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Abstract

Objective: To evaluate the in vivo relevance of the inhibitory effect of tenofovir upon telomerase activity observed in vitro. Design: Cross sectional study of HIV-infected patients with suppressed virological replication (HIV RNA < 50 copies/mL for more than one year) Methods: Telomere length in whole blood was measured by quantitative real time PCR. We performed a multivariate analysis to elucidate variables associated with telomere length and also evaluated the association between telomere length and use of tenofovir difumarate (TDF) adjusted by significant confounders RESULTS:: 200 patients included, 72% male, median age 49 (IQR 45-54.5), 103 with exposure to a TDF containing ART regimen (69.9% for more than 5 years) and 97 never exposed to a TDF containing ART regimen. In the multivariate analysis significant predictors of shorter telomere length were older age (p = 0.008), parental age at birth (p = 0.038), Caucasian race (p = 0.048) and longer time of known HIV infection (10-20 years and ≥20 years compared with <10 years, p = 0.003 and p = 0.056 respectively). There was no association between TDF exposure and telomere length after adjusting for possible confounding factors (age, parental age at birth, race and time of HIV infection). Total time receiving ART and duration of treatment with nucleoside reverse transcriptase inhibitors were associated with shorter telomere length but these associations were explained by time of known HIV infection CONCLUSIONS:: Our data do not suggest that telomerase activity inhibition caused by TDF in vitro, leads to telomere shortening in peripheral blood of HIV infected patients.

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... In HIVinfected patients, telomere shortening is caused by inhibition of human telomerase by antiretroviral drugs, more specifically NRTIs [13,[33][34][35]. Tenofovir (TFV) at therapeutic concentrations is a potent inhibitor of telomerase activity, causing telomere shortening in vitro [13,[34][35][36]. TFV is a more potent inhibitor of telomerase than abacavir, lamivudine or emtricitabine [13,36]. ...
... Tenofovir (TFV) at therapeutic concentrations is a potent inhibitor of telomerase activity, causing telomere shortening in vitro [13,[34][35][36]. TFV is a more potent inhibitor of telomerase than abacavir, lamivudine or emtricitabine [13,36]. NRTIs, including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro [12]. ...
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The widespread use of combination antiretroviral therapy (cART) has led to the accelerated aging of the HIV-infected population, and these patients continue to have a range of mild to moderate HIV-associated neurocognitive disorders (HAND). Infection results in altered mitochondrial function. The HIV-1 viral protein Tat significantly alters mtDNA content and enhances oxidative stress in immune cells. Microglia are the immune cells of the central nervous system (CNS) that exhibit a significant mitotic potential and are thus susceptible to telomere shortening. HIV disrupts the normal interplay between microglia and neurons, thereby inducing neurodegeneration. HIV cART contributes to the inhibition of telomerase activity and premature telomere shortening in activated peripheral blood mononuclear cells (PBMC). However, limited information is available on the effect of cART on telomere length (TL) in microglia. Although it is well established that telomere shortening induces cell senescence and contributes to the development of age-related neuro-pathologies, the effect of HIV-Tat on telomere length in human microglial cells and its potential contribution to HAND are not well understood. It is speculated that in HAND intrinsic molecular mechanisms that control energy production underlie microglia-mediated neuronal injury. TL, telomerase and mtDNA expression were quantified in microglial cells using real time PCR. Cellular energetics were measured using the Seahorse assay. The changes in mitochondrial function were examined by Raman Spectroscopy. We have also examined TL in the PBMC obtained from HIV-1 infected rapid progressors (RP) on cART and those who were cART naïve, and observed a significant decrease in telomere length in RP on cART as compared to RP’s who were cART naïve. We observed a significant decrease in telomerase activity, telomere length and mitochondrial function, and an increase in oxidative stress in human microglial cells treated with HIV Tat. Neurocognitive impairment in HIV disease may in part be due to accelerated neuro-pathogenesis in microglial cells, which is attributable to increased oxidative stress and mitochondrial dysfunction.
... Genomic DNA was isolated from whole-blood specimens, using the MagPurix Blood DNA Extraction Kit 1200 (Zinexts Life Science, New Tapei City, Taiwan) according to the manufacturer's instructions. Relative TL, expressed as the ratio of the telomere amplification product (T) to that of a single-copy gene (S), was determined by monochrome quantitative multiplex polymerase chain reaction (PCR) assay as described in our previous study [11]. A standard curve with genomic DNA from a pool of 3 HIV-infected participants was prepared by serial dilutions and included in each run, together with a reference sample and negative control. ...
... In a previous cross-sectional study [11], we did not find an association between history of tenofovir disoproxil fumarate exposure and blood TL. Importantly, in that study 43% of participants with a history of tenofovir disoproxil fumarate use had switched to boosted protease inhibitor monotherapy at the time of the cross-sectional analysis. ...
Article
Background: Tenofovir is a potent inhibitor of human telomerase. The clinical relevance of this inhibition is unknown. Methods: Prospective cohort of HIV-1 infected participants with suppressed virological replication, comparing whole blood telomere length (measured by quantitative multiplex PCR) of participants with current exposure versus never exposed to tenofovir disoproxil fumarate. Results: 172 participants included: 67 in the tenofovir disoproxil fumarate (TDF)-group and 105 in the non-TDF group [75 receiving two nucleosides (69 abacavir), 25 receiving a completely nucleos(t)ide sparing regimen, and 5 receiving lamivudine as the only nucleoside]. After two years mean blood telomere length increased significantly in both groups. The TDF group had significantly smaller gains in telomere length than the non-TDF group. In the analysis restricted to participants receiving nucleos(t)ide reverse transcriptase inhibitors, TDF exposure was not associated with an independent negative effect. In the non-TDF group participants treated with two nucleosides had also significantly smaller gains in telomere length than those receiving completely nucleos(t)ide reverse transcriptase sparing regimens or lamivudine as the only nucleoside. Discussion: in HIV-infected adults with prolonged virological suppression treatment with TDF or abacavir was associated with smaller gains in blood telomere length after two years of follow-up.
... