Neil A.; Omar, Tanvier

    2013-01-01

    Objective To determine the impact of HAART on incidence, regression, and progression of cytopathological abnormalities in HIV-infected women. Design Prospective cohort. Methods HIV-infected women (N=1123) from Soweto, South Africa underwent serial cervical smears that were analyzed and reported using the Bethesda System. The results of HAART and non-HAART users were compared using two statistical approaches: a survival analysis assessing risk of incident smear abnormality among women with baseline normal smear results; and analysis with marginal models assessing for an association between HAART use and likelihood of regression/progression in consecutive smears. Results After multivariate survival analysis, women using HAART with a normal baseline smear were 38% less likely to have an incident smear abnormality during follow-up than nonusers [confidence interval (CI) 0.42–0.91; P=0.01]. Multivariate marginal models analysis identified a significantly increased likelihood (odds ratio 2.61; CI 1.75–3.89; P< 0.0001) of regression of cervical lesions among women on HAART. Conclusion Our large prospective cohort study adds significant weight to the side of the balance of clinical research supporting the positive impact of HAART on the natural history of human papillomavirus-related cervical disease in HIV-infected women. PMID:22555167

  16. Increased frequency of circulating Tc22/Th22 cells and polyfunctional CD38(-) T cells in HIV-exposed uninfected subjects.

    PubMed

    Oliveira, Luanda M S; Lima, Josenilson F; Cervantes, Cesar A C; Casseb, Jorge S; Mendonça, Marcelo; Duarte, Alberto J S; Sato, Maria N

    2015-09-08

    Some individuals are resistant to HIV-1 infection despite repeated exposure to the virus, suggesting the presence of a complex antiviral response. Innate factors like IL-22 exert gut mucosal protection and polyfunctional T cells have been associated with low progression in HIV infection; therefore, we evaluated the frequencies of CD4+ and CD8+ T cell-secreting cytokines, including Tc22/Th22 cells and polyfunctional T cells in HIV-1-exposed uninfected individuals (EUs), their HIV-1-infected partners and healthy controls. EUs exhibited an increased frequency of p15 Gag CD4+ IL-22+ secreting T cells, whereas HIV-infected partners demonstrated a high frequency of CD4+ IL-17+ T cells in response to p24. Similar responses of Th22 and Tc22 cells to Gag peptides and Staphylococcal enterotoxin B (SEB) stimulation were detected in the serodiscordant couples. However, polyfunctionality in HIV subjects was associated with an HIV Gag response of CD38+ T cells, whereas polyfunctionality for EUs was induced upon SEB stimulation by CD38- T cells. EUs demonstrated the presence of Tc22/Th22 cells and polyfunctional CD38- T cells with a low activation profile. These data suggest that SEB-induced polyfunctional CD4+ and CD8+ T cells together with Tc22/Th22 cells in EU individuals can provide an immunological advantage in the response to pathogens such as HIV-1.

  17. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    PubMed

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

  18. Increased frequency and function of KIR2DL1-3+ NK cells in primary HIV-1 infection are determined by HLA-C group haplotypes

    PubMed Central

    Körner, Christian; Granoff, Mitchell E.; Amero, Molly A.; Sirignano, Michael N.; Vaidya, Sagar A.; Jost, Stephanie; Allen, Todd M.; Rosenberg, Eric S.; Altfeld, Marcus

    2014-01-01

    Acquisition and maintenance of NK cell function is mediated by inhibitory killer-cell immunoglobulin-like receptors (KIR) through the interaction with HLA class I molecules. Recently, HLA-C expression levels were shown to be correlated with protection against multiple outcomes of HIV-1 infection; however the underlying mechanisms are poorly understood. As HLA-C is the natural ligand for the inhibitory receptors KIR2DL1 and KIR2DL2/3, we sought to determine whether HLA-C group haplotypes affect NK cell responses during primary HIV-1 infection. The phenotypes and functional capacity of NK cells derived from HIV-1(+) and HIV-1(-) individuals were assessed (N=42 and N=40, respectively). HIV-1 infection was associated with an increased frequency of KIR2DL1-3+ NK cells. Further analysis showed that KIR2DL1+ NK cells were selectively increased in individuals homozygous for HLA-C2, while HLA-C1-homozygous individuals displayed increased proportions of KIR2DL2/3+ NK cells. KIR2DL1-3+ NK cells were furthermore more polyfunctional during primary HIV-1 infection in individuals also encoding for their cognate HLA-C group haplotypes as measured by degranulation and cytokine production. These results identify a novel relationship between HLA-C and KIR2DL+ NK cell subsets and demonstrate that HLA-C-mediated licensing modulates NK cell responses to primary HIV-1 infection. PMID:25043727

  19. Short communication: genital tumor growth factor-β1 levels in HIV-infected Indian women are associated with reduced levels of innate antimicrobial products and increased HIV shedding.

    PubMed

    Thakar, Madhuri; Patil, Rahul; Shukre, Subodh; Bichare, Shubhangi; Kadam, Poonam; Khopkar, Priyanka; Ghate, Manisha; Paranjape, Ramesh

    2014-07-01

    Tumor growth factor (TGF)-β1 is a cytokine with potent immunoinhibitory functions and is known to be secreted by vaginal epithelial cells. The present study was designed to determine the association of cervicovaginal levels of TGF- β1 with various innate immune secretions such as cytokines and antimicrobial polypeptides [Trappin-2/Elafin and secretory leukocyte protease inhibitor (SLPI)] and cervical HIV shedding in HIV-infected Indian women. TGF- β1, antimicrobial polypeptides, and cytokine levels were estimated in the cervicovaginal lavages (CVLs) of 36 age-matched HIV-infected and 31 HIV-uninfected asymptomatic Indian women using an ELISA and Bio-Plex Assay, respectively. The nonparametric Mann-Whitney test and Spearman's test were used to compare the levels from both the groups and to determine the association of the TGF-β1 levels with cervical viral shedding and antimicrobial peptides. The levels of Trappin-2/Elafin and SLPI were similar in the CVLs of HIV-infected and HIV-uninfected women, but were significantly associated with a low cervical viral load (r=-0.501, p=0.005 for Trappin-2/Elafin and r=-0.488, p=0.007 for SLPI). Eleven (30.5%) of the 36 HIV-infected women showed 5- to 30-fold higher levels of TGF-β1 as compared to the levels in uninfected women. The TGF-β1 levels were significantly associated with higher cervical viral load (r=0.425, p=0.03) and with lower levels of Trappin-2/Elafin (r=-0.407, p=0.03) and SLPI (r=-0.405, p=0.04). The findings indicate a possible interdependent mechanism driving the identified higher TGF-β1 and lower antimicrobial peptide (Trappin-2/Elafin and SLPI) levels at the genital mucosa surface in HIV-infected women. We postulate that a combination of increased TGF-β1 secretion and altered levels of Trappin-2/Elafin and SLPI contributes to increased HIV shedding. The observation warrants further studies to identify the underlying mechanisms linking increased mucosal TGF-β1 levels and genital HIV shedding

  20. Increased cancer risk of augmentation cystoplasty: possible role for hyperosmolal microenvironment on DNA damage recognition

    PubMed Central

    Dixon, Bradley P.; Chu, Albert; Henry, Jeff; Kim, Rebecca; Bissler, John J.

    2009-01-01

    Patients who have had surgical bladder augmentation have an increased risk of bladder malignancy, though the mechanism for this increased risk is unknown. Hyperosmolal microenvironments such as the bladder may impair DNA damage signaling and repair; this effect may be more pronounced in tissues not normally exposed to such conditions. Comparing gastric and colon epithelial cell lines to transitional epithelial cell lines gradually adapted to an osmolality of 600mOsm/kg with either sodium chloride or urea, cell lines of gastrointestinal origin were inhibited in their ability to activate ATM and downstream effectors of DNA damage signaling and repair such as p53, Nbs1, replication protein A (RPA), and γH2AX following the induction of DNA damage with etoposide. In contrast, bladder cell lines demonstrated a preserved ability to phosphorylate ATM and its effectors under conditions of hyperosmolal urea, and to a lesser extent with sodium chloride. The bladder cell lines’ ability to respond to DNA damage under hyperosmolal conditions may be due in part to protective mechanisms such as the accumulation of intracellular organic osmolytes and the uroplakin-containing asymmetric unit membrane as found in transitional epithelial cells, but not in gastrointestinal cells. Failure of such protective adaptations in the tissues used for augmentation cystoplasties may place these tissues at increased risk for malignancy. PMID:19647003

  1. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice.

    PubMed

    Jafarpour, Nazli; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Kohram, Fatemeh; Aghababa, Haniyeh; Khoramabadi, Nima; Mahdavi, Mehdi

    2014-08-01

    Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine. PMID:24842263

  2. Spatiotemporal dynamics of HIV infection

    NASA Astrophysics Data System (ADS)

    Strain, Matthew Carl

    Mathematical models of the dynamics of infection with the human immunodeficiency virus (HIV) have contributed to tremendous advances over the past 20 years. This thesis extends this previous work by exploring the importance of spatial heterogeneity in HIV infection both in vitro and in vivo in patients treated with highly-active antiretroviral therapy. Viral infections propagate locally in space, yet HIV infection has been widely regarded as equilibrated over the entire body of an infected patient. This dissertation constructs and explores a cellular automata model of viral spread at the cellular level. Coupling the automata to a blood compartment represented by a differential equation leads to a whole-body model of HIV infection that explicitly includes spatial effects at both the cellular and tissue levels. These models are tested by comparison with experimental data. A central prediction of the spatial model is that, due to competition between Brownian motion and viral lability, HIV infectivity increases with target cell density. This production is verified in a series of in vitro experiments in cell culture. The predicted independence of inhibitory concentrations of antiretoviral agents is verified for nevirapine, but azidothymidine inhibits HIV replication less efficiently in more dense cultures. These in vitro results suggest that systems allowing cell concentrations closer to tissue densities would better reflect virus replication kinetics, although standard measures of relative drug susceptibility may accurately reflect in vivo conditions. The coupled spatial model of in vivo dynamics is compared with novel mathematical analysis of experiments in HIV-infected patients. These analyses indicate that HIV DNA provides a useful marker of the size of long-lived cellular reservoirs of HIV. Levels of HIV DNA in peripheral blood are predictive of the average rate of residual virus production after years of treatment, regardless of whether patients initiate therapy

  3. DNA injury is acutely enhanced in response to increasing bulks of aerobic physical exercise.

    PubMed

    Lippi, Giuseppe; Buonocore, Ruggero; Tarperi, Cantor; Montagnana, Martina; Festa, Luca; Danese, Elisa; Benati, Marco; Salvagno, Gian Luca; Bonaguri, Chiara; Roggenbuck, Dirk; Schena, Federico

    2016-09-01

    The aim of this study was to evaluate DNA damage in response to increasing bulks of aerobic physical exercise. Fifteen adult and trained athletes performed four sequential trials with increasing running distance (5-, 10-, 21- and 42-km) in different periods of the year. The γ-H2AX foci parameters were analyzed before and 3h after the end of each trial. The values of all γ-H2AX foci parameters were enhanced after the end of each trial, with values gradually increasing from the 5- to the 42-km trial. Interestingly, a minor increase of γ-H2AX foci was still evident after 5- to 10-km running, but a much higher increase occurred when the running distance exceeded 21km. The generation of DNA injury was then magnified by running up to 42-km. The increase of each γ-H2AX foci parameter was then found to be associated with both running distance and average intensity. In multivariate linear regression analysis, the running distance was significantly associated with average intensity and post-run variation in the percentage of cells with γ-H2AX foci. We can hence conclude that aerobic exercise may generate an acute DNA damage in trained athletes, which is highly dependent upon running distance and average intensity. PMID:27374303

  4. Anti-Retroviral Therapy Increases the Prevalence of Dyslipidemia in South African HIV-Infected Patients

    PubMed Central

    Dave, Joel A.; Levitt, Naomi S.; Ross, Ian L.; Lacerda, Miguel; Maartens, Gary; Blom, Dirk

    2016-01-01

    Purpose Data on the prevalence of dyslipidaemia and associated risk factors in HIV-infected patients from sub-Saharan Africa is sparse. We performed a cross-sectional analysis in a cohort of HIV-infected South African adults. Methods We studied HIV-infected patients who were either antiretroviral therapy (ART)-naive or receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-based or protease inhibitor (PI)-based ART. Evaluation included fasting lipograms, oral glucose tolerance tests and clinical anthropometry. Dyslipidemia was defined using the NCEP ATPIII guidelines. Results The median age of the participants was 34 years (range 19–68 years) and 78% were women. The prevalence of dyslipidemia in 406 ART-naive and 551 participants on ART was 90.0% and 85%, respectively. Low HDL-cholesterol (HDLC) was the most common abnormality [290/406 (71%) ART-naïve and 237/551 (43%) ART- participants]. Participants on ART had higher triglycerides (TG), total cholesterol (TC), LDL-cholesterol (LDLC) and HDLC than the ART-naïve group. Severe dyslipidaemia, (LDLC> 4.9 mmol/L or TG >5.0 mmol/L) was present in <5% of participants. In multivariate analyses there were complex associations between age, gender, type and duration of ART and body composition and LDLC, HDLC and TG, which differed between ART-naïve and ART-participants. Conclusion Participants on ART had higher TG, TC, LDLC and HDLC than those who were ART-naïve but severe lipid abnormalities requiring evaluation and treatment were uncommon. PMID:26986065

  5. Modelling cross-hybridization on phylogenetic DNA microarrays increases the detection power of closely related species.

    PubMed

    Engelmann, Julia C; Rahmann, Sven; Wolf, Matthias; Schultz, Jörg; Fritzilas, Epameinondas; Kneitz, Susanne; Dandekar, Thomas; Müller, Tobias

    2009-01-01

    DNA microarrays are a popular technique for the detection of microorganisms. Several approaches using specific oligomers targeting one or a few marker genes for each species have been proposed. Data analysis is usually limited to call a species present when its oligomer exceeds a certain intensity threshold. While this strategy works reasonably well for distantly related species, it does not work well for very closely related species: Cross-hybridization of nontarget DNA prevents a simple identification based on signal intensity. The majority of species of the same genus has a sequence similarity of over 90%. For biodiversity studies down to the species level, it is therefore important to increase the detection power of closely related species. We propose a simple, cost-effective and robust approach for biodiversity studies using DNA microarray technology and demonstrate it on scenedesmacean green algae. The internal transcribed spacer 2 (ITS2) rDNA sequence was chosen as marker because it is suitable to distinguish all eukaryotic species even though parts of it are virtually identical in closely related species. We show that by modelling hybridization behaviour with a matrix algebra approach, we are able to identify closely related species that cannot be distinguished with a threshold on signal intensity. Thus this proof-of-concept study shows that by adding a simple and robust data analysis step to the evaluation of DNA microarrays, species detection can be significantly improved for closely related species with a high sequence similarity.

  6. Increasing DNA extraction yield from saliva stains with a modified Chelex method.

    PubMed

    Sweet, D; Lorente, M; Valenzuela, A; Lorente, J A; Alvarez, J C

    1996-12-27

    Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

  7. Mammalian Brain Development is Accompanied by a Dramatic Increase in Bipolar DNA Methylation

    PubMed Central

    Sun, Ming-an; Sun, Zhixiong; Wu, Xiaowei; Rajaram, Veena; Keimig, David; Lim, Jessica; Zhu, Hongxiao; Xie, Hehuang

    2016-01-01

    DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders. PMID:27585862

  8. Mammalian Brain Development is Accompanied by a Dramatic Increase in Bipolar DNA Methylation.

    PubMed

    Sun, Ming-An; Sun, Zhixiong; Wu, Xiaowei; Rajaram, Veena; Keimig, David; Lim, Jessica; Zhu, Hongxiao; Xie, Hehuang

    2016-01-01

    DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders. PMID:27585862

  9. Do accurate HIV and antiretroviral therapy knowledge, and previous testing experiences increase the uptake of HIV voluntary counselling and testing? Results from a cohort study in rural Tanzania

    PubMed Central

    2013-01-01

    Background Despite the introduction of free antiretroviral therapy (ART), the use of voluntary counselling and testing (VCT) services remains persistently low in many African countries. This study investigates how prior experience of HIV and VCT, and knowledge about HIV and ART influence VCT use in rural Tanzania. Methods In 2006–7, VCT was offered to study participants during the fifth survey round of an HIV community cohort study that includes HIV testing for research purposes without results disclosure, and a questionnaire covering knowledge, attitudes and practices around HIV infection and HIV services. Categorical variables were created for HIV knowledge and ART knowledge, with “good” HIV and ART knowledge defined as correctly answering at least 4/6 and 5/7 questions about HIV and ART respectively. Experience of HIV was defined as knowing people living with HIV, or having died from AIDS. Logistic regression methods were used to assess how HIV and ART knowledge, and prior experiences of HIV and VCT were associated with VCT uptake, with adjustment for HIV status and socio-demographic confounders. Results 2,695/3,886 (69%) men and 2,708/5,575 women (49%) had “good” HIV knowledge, while 613/3,886 (16%) men and 585/5575 (10%) women had “good” ART knowledge. Misconceptions about HIV transmission were common, including through kissing (55% of women, 43% of men), or mosquito bites (42% of women, 34% of men). 19% of men and 16% of women used VCT during the survey. After controlling for HIV status and socio-demographic factors, the odds of VCT use were lower among those with poor HIV knowledge (aOR = 0.5; p = 0.01 for men and aOR = 0.6; p < 0.01 for women) and poor ART knowledge (aOR = 0.8; p = 0.06 for men, aOR = 0.8; p < 0.01 for women), and higher among those with HIV experience (aOR = 1.3 for men and aOR = 1.6 for women, p < 0.01) and positive prior VCT experience (aOR = 2.0 for all men and aOR = 2

  10. Somatic mitochondrial DNA mutations do not increase neuronal vulnerability to MPTP in young POLG mutator mice.

    PubMed

    Dai, Ying; Clark, Joanne; Zheng, Kangni; Kujoth, Gregory C; Prolla, Tomas A; Simon, David K

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations are hypothesized to play a pathogenic role in aging and age-related neurodegenerative diseases such as Parkinson's disease (PD). In support of this, high levels of somatic mtDNA mutations in “POLG mutator” mice carrying a proofreading-deficient form of mtDNA polymerase ã (Polg(D257A)) lead to a premature aging phenotype. However, the relevance of this finding to the normal aging process has been questioned as the number of mutations is greater even in young POLG mutator mice, which shows no overt phenotype, than levels achieved during normal aging in mice. Vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) increases with age, and we hypothesized that this may result in part from the accumulation with age of somatic mtDNA mutations. If correct, then levels of mutations in young (2–3 month old) POLG mutator mice should be sufficient to increase vulnerability to MPTP. In contrast, we find that susceptibility to MPTP in both heterozygous and homozygous POLG mutator mice at this young age is not different from that of wild type littermate controls as measured by levels of tyrosine hydroxylase positive (TH+) striatal terminals, striatal dopamine and its metabolites, a marker of oxidative damage, or stereological counts of TH+ and total substantia nigra neurons. These unexpected results do not support the hypothesis that somatic mtDNA mutations contribute to the age-related vulnerability of dopaminergic neurons to MPTP. It remains possible that somatic mtDNA mutations influence vulnerability to other stressors, or require additional time for the deleterious consequences to manifest. Furthermore, the impact of the higher levels of mutations present at older ages in these mice was not assessed in our study, although a prior study also failed to detect an increase in vulnerability to MPTP in older mice. With these caveats, the current data do not provide evidence for a role of somatic mtDNA

  11. Four-tiered {pi} interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity

    SciTech Connect

    Al-Mawsawi, Laith Q.; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

    2008-08-01

    HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive {pi} electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact {pi} electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact {pi} electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished {pi} interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the {pi} interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this {pi}-{pi} interaction should lead to powerful anti-retroviral drugs.

  12. DNA topoisomerase IIα inhibitory and anti-HIV-1 flavones from leaves and twigs of Gardenia carinata.

    PubMed

    Kongkum, Naowarat; Tuchinda, Patoomratana; Pohmakotr, Manat; Reutrakul, Vichai; Piyachaturawat, Pawinee; Jariyawat, Surawat; Suksen, Kanoknetr; Yoosook, Chalobon; Kasisit, Jitra; Napaswad, Chanita

    2012-03-01

    Four new flavones, 5,2'-dihydroxy-7,3',4',5'-tetramethoxyflavone (1), 5,2',5'-trihydroxy-7,3',4'-trimethoxyflavone (2), 5,7,2',5'-tetrahydroxy-6,3',4'-trimethoxyflavone (3) and 5,2',5'-trihydroxy-6,7,3',4'-tetramethoxyflavone (4), along with the known 5,3'-dihydroxy-6,7,4',5'-tetramethoxyflavone (5), 5,7,3',5'-tetrahydroxy-6,4'-dimethoxyflavone (6), syringaldehyde, vanillic acid and scopoletin were isolated from the leaves and twigs of Gardenia carinata (Rubiaceae). Their structures were determined by spectroscopic methods. Flavone 2 exhibited cytotoxic activity against P-388 and MCF-7 cell lines, while 3, 5 and 6 were active only in P-388 cell line. All active compounds were found to inhibit DNA topoisomerase IIα activity, which may be responsible for the observed cytotoxicity. Flavones 1-3, 5 and 6 also exhibited anti-HIV-1 activity in the anti-syncytium assay using (∆Tat/rev)MC99 virus and 1A2 cell line system; 2 was most potent. Only flavones 1 and 6 showed considerably activity against HIV-1 reverse transcriptase.

  13. Increased Escherichia coli-Induced Interleukin-23 Production by CD16+ Monocytes Correlates with Systemic Immune Activation in Untreated HIV-1-Infected Individuals

    PubMed Central

    Manuzak, Jennifer A.; Dillon, Stephanie M.; Lee, Eric J.; Dong, Zachary M.; Hecht, Daniel K.

    2013-01-01

    The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16+ monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14+ monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16+ monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16+ monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals. PMID:24067979

  14. Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

    PubMed Central

    Deuzing, Ilona P.; Charpentier, Charlotte; Wright, David W.; Matheron, Sophie; Paton, Jack; Frentz, Dineke; van de Vijver, David A.; Coveney, Peter V.; Descamps, Diane; Boucher, Charles A. B.

    2014-01-01

    ABSTRACT Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity. IMPORTANCE Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug

  15. TOGETHER Project to Increase Understanding of the HIV Epidemic Among Sub-Saharan African Migrants: Protocol of Community-Based Participatory Mixed-Method Studies

    PubMed Central

    Vuylsteke, Bea; Manirankunda, Lazare; Deblonde, Jessika; Kint, Ilse; Namanya, Fiona; Fransen, Katrien; Colebunders, Robert; Laga, Marie; Adobea, Dorothy; Nöstlinger, Christiana

    2016-01-01

    Background Sub-Saharan African Migrants (SAM) are the second largest group affected by HIV/AIDS in Belgium and the rest of Western Europe. Increasing evidence shows that, more than previously thought, SAM are acquiring HIV in their host countries. This calls for a renewed focus on primary prevention. Yet, knowledge on the magnitude of the HIV epidemic among SAM (HIV prevalence estimates and proportions of undiagnosed HIV infections) and underlying drivers are scarce and limit the development of such interventions. Objective By applying a community-based participatory and mixed-methods approach, the TOGETHER project aims to deepen our understanding of HIV transmission dynamics, as well as inform future primary prevention interventions for this target group. Methods The TOGETHER project consists of a cross-sectional study to assess HIV prevalence and risk factors among SAM visiting community settings in Antwerp city, Belgium, and links an anonymous electronic self-reported questionnaire to oral fluid samples. Three formative studies informed this method: (1) a social mapping of community settings using an adaptation of the PLACE method; (2) a multiple case study aiming to identify factors that increase risk and vulnerability for HIV infection by triangulating data from life history interviews, lifelines, and patient files; and (3) an acceptability and feasibility study of oral fluid sampling in community settings using participant observations. Results Results have been obtained from 4 interlinked studies and will be described in future research. Conclusions Combining empirically tested and innovative epidemiological and social science methods, this project provides the first HIV prevalence estimates for a representative sample of SAM residing in a West European city. By triangulating qualitative and quantitative insights, the project will generate an in-depth understanding of the factors that increase risk and vulnerability for HIV infection among SAM. Based on this

  16. Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

    PubMed Central

    Wang, Dong; Lloyd, Andrew H.; Timmis, Jeremy N.

    2012-01-01

    Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally. PMID:22308419

  17. Glucopyranosyl lipid A adjuvant significantly enhances HIV specific T and B cell responses elicited by a DNA-MVA-protein vaccine regimen.

    PubMed

    McKay, Paul F; Cope, Alethea V; Mann, Jamie F S; Joseph, Sarah; Esteban, Mariano; Tatoud, Roger; Carter, Darrick; Reed, Steven G; Weber, Jonathan; Shattock, Robin J

    2014-01-01

    Using a unique vaccine antigen matched and single HIV Clade C approach we have assessed the immunogenicity of a DNA-poxvirus-protein strategy in mice and rabbits, administering MVA and protein immunizations either sequentially or simultaneously and in the presence of a novel TLR4 adjuvant, GLA-AF. Mice were vaccinated with combinations of HIV env/gag-pol-nef plasmid DNA followed by MVA-C (HIV env/gag-pol-nef) with HIV CN54gp140 protein (+/-GLA-AF adjuvant) and either co-administered in different muscles of the same animal with MVA-C or given sequentially at 3-week intervals. The DNA prime established a population of B cells that were able to mount a statistically significant anamnestic response to the boost vaccines. The greatest antigen-specific antibody response was observed in animals that received all vaccine components. Moreover, a high proportion of the total mucosal IgG (20 - 50%) present in the vaginal vault of these vaccinated animals was vaccine antigen-specific. The potent elicitation of antigen-specific immune responses to this vaccine modality was also confirmed in rabbits. Importantly, co-administration of MVA-C with the GLA-AF adjuvanted HIV CN54gp140 protein significantly augmented the antigen-specific T cell responses to the Gag antigen, a transgene product expressed by the MVA-C vector in a separate quadriceps muscle. We have demonstrated that co-administration of MVA and GLA-AF adjuvanted HIV CN54gp140 protein was equally effective in the generation of humoral responses as a sequential vaccination modality thus shortening and simplifying the immunization schedule. In addition, a significant further benefit of the condensed vaccination regime was that T cell responses to proteins expressed by the MVA-C were potently enhanced, an effect that was likely due to enhanced immunostimulation in the presence of systemic GLA-AF.

  18. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    SciTech Connect

    Chowdhury, E.H.

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  19. 17β-Estradiol Protects Primary Macrophages Against HIV Infection Through Induction of Interferon-Alpha

    PubMed Central

    Tasker, Carley; Ding, Jian; Schmolke, Mirco; Rivera-Medina, Amariliz; García-Sastre, Adolfo

    2014-01-01

    Abstract Estrogen has been shown to increase resistance to HIV/SIV transmission by increasing the thickness of the genital epithelium. The immunological role of estrogen in HIV infection of primary target cells is less well characterized. We have found that primary macrophages are a target for anti-HIV activity of 17β-estradiol (E2). E2 did not affect surface expression of CD4 and HIV co-receptors nor HIV attachment to monocyte-derived macrophages (MDMs). In addition, E2 treatment blocked infection by a co-receptor-independent HIV-1VSV-G pseudotyped virus. Quantitative polymerase chain reaction analysis of HIV reverse transcribed DNA products indicated that E2 blocked HIV reverse transcription. E2 upregulated gene expression of interferons (IFNs) in MDMs from multiple donors. However, induction of host restriction factors APOBEC3G, APOBEC3F, or SAMHD1 was not consistent, with exception of APOBEC3A. Anti-HIV activity of E2 was abolished in the presence of IFN-α neutralizing antibody, and was absent in bone marrow–derived macrophages from IFN-α receptor deficient mice. Interestingly, HIV overcame E2-mediated HIV inhibition by suppressing induction of IFNs when MDMs were exposed to HIV before E2 treatment. These results offer a new mechanism of E2 on HIV inhibition. Future studies on the interplay between HIV and E2-mediated innate immune responses will likely provide insights relevant for development of effective strategies for HIV prevention. PMID:24801776

  20. Simultaneous runs of the Bayer VERSANT HIV-1 version 3.0 and HCV bDNA version 3.0 quantitative assays on the system 340 platform provide reliable quantitation and improved work flow.

    PubMed

    Elbeik, Tarek; Markowitz, Norman; Nassos, Patricia; Kumar, Uday; Beringer, Scott; Haller, Barbara; Ng, Valerie

    2004-07-01

    Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63 degrees C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround. PMID:15243070

  1. Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch collaborative study.

    PubMed

    Tu, Elise; Swenson, Luke C; Land, Sally; Pett, Sarah; Emery, Sean; Marks, Kat; Kelleher, Anthony D; Kaye, Steve; Kaiser, Rolf; Schuelter, Eugene; Harrigan, Richard

    2013-07-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism

  2. Rapid and Slow Progressors Show Increased IL-6 and IL-10 Levels in the Pre-AIDS Stage of HIV Infection

    PubMed Central

    de Medeiros, Rúbia M.; Valverde-Villegas, Jacqueline M.; Junqueira, Dennis M.; Gräf, Tiago; Lindenau, Juliana D.; de Mello, Marineide G.; Vianna, Priscila; Almeida, Sabrina E. M.; Chies, Jose Artur B.

    2016-01-01

    Cytokines are intrinsically related to disease progression in HIV infection. We evaluated the plasma levels of Th1/Th2/Th17 cytokines in extreme progressors, including slow (SPs) and rapid (RPs) progressors, who were thus classified based on clinical and laboratory follow-up covering a period of time before the initiation of HAART, ranging from 93–136.5 months for SPs and 7.5–16.5 months for RPs. Analyses were also performed based on the different stages of HIV infection (chronic, pre-HAART individuals—subjects sampled before initiating HAART but who initiated therapy from 12 to 24 months—and those receiving HAART). The plasma cytokine levels of 16 HIV-infected rapid progressors and 25 slow progressors were measured using a Human Th1/Th2/Th17 CBA kit. The IL-6 and IL-10 plasma levels differed significantly between the stages of HIV infection. The IL-6 levels were higher in slow progressors pre-HAART than in chronically infected SPs and HIV-seronegative individuals. The IL-10 levels were higher in slow progressors pre-HAART than in slow progressors receiving HAART and HIV-seronegative controls, and in rapid progressors, the IL-10 levels were higher in pre-HAART subjects than in HIV-seronegative controls. The results reflect the changes in the cytokine profile occurring during different clinical stages in HIV+ subjects. Our results suggest an association between increased IL-6 and IL-10 levels and pre-HAART stages independent of the slow or rapid progression status of the subjects. Thus, increased IL-6 and IL-10 levels could indicate a global inflammatory status and could be used as markers of the disease course in HIV-infected individuals. PMID:27214135

  3. Microgravity increases DNA damage response in Caenorhabditis elegans during Shenzhou-8 spaceflight

    NASA Astrophysics Data System (ADS)

    Gao, Ying; Sun, Yeqing; Xu, Dan; Zhao, Lei; Xu, Jiamin

    DNA damage response (DDR) plays an important role in genome maintenance through cell cycle arrest followed by DNA repair and/or apoptosis. Perturbing DDR may elicit genomic instability, carcinogenesis, even cell death. Space radiation and microgravity both have been reported to cause DDR in mammal cells,while, in the space environment, the interaction of space radiation and microgravity on DDR is still controversial. To clarify the interaction, dauer larva of Caenorhabditis elegans were employed in Shenzhou-8 space mission and suffered space synthetic environment (RM) and space radiation (R) during 16.5-day spaceflight. mRNA microarray, qPCR and miRNA microarray were performed individually to detect the differences of transcriptome and microRNome affected by two environments. The results showed that, two fold genes were regulated more significantly by RM than by R. These regulated genes were involved in different physiological activities from each environment, which mainly involve in protein metabolic and modification processes in RM, and energy metabolic process in R. 21 of 500 DDR genes were extracted as significantly different expression in two space environments. DNA repair and apoptosis were enhanced by microgravity, since 18 of 21 genes were altered by RM specifically, including six “Response to DNA damage stimulus” genes, four “DNA repair” genes and eight “apoptosis process” genes. miRNAome also showed changes in response to microgravity. miRNA-81, 82, 124 and 795 were predicted to respond to RM and regulate DDR in C.elegans for the first time. These results suggest that microgravity increases the physiological activities to the space environment, especially enhance DNA damage response on transcription and post-transcriptional regulation in metazoan. We expect the finding provides new informations on synergetic effects between microgravity and radiation, and may be helpful for space risk assessment.

  4. Choline deficiency increases lymphocyte apoptosis and DNA damage in humans2,3

    PubMed Central

    da Costa, Kerry-Ann; Niculescu, Mihai D; Craciunescu, Corneliu N; Fischer, Leslie M; Zeisel, Steven H

    2008-01-01

    Background: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. Objective: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Design: Fifty-one men and women aged 18–70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138–550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase–mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. Results: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. Conclusions: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dys-function also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected. PMID:16825685

  5. Incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

    SciTech Connect

    Pardo, E.G.; Hernandez, P.; Gutierrez, C.

    1987-02-01

    The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10/sup -9/-5 x 10/sup -7/ M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10/sup -1/ M. However, at the highest FdUrd dose tested (10/sup -7/ M), 10/sup -4/ M dUrd could not reverse the FdUrd effect in long-term experiments as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with (6-/sup 3/H)dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label in the form of (6-/sup 3/H)dUMP. Thus the authors conclude that under the experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. By analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, they has scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.

  6. Fatal Pediatric Cerebral Malaria Is Associated with Intravascular Monocytes and Platelets That Are Increased with HIV Coinfection.

    PubMed

    Hochman, Sarah E; Madaline, Theresa F; Wassmer, Samuel C; Mbale, Emmie; Choi, Namjong; Seydel, Karl B; Whitten, Richard O; Varughese, Julie; Grau, Georges E R; Kamiza, Steve; Molyneux, Malcolm E; Taylor, Terrie E; Lee, Sunhee; Milner, Danny A; Kim, Kami

    2015-09-22

    Cerebral malaria (CM) is a major contributor to malaria deaths, but its pathophysiology is not well understood. While sequestration of parasitized erythrocytes is thought to be critical, the roles of inflammation and coagulation are controversial. In a large series of Malawian children hospitalized with CM, HIV coinfection was more prevalent than in pediatric population estimates (15% versus 2%, P < 0.0001, chi-square test), with higher mortality than that seen in HIV-uninfected children (23% versus 17%, P = 0.0178, chi-square test). HIV-infected (HIV(+)) children with autopsy-confirmed CM were older than HIV-uninfected children (median age, 99 months versus 32 months, P = 0.0007, Mann-Whitney U test) and appeared to lack severe immunosuppression. Because HIV infection is associated with dysregulated inflammation and platelet activation, we performed immunohistochemistry analysis for monocytes, platelets, and neutrophils in brain tissue from HIV(+) and HIV-uninfected children with fatal CM. Children with autopsy-confirmed CM had significantly (>9 times) more accumulations of intravascular monocytes and platelets, but not neutrophils, than did children with nonmalarial causes of coma. The monocyte and platelet accumulations were significantly (>2-fold) greater in HIV(+) children than in HIV-uninfected children with autopsy-confirmed CM. Our findings indicate that HIV is a risk factor for CM and for death from CM, independent of traditional measures of HIV disease severity. Brain histopathology supports the hypotheses that inflammation and coagulation contribute to the pathogenesis of pediatric CM and that immune dysregulation in HIV(+) children exacerbates the pathological features associated with CM. IMPORTANCE : There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing

  7. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation.

    PubMed

    Korzeneva, Inna B; Kostuyk, Svetlana V; Ershova, Liza S; Osipov, Andrian N; Zhuravleva, Veronika F; Pankratova, Galina V; Porokhovnik, Lev N; Veiko, Natalia N

    2015-09-01

    The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly - at survival of the cells, including the cells with the damaged DNA. PMID:26113293

  8. Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy

    PubMed Central

    Lee, Sulggi A.; Bacchetti, Peter; Chomont, Nicolas; Fromentin, Remi; Lewin, Sharon R.; O’Doherty, Una; Palmer, Sarah; Richman, Douglas D.; Siliciano, Janet D.; Yukl, Steven A.; Deeks, Steven G.; Burbelo, Peter D.

    2016-01-01

    Background A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the “reservoir”). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART). Methods We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables. Results Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results. Conclusions Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV

  9. Intrahepatic DNA vaccination: unexpected increased resistance against murine cysticercosis induced by non-specific enhanced immunity.

    PubMed

    Cruz-Revilla, C; Sonabend, A M; Rosas, G; Toledo, A; Meneses, G; Lopez-Casillas, F; Hernández, B; Fragoso, G; Sciutto, E

    2006-06-01

    Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model with which to test the efficacy of new vaccine candidates and delivery systems against pig cysticercosis. A high level of protection against murine cysticercosis was previously observed by intramuscular or intradermal DNA immunization with the use of the sequence of the recombinant KETc7 antigen cloned in pcDNA3 (pTc-sp7). To determine the effect of KETc7 differential expression in DNA vaccination, KETc7 was cloned in pGEM 11Zf(+) under the control of the tissue-specific regulatory promoter phosphoenolpyruvate carboxykinase (pPc-sp7). A high level of protection was induced by intrahepatic immunization with pPc-sp7, pTc-sp7 and the empty vector in the absence of any specific immunity. The empty vector pGEM 11Zf(+), the plasmid with the highest content of CpG sequences, provided to the most efficient protection. This protection was related to an increased number of splenocytes, enhanced nonspecific splenocyte proliferation, and intensified intrahepatic INF-gamma production. Overall, intrahepatic plasmid CpG-DNA immunization provokes an exacerbated nonspecific immune response that can effectively control Taenia crassiceps cysticercosis.

  10. Mung Bean Nuclease Treatment Increases Capture Specificity of Microdroplet-PCR Based Targeted DNA Enrichment

    PubMed Central

    Yu, Zhenming; Cao, Kajia; Tischler, Tanya; Stolle, Catherine A.; Santani, Avni B.

    2014-01-01

    Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial. PMID:25058678

  11. Increased Hb A2 values in an HIV-1-infected patient receiving antiretroviral drugs: a pitfall for thalassemia antenatal diagnosis.

    PubMed

    Pornprasert, Sakorn; Sukunthamala, Kanyakan; Leechanachai, Pranee; Sanguansermsri, Torpong

    2009-01-01

    We report a human immunodeficiency virus-1 (HIV-1)-infected couple, where the woman in the 11th week of gestation, carried a Hb E trait. She and her spouse were referred to the hemoglobinopathy counselors. Her spouse's blood was subsequently tested and showed an increased Hb A(2) value. However, his red cell indices and osmotic fragility test were different from those found in beta-thalassemia (beta-thal) carriers. The beta-thal genes were investigated further and no mutations were observed. Therefore, it is unlikely that he is a beta-thal carrier and the increased Hb A(2) value is a result of receiving antiretroviral drugs. As antenatal thalassemia screening becomes more widespread, measuring the Hb A(2) values should be taken in all HIV-1-infected couples before the initiation of antiretroviral drugs to rule out misdiagnosis of beta-thal. However, if these tests are not available, the results of the red cell indices and osmotic fragility test should be considered as they may provide great value for beta-thal investigations.

  12. AIDS Impact/SEISIDA Special Issue: Using Social and Behavior Change Communication to Increase HIV Testing and Condom Use: The Malawi BRIDGE Project

    PubMed Central

    Kaufman, Michelle R.; Rimal, Rajiv N.; Carrasco, Maria; Fajobi, OlaOluwa; Soko, Anthony; Limaye, Rupali; Mkandawire, Glory

    2015-01-01

    While overall HIV prevalence in Malawi has decreased, it is still high in the Southern region of the country. Behavioral prevention activities are crucial to continue the reduction in HIV prevalence. Behavior change is influenced by many factors. Previous work indicates knowledge about HIV transmission, self-efficacy to protect oneself from exposure, and accurate risk perception of one’s susceptibility all impact sexual behavior. The current study looks at the effects of a behavior change communication program in Malawi called the BRIDGE II Project on psychosocial and behavioral variables. The program sought to address barriers to individual action and confront societal norms related to sexual risk behavior through a mix of community-based activities and mass media messages delivered through local radio stations. Using cohort data (n = 594), we examined the effect of BRIDGE exposure on three variables that affect HIV behaviors: knowledge, self-efficacy, and risk perception, as well as two behavioral outcomes: HIV testing and condom use at last sex. Data were collected at baseline and for a midterm evaluation. Regression analyses showed exposure to BRIDGE was significantly associated with knowledge level (β = 0.20, p < .001) and self-efficacy (β = 0.35, p < .001) at midterm when controlling for baseline scores, but not risk perception. Psychosocial variables did not show a significant relationship to either behavioral outcome. However, program exposure was a significant predictor of both HIV testing in the past year (OR = 1.40, p < .001) and condom use at last sex (OR = 1.26, p < .05). This study suggests such a communication intervention may play an important role in not only affecting HIV-related behaviors themselves, but also critical factors that affect HIV behaviors, including knowledge and self-efficacy. It is recommended that communication efforts around HIV risk reduction be increased. PMID:24735337

  13. Persistent CSF but not plasma HIV RNA is associated with increased risk of new-onset moderate-to-severe depressive symptoms; a prospective cohort study.

    PubMed

    Hammond, Edward R; Crum, Rosa M; Treisman, Glenn J; Mehta, Shruti H; Clifford, David B; Ellis, Ronald J; Gelman, Benjamin B; Grant, Igor; Letendre, Scott L; Marra, Christina M; Morgello, Susan; Simpson, David M; Mcarthur, Justin C

    2016-08-01

    Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up of 674 participants. We fit linear mixed models (N = 233) and discrete-time survival models (N = 154; 832 observations) to evaluate trajectories of Beck Depression Inventory (BDI) II scores and the incidence of new-onset moderate-to-severe depressive symptoms (BDI ≥ 17) among participants on combination antiretroviral therapy (cART), who were free of depression at study entry and received a minimum of three CSF examinations over 2496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR) = 4.76, (95 % CI 1.58-14.3); P = 0.006. Depression (BDI) scores were 2.53 points higher (95 % CI 0.47-4.60; P = 0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of CART, and lifetime depression diagnosis by Diagnostic Statistical Manual (DSM-IV) criteria. Persistent CSF but not plasma HIV RNA is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association. PMID:26727907

  14. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    SciTech Connect

    Mefford, Megan E.; Kunstman, Kevin; Wolinsky, Steven M.; Gabuzda, Dana

    2015-07-15

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.

  15. Persistent CSF but not plasma HIV RNA is associated with increased risk of new-onset moderate-to-severe depressive symptoms; a prospective cohort study.

    PubMed

    Hammond, Edward R; Crum, Rosa M; Treisman, Glenn J; Mehta, Shruti H; Clifford, David B; Ellis, Ronald J; Gelman, Benjamin B; Grant, Igor; Letendre, Scott L; Marra, Christina M; Morgello, Susan; Simpson, David M; Mcarthur, Justin C

    2016-08-01

    Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up of 674 participants. We fit linear mixed models (N = 233) and discrete-time survival models (N = 154; 832 observations) to evaluate trajectories of Beck Depression Inventory (BDI) II scores and the incidence of new-onset moderate-to-severe depressive symptoms (BDI ≥ 17) among participants on combination antiretroviral therapy (cART), who were free of depression at study entry and received a minimum of three CSF examinations over 2496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR) = 4.76, (95 % CI 1.58-14.3); P = 0.006. Depression (BDI) scores were 2.53 points higher (95 % CI 0.47-4.60; P = 0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of CART, and lifetime depression diagnosis by Diagnostic Statistical Manual (DSM-IV) criteria. Persistent CSF but not plasma HIV RNA is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association.

  16. Low-dose aspirin (acetylsalicylate) prevents increases in brain PGE2, 15-epi-lipoxin A4 and 8-isoprostane concentrations in 9 month-old HIV-1 transgenic rats, a model for HIV-1 associated neurocognitive disorders

    PubMed Central

    Blanchard, Helene C.; Taha, Ameer Y.; Rapoport, Stanley I; Yuan, Zhi-Xin

    2015-01-01

    Background Older human immunodeficiency virus (HIV)-1 transgenic rats are a model for HIV-1 associated neurocognitive disorders (HAND). They show behavioral changes, neuroinflammation, neuronal loss, and increased brain arachidonic acid (AA) enzymes. Aspirin (acetylsalicylate, ASA) inhibits AA oxidation by cyclooxygenase (COX)-1 and COX-2. Hypothesis Chronic low-dose ASA will downregulate brain AA metabolism in HIV-1 transgenic rats. Methods Nine month-old HIV-1 transgenic and wildtype rats were given 42 days of 10 mg/kg/day ASA or nothing in drinking water; eicosanoids were measured using ELISAs on microwaved brain extracts. Results Brain 15-epi-lipoxin A4 and 8-isoprostane concentrations were significantly higher in HIV-1 transgenic than wildtype rats; these differences were prevented by ASA. ASA reduced prostaglandin E2 and leukotriene B4 concentrations in HIV-1 Tg but not wildtype rats. Thromboxane B2, 15-HETE, lipoxin A4 and resolvin D1 concentrations were unaffected by genotype or treatment. Conclusion Chronic low-dose ASA reduces AA-metabolite markers of neuroinflammation and oxidative stress in a rat model for HAND. PMID:25638779

  17. DNA Methylation is Associated with an Increased Level of Conservation at Nondegenerate Nucleotides in Mammals

    PubMed Central

    Chuang, Trees-Juen; Chen, Feng-Chi

    2014-01-01

    DNA methylation at CpG dinucleotides can significantly increase the rate of cytosine-to-thymine mutations and the level of sequence divergence. Although the correlations between DNA methylation and genomic sequence evolution have been widely studied, an unaddressed yet fundamental question is how DNA methylation is associated with the conservation of individual nucleotides in different sequence contexts. Here, we demonstrate that in mammalian exons, the correlations between DNA methylation and the conservation of individual nucleotides are dependent on the type of exonic sequence (coding or untranslated), the degeneracy of coding nucleotides, background selection pressure, and the relative position (first or nonfirst exon in the transcript) where the nucleotides are located. For untranslated and nonzero-fold degenerate nucleotides, methylated sites are less conserved than unmethylated sites regardless of background selection pressure and the relative position of the exon. For zero-fold degenerate (or nondegenerate) nucleotides, however, the reverse trend is observed in nonfirst coding exons and first coding exons that are under stringent background selection pressure. Furthermore, cytosine-to-thymine mutations at methylated zero-fold degenerate nucleotides are predicted to be more detrimental than those that occur at unmethylated nucleotides. As zero-fold and nonzero-fold degenerate nucleotides are very close to each other, our results suggest that the “functional resolution” of DNA methylation may be finer than previously recognized. In addition, the positive correlation between CpG methylation and the level of conservation at zero-fold degenerate nucleotides implies that CpG methylation may serve as an “indicator” of functional importance of these nucleotides. PMID:24157417

  18. Inhibition of catechol-O-methyltransferase increases estrogen-DNA adduct formation

    PubMed Central

    Zahid, Muhammad; Saeed, Muhammad; Lu, Fang; Gaikwad, Nilesh; Rogan, Eleanor; Cavalieri, Ercole

    2007-01-01

    The association found between breast cancer development and prolonged exposure to estrogens suggests that this hormone is of etiologic importance in the causation of the disease. Studies on estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA forms depurinating adducts [4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua]. These adducts cause mutations leading to the initiation of breast cancer. Catechol-O-methyltransferase (COMT) is considered an important enzyme that protects cells from the genotoxicity and cytotoxicity of catechol estrogens, by preventing their conversion to quinones. The goal of the present study was to investigate the effect of COMT inhibition on the formation of depurinating estrogen-DNA adducts. Immortalized human breast epithelial MCF-10F cells were treated with 4-OHE2 (0.2 or 0.5 μM) for 24 h at 120, 168, 216, and 264 h post-plating or one time at 1–30 μM 4-OHE2 with or without the presence of COMT inhibitor (Ro41-0960). The culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector. The results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. The COMT inhibitor Ro41-0960 blocked the methoxylation of catechol estrogens, with concomitant 3–4 fold increases in the levels of the depurinating adducts. Thus, low activity of COMT leads to higher levels of depurinating estrogen-DNA adducts that can induce mutations and initiate cancer. PMID:17964424

  19. Increased Sperm DNA Damage in Experimental Rat Varicocele Model and The Beneficial Effect of Varicocelectomy

    PubMed Central

    Öztürk, Metin İshak; Koca, Orhan; Keleş, Muzaffer Oğuz; Yılmaz, Seda; Karaman, Muhammet Ihsan

    2012-01-01

    Background Varicocele, the abnormal dilatation of the veins in the pampiniform plexus is commonly seen in infertile patients. In this study, we aim to examine sperm DNA damage after the creation of experimental varicocele in rats and to observe the change of this damage after a varicocelectomy. Materials and Methods In this experimental study, a total of 30 adult male Wistar albino rats were divided into three groups. The 10 rats in group 1 underwent a sham operation, an experimental varicocele was created in both the10 rats in group 2 and the 10 rats in group 3 (a total of 20 rats). While the rats of group 2 were sacrificed after four weeks, the rats in group 3 underwent a varicocelectomy after four weeks and were sacrificed four weeks after the varicocelectomy to observe its effects. Sperm DNA fragmentation was assessed with a Halomax® kit. The DNA Fragmentation Index (DFI) was calculated and the groups were compared according to their DFI. Statistical analysis was performed using the Mann-Whitney U test. Results Median sperm DFI was 17.6 (range: 7.6) in the right testicle and 18.3 (range: 6.8) in the left testicle in the control group; 30.7 (range: 8.8) in the right testicle and 31.8 (range: 9.6) in the left testicle in the varicocele group; 27.1 (range: 8.1) in the right testicle and 28.6 (range: 8.9) in the left testicle in the varicocelectomy group. DNA damage in both right and left testicles was statistically significant between the three groups (p<0.05). Conclusion The results of this study show that varicocele leads to increased sperm DNA damage and this damage is decreased by varicocelectomy. PMID:25493165

  20. Homophobia is associated with sexual behavior that increases risk of acquiring and transmitting HIV infection among black men who have sex with men.

    PubMed

    Jeffries, William L; Marks, Gary; Lauby, Jennifer; Murrill, Christopher S; Millett, Gregorio A

    2013-05-01

    We investigated whether the experience of homophobic events increases the odds of engaging in unprotected anal intercourse (UAI) among black men who have sex with men (MSM) and whether social integration level buffered the association. Participants (N = 1,154) reported homophobic events experienced in the past 12 months. Social integration measures included social support, closeness with family members and friends, attachment to the black gay community, openness about sexuality within religious communities, and MSM social network size. Logistic regression analyses indicated that experiencing homophobia was associated with (1) UAI among men not previously diagnosed with HIV and (2) sexual HIV transmission risk behavior among men who knew they were HIV-infected. None of the social integration measures buffered these associations. Homophobia may promote acquisition and transmission of HIV infection among black MSM. Interventions are needed to reduce homophobia experienced by black MSM.

  1. Increasing the Morphological Stability of DNA-Templated Nanostructures with Surface Hydrophobicity.

    PubMed

    Lermusiaux, Laurent; Bidault, Sébastien

    2015-11-11

    DNA has been extensively used as a versatile template to assemble inorganic nanoparticles into complex architectures; thanks to its programmability, stability, and long persistence length. But the geometry of self-assembled nanostructures depends on a complex combination of attractive and repulsive forces that can override the shape of a molecular scaffold. In this report, an approach to increase the morphological stability of DNA-templated gold nanoparticle (AuNP) groupings against electrostatic interactions is demonstrated by introducing hydrophobicity on the particle surface. Using single nanostructure spectroscopy, the nanometer-scale distortions of 40 nm diameter AuNP dimers are compared with different hydrophilic, amphiphilic, neutral, and negatively charged surface chemistries, when modifying the local ionic strength. It is observed that, with most ligands, a majority of studied nanostructures deform freely from a stretched geometry to touching particles when increasing the salt concentration while hydrophobicity strongly limits the dimer distortions. Furthermore, an amphiphilic surface chemistry provides DNA-linked AuNP dimers with a high long-term stability against internal aggregation. PMID:26395441

  2. CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

    PubMed

    Kava, Hieronimus W; Murray, Vincent

    2016-10-01

    This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use. PMID:27567075

  3. Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae

    PubMed Central

    Donigan, Katherine A.; Cerritelli, Susana M.; McDonald, John P.; Vaisman, Alexandra; Crouch, Robert J.; Woodgate, Roger

    2015-01-01

    DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of S. cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related polι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S. cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNaseH2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. PMID:26340535

  4. Closing the digital divide in HIV/AIDS care: development of a theory-based intervention to increase Internet access.

    PubMed

    Kalichman, S C; Weinhardt, L; Benotsch, E; Cherry, C

    2002-08-01

    Advances in information technology are revolutionizing medical patient education and the Internet is becoming a major source of information for people with chronic medical conditions, including HIV/AIDS. However, many AIDS patients do not have equal access to the Internet and are therefore at an information disadvantage, particularly minorities, persons of low-income levels and individuals with limited education. This paper describes the development and pilot testing of a workshop-style intervention designed to close the digital divide in AIDS care. Grounded in the Information-Motivation-Behavioral Skills (IMB) model of health behaviour change, we developed an intervention for persons with no prior history of using the Internet. The intervention included instruction in using hardware and search engines, motivational enhancement to increase interest and perceived relevance of the Internet, and skills for critically evaluating and using health information accessed via the Internet. Participants were also introduced to communication and support functions of the Internet including e-mail, newsgroups and chat groups. Pilot testing demonstrated feasibility, acceptability and promise for closing the digital divide in HIV/AIDS care using a relatively brief and intensive theory-based intervention that could be implemented in community settings. PMID:12204154

  5. National Income Inequality and Declining GDP Growth Rates Are Associated with Increases in HIV Diagnoses among People Who Inject Drugs in Europe: A Panel Data Analysis

    PubMed Central

    Nikolopoulos, Georgios K.; Fotiou, Anastasios; Kanavou, Eleftheria; Richardson, Clive; Detsis, Marios; Pharris, Anastasia; Suk, Jonathan E.; Semenza, Jan C.; Costa-Storti, Claudia; Paraskevis, Dimitrios; Sypsa, Vana; Malliori, Melpomeni-Minerva; Friedman, Samuel R.; Hatzakis, Angelos

    2015-01-01

    Background There is sparse evidence that demonstrates the association between macro-environmental processes and drug-related HIV epidemics. The present study explores the relationship between economic, socio-economic, policy and structural indicators, and increases in reported HIV infections among people who inject drugs (PWID) in the European Economic Area (EEA). Methods We used panel data (2003–2012) for 30 EEA countries. Statistical analyses included logistic regression models. The dependent variable was taking value 1 if there was an outbreak (significant increase in the national rate of HIV diagnoses in PWID) and 0 otherwise. Explanatory variables included the growth rate of Gross Domestic Product (GDP), the share of the population that is at risk for poverty, the unemployment rate, the Eurostat S80/S20 ratio, the Gini coefficient, the per capita government expenditure on health and social protection, and variables on drug control policy and drug-using population sizes. Lags of one to three years were investigated. Findings In multivariable analyses, using two-year lagged values, we found that a 1% increase of GDP was associated with approximately 30% reduction in the odds of an HIV outbreak. In GDP-adjusted analyses with three-year lagged values, the effect of the national income inequality on the likelihood of an HIV outbreak was significant [S80/S20 Odds Ratio (OR) = 3.89; 95% Confidence Interval (CI): 1.15 to 13.13]. Generally, the multivariable analyses produced similar results across three time lags tested. Interpretation Given the limitations of ecological research, we found that declining economic growth and increasing national income inequality were associated with an elevated probability of a large increase in the number of HIV diagnoses among PWID in EEA countries during the last decade. HIV prevention may be more effective if developed within national and European-level policy contexts that promote income equality, especially among vulnerable

  6. Increased DNA and RNA damage by oxidation in patients with bipolar I disorder.

    PubMed

    Jacoby, A S; Vinberg, M; Poulsen, H E; Kessing, L V; Munkholm, K

    2016-01-01

    The mechanisms underlying bipolar disorder (BD) and the associated medical burden are unclear. Damage generated by oxidation of nucleosides may be implicated in BD pathophysiology; however, evidence from in vivo studies is limited and the extent of state-related alterations is unclear. This prospective study investigated for we believe the first time the damage generated by oxidation of DNA and RNA strictly in patients with type I BD in a manic or mixed state and subsequent episodes and remission compared with healthy control subjects. Urinary excretion of 8-oxo-deoxyguanosine (8-oxodG) and 8-oxo-guanosine (8-oxoGuo), valid markers of whole-body DNA and RNA damage by oxidation, respectively, was measured in 54 patients with BD I and in 35 healthy control subjects using a modified ultraperformance liquid chromatography and mass spectrometry assay. Repeated measurements were evaluated in various affective phases during a 6- to 12-month period and compared with repeated measurements in healthy control subjects. Independent of lifestyle and demographic variables, a 34% (P<0.0001) increase in RNA damage by oxidation across all affective states, including euthymia, was found in patients with BD I compared with healthy control subjects. Increases in DNA and RNA oxidation of 18% (P<0.0001) and 8% (P=0.02), respectively, were found in manic/hypomanic states compared with euthymia, and levels of 8-oxodG decreased 15% (P<0.0001) from a manic or mixed episode to remission. The results indicate a role for DNA and RNA damage by oxidation in BD pathophysiology and a potential for urinary 8-oxodG and 8-oxoGuo to function as biological markers of diagnosis, state and treatment response in BD. PMID:27505230

  7. Increased DNA and RNA damage by oxidation in patients with bipolar I disorder

    PubMed Central

    Jacoby, A S; Vinberg, M; Poulsen, H E; Kessing, L V; Munkholm, K

    2016-01-01

    The mechanisms underlying bipolar disorder (BD) and the associated medical burden are unclear. Damage generated by oxidation of nucleosides may be implicated in BD pathophysiology; however, evidence from in vivo studies is limited and the extent of state-related alterations is unclear. This prospective study investigated for we believe the first time the damage generated by oxidation of DNA and RNA strictly in patients with type I BD in a manic or mixed state and subsequent episodes and remission compared with healthy control subjects. Urinary excretion of 8-oxo-deoxyguanosine (8-oxodG) and 8-oxo-guanosine (8-oxoGuo), valid markers of whole-body DNA and RNA damage by oxidation, respectively, was measured in 54 patients with BD I and in 35 healthy control subjects using a modified ultraperformance liquid chromatography and mass spectrometry assay. Repeated measurements were evaluated in various affective phases during a 6- to 12-month period and compared with repeated measurements in healthy control subjects. Independent of lifestyle and demographic variables, a 34% (P<0.0001) increase in RNA damage by oxidation across all affective states, including euthymia, was found in patients with BD I compared with healthy control subjects. Increases in DNA and RNA oxidation of 18% (P<0.0001) and 8% (P=0.02), respectively, were found in manic/hypomanic states compared with euthymia, and levels of 8-oxodG decreased 15% (P<0.0001) from a manic or mixed episode to remission. The results indicate a role for DNA and RNA damage by oxidation in BD pathophysiology and a potential for urinary 8-oxodG and 8-oxoGuo to function as biological markers of diagnosis, state and treatment response in BD. PMID:27505230

  8. Opposite transcriptional regulation of integrated vs unintegrated HIV genomes by the NF-κB pathway

    PubMed Central

    Thierry, Sylvain; Thierry, Eloïse; Subra, Frédéric; Deprez, Eric; Leh, Hervé; Bury-Moné, Stéphanie; Delelis, Olivier

    2016-01-01

    Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence. PMID:27167871

  9. Detection of cytomegalovirus DNA on dried blood spots collected from infants infected with HIV: an in-house method adaptable in resource-limited settings.

    PubMed

    Leruez-Ville, Marianne; Ngin, Sopeak; Guilleminot, Tiffany; Kfutwah, Anfumbom; Moussa, Sandrine; Tran, Ton; Nerrienet, Eric

    2013-11-01

    In countries with limited resources, infants infected with HIV are highly exposed to CMV co-infection which probably represents a major risk factor for disease progression in this population. This study aimed to evaluate the performance of a low cost CMV DNA extraction method from DBS and the feasibility of its implementation in laboratories of 4 countries with limited resources. DNA was extracted from DBS with a cationic resin (chelex 100) and amplified with an "in house" real time CMV PCR. Dilutions of a quantified whole blood sample were spotted on paper to evaluate the 95% detection limit. A DBS quality control panel was analyzed in all laboratories. CMV PCR was compared between DBS and liquid whole blood (gold standard) in 2 populations: 418 transplanted patients and 59 infants infected with HIV (median age of 2 months). The CMV PCR 95% detection limit in DBS was 3.87 log10 copies/mL. Its positive and negative predictive values for CMV diagnosis in infants infected with HIV were 100% and 87.5% respectively. Quality control panels gave consistent qualitative results in all laboratories. This assay had high predictive values for CMV diagnosis in infants infected with HIV and its implementation in resource-limited countries with limited resources is feasible. PMID:23891874

  10. Declining Inconsistent Condom Use but Increasing HIV and Syphilis Prevalence Among Older Male Clients of Female Sex Workers: Analysis From Sentinel Surveillance Sites (2010-2015), Guangxi, China.

    PubMed

    Chen, Yi; Abraham Bussell, Scottie; Shen, Zhiyong; Tang, Zhenzhu; Lan, Guanghua; Zhu, Qiuying; Liu, Wei; Tang, Shuai; Li, Rongjian; Huang, Wenbo; Huang, Yuman; Liang, Fuxiong; Wang, Lu; Shao, Yiming; Ruan, Yuhua

    2016-05-01

    Clients of female sex workers (CFSWs) are a bridge population for the spread of HIV and syphilis to low or average risk heterosexuals. Most studies have examined the point prevalence of these infections in CFSWs. Limited evidence suggests that older age CFSWs are at a higher risk of acquiring sexually transmitted diseases compared with younger clients. Thus, we sought to describe long-term trends in HIV, syphilis, and hepatitis C (HCV) to better understand how these infections differ by sex worker classification and client age. We also examined trends in HIV, syphilis, and HCV among categories of female sex workers (FSWs).We conducted serial cross-sectional studies from 2010 to 2015 in Guangxi autonomous region, China. We collected demographic and behavior variables. FSWs and their clients were tested for HIV, syphilis, and HCV antibodies. Positive HIV and syphilis serologies were confirmed by Western blot and rapid plasma regain, respectively. Clients were categorized as middle age (40-49 years) and older clients (≥50 years). FSWs were categorized as high-tier, middle-tier, or low-tier based on the payment amount charged for sex and their work venue. Chi-square test for trends was used for testing changes in prevalence over time.By 2015, low-tier FSWs (LTFSWs) accounted for almost half of all FSWs; and they had the highest HIV prevalence at 1.4%. HIV prevalence declined significantly for FSWs (high-tier FSW, P = 0.003; middle-tier FSWs; P = 0.021; LTFSWs, P < 0.001). Syphilis infections significantly declined for FSWs (P < 0.001) but only to 7.3% for LTFSWs. HCV and intravenous drug use were uncommon in FSWs. HIV prevalence increased for older age clients (1.3%-2.0%, P = 0.159) while syphilis prevalence remained stable. HCV infections were halved among older clients in 3 years (1.7%-0.8%, P < 0.001). Condom use during the last sexual encounter increased for FSWs and CFSWs. Few clients reported sex with men or intravenous drug use. Clients

  11. Does Older Age Confer an Increased Risk of Incident Neurocognitive Disorders Among Persons Living with HIV Disease?

    PubMed Central

    Sheppard, David P.; Woods, Steven Paul; Bondi, Mark W.; Gilbert, Paul E.; Massman, Paul J.; Doyle, Katie L.

    2015-01-01

    Objective This study aimed to determine the combined effects of age and HIV infection on the risk of incident neurocognitive disorders. Method A total of 146 neurocognitively normal participants were enrolled at baseline into one of four groups based on age (≤ 40 years and ≥ 50 years) and HIV serostatus resulting in 24 younger HIV−, 27 younger HIV+, 39 older HIV−, and 56 older HIV+ individuals. All participants were administered a standardized clinical neuropsychological battery at baseline and 14.3 ±0.2 months later. Results A logistic regression predicting incident neurocognitive disorders from HIV, age group, and their interaction was significant (χ2[4] = 13.56, p = .009), with a significant main effect of HIV serostatus (χ2[1] = 5.01, p = .025), but no main effect of age or age by HIV interaction (ps > .10). Specifically, 15.7 percent of the HIV+ individuals had an incident neurocognitive disorder as compared to 3.2 percent of the HIV− group (odds ratio = 4.8 [1.2, 32.6]). Among older HIV+ adults, lower baseline cognitive reserve, prospective memory, and verbal fluency each predicted incident neurocognitive disorders at follow-up. Conclusions Independent of age, HIV infection confers a nearly 5-fold risk for developing a neurocognitive disorder over approximately one year. Individuals with lower cognitive reserve and mild weaknesses in higher-order neurocognitive functions may be targeted for closer clinical monitoring and preventative measures. PMID:26367342

  12. Mathematical Modeling Predicts that Increased HSV-2 Shedding in HIV-1 Infected Persons Is Due to Poor Immunologic Control in Ganglia and Genital Mucosa.

    PubMed

    Schiffer, Joshua T; Swan, David A; Magaret, Amalia; Schacker, Timothy W; Wald, Anna; Corey, Lawrence

    2016-01-01

    A signature feature of HIV infection is poor control of herpes virus infections, which reactivate from latency and cause opportunistic infections. While the general mechanism underlying this observation is deficient CD4+T-cell function, it is unknown whether increased severity of herpes virus infections is due primarily to poor immune control in latent or lytic sites of infection, or whether CD4+ immunodeficiency leads to more critical downstream deficits in humoral or cell-mediated immunologic responses. Here we compare genital shedding patterns of herpes simplex virus-2 (HSV-2) in 98 HIV infected and 98 HIV uninfected men matched on length of infection, HSV-1 serostatus and nationality. We demonstrate that high copy HSV-2 shedding is more frequent in HIV positive men, particularly in participants with CD4+ T-cell count <200/μL. Genital shedding is more frequent due to higher rate of shedding episodes, as well as a higher proportion of prolonged shedding episodes. Peak episode viral load was not found to differ between HIV infected and uninfected participants regardless of CD4+ T-cell count. We simulate a mathematical model which recapitulates these findings and identifies that rate of HSV-2 release from neural tissue increases, duration of mucosal cytolytic immune protection decreases, and cell-free viral lifespan increases in HIV infected participants. These results suggest that increased HSV-2 shedding in HIV infected persons may be caused by impaired immune function in both latent and lytic tissue compartments, with deficits in clearance of HSV-2 infected cells and extracellular virus.

  13. Mathematical Modeling Predicts that Increased HSV-2 Shedding in HIV-1 Infected Persons Is Due to Poor Immunologic Control in Ganglia and Genital Mucosa

    PubMed Central

    Schiffer, Joshua T.; Swan, David A.; Magaret, Amalia; Schacker, Timothy W.; Wald, Anna; Corey, Lawrence

    2016-01-01

    A signature feature of HIV infection is poor control of herpes virus infections, which reactivate from latency and cause opportunistic infections. While the general mechanism underlying this observation is deficient CD4+T-cell function, it is unknown whether increased severity of herpes virus infections is due primarily to poor immune control in latent or lytic sites of infection, or whether CD4+ immunodeficiency leads to more critical downstream deficits in humoral or cell-mediated immunologic responses. Here we compare genital shedding patterns of herpes simplex virus-2 (HSV-2) in 98 HIV infected and 98 HIV uninfected men matched on length of infection, HSV-1 serostatus and nationality. We demonstrate that high copy HSV-2 shedding is more frequent in HIV positive men, particularly in participants with CD4+ T-cell count <200/μL. Genital shedding is more frequent due to higher rate of shedding episodes, as well as a higher proportion of prolonged shedding episodes. Peak episode viral load was not found to differ between HIV infected and uninfected participants regardless of CD4+ T-cell count. We simulate a mathematical model which recapitulates these findings and identifies that rate of HSV-2 release from neural tissue increases, duration of mucosal cytolytic immune protection decreases, and cell-free viral lifespan increases in HIV infected participants. These results suggest that increased HSV-2 shedding in HIV infected persons may be caused by impaired immune function in both latent and lytic tissue compartments, with deficits in clearance of HSV-2 infected cells and extracellular virus. PMID:27285483

  14. Mathematical Modeling Predicts that Increased HSV-2 Shedding in HIV-1 Infected Persons Is Due to Poor Immunologic Control in Ganglia and Genital Mucosa.

    PubMed

    Schiffer, Joshua T; Swan, David A; Magaret, Amalia; Schacker, Timothy W; Wald, Anna; Corey, Lawrence

    2016-01-01

    A signature feature of HIV infection is poor control of herpes virus infections, which reactivate from latency and cause opportunistic infections. While the general mechanism underlying this observation is deficient CD4+T-cell function, it is unknown whether increased severity of herpes virus infections is due primarily to poor immune control in latent or lytic sites of infection, or whether CD4+ immunodeficiency leads to more critical downstream deficits in humoral or cell-mediated immunologic responses. Here we compare genital shedding patterns of herpes simplex virus-2 (HSV-2) in 98 HIV infected and 98 HIV uninfected men matched on length of infection, HSV-1 serostatus and nationality. We demonstrate that high copy HSV-2 shedding is more frequent in HIV positive men, particularly in participants with CD4+ T-cell count <200/μL. Genital shedding is more frequent due to higher rate of shedding episodes, as well as a higher proportion of prolonged shedding episodes. Peak episode viral load was not found to differ between HIV infected and uninfected participants regardless of CD4+ T-cell count. We simulate a mathematical model which recapitulates these findings and identifies that rate of HSV-2 release from neural tissue increases, duration of mucosal cytolytic immune protection decreases, and cell-free viral lifespan increases in HIV infected participants. These results suggest that increased HSV-2 shedding in HIV infected persons may be caused by impaired immune function in both latent and lytic tissue compartments, with deficits in clearance of HSV-2 infected cells and extracellular virus. PMID:27285483

  15. Comparison of careHPV and hybrid capture 2 assays for detection of high-risk human Papillomavirus DNA in cervical samples from HIV-1-infected African women.

    PubMed

    Ngou, Jean; Magooa, Mahlape P; Gilham, Clare; Djigma, Florencia; Didelot, Marie-Noelle; Kelly, Helen; Yonli, Albert; Sawadogo, Bernard; Lewis, David A; Delany-Moretlwe, Sinead; Mayaud, Philippe; Segondy, Michel

    2013-12-01

    The careHPV and HC2 assays were compared for high-risk human papillomavirus (HR-HPV) DNA detection in cervical samples from 149 HIV-1-infected African women. The HR-HPV DNA detection rates were 37.6% and 34.9% for careHPV and HC2, respectively. Agreement between the two tests was 94.6% (95% confidence interval [CI], 89.7% to 97.7%) with a kappa value of 0.88 (95% CI, 0.81 to 0.96), indicating an excellent agreement. careHPV may be considered as suitable as HC2 for cervical cancer screening among HIV-infected African women.

  16. Measuring Turnover of SIV DNA in Resting CD4+ T Cells Using Pyrosequencing: Implications for the Timing of HIV Eradication Therapies

    PubMed Central

    Reece, Jeanette C.; Martyushev, Alexey; Petravic, Janka; Grimm, Andrew; Gooneratne, Shayarana; Amaresena, Thakshila; De Rose, Robert; Loh, Liyen; Davenport, Miles P.; Kent, Stephen J.

    2014-01-01

    Resting CD4+ T cells are a reservoir of latent HIV-1. Understanding the turnover of HIV DNA in these cells has implications for the development of eradication strategies. Most studies of viral latency focus on viral persistence under antiretroviral therapy (ART). We studied the turnover of SIV DNA resting CD4+ T cells during active infection in a cohort of 20 SIV-infected pigtail macaques. We compared SIV sequences at two Mane-A1*084:01-restricted CTL epitopes using serial plasma RNA and resting CD4+ T cell DNA samples by pyrosequencing, and used a mathematical modeling approach to estimate SIV DNA turnover. We found SIV DNA turnover in resting CD4+ T cells was slow in animals with low chronic viral loads, consistent with the long persistence of latency seen under ART. However, in animals with high levels of chronic viral replication, turnover was high. SIV DNA half-life within resting CD4 cells correleated with viral load (p = 0.0052) at the Gag KP9 CTL epitope. At a second CTL epitope in Tat (KVA10) there was a trend towards an association of SIV DNA half-life in resting CD4 cells and viral load (p = 0.0971). Further, we found that the turnover of resting CD4+ T cell SIV DNA was higher for escape during early infection than for escape later in infection (p = 0.0084). Our results suggest viral DNA within resting CD4 T cells is more labile and may be more susceptible to reactivation/eradication treatments when there are higher levels of virus replication and during early/acute infection. PMID:24710023

  17. Retention Strategies and Factors Associated with Missed Visits Among Low Income Women at Increased Risk of HIV Acquisition in the US (HPTN 064)

    PubMed Central

    Lucas, Jonathan; Golin, Carol E.; Wang, Jing; Hughes, James P.; Emel, Lynda; El-Sadr, Wafaa; Frew, Paula M.; Just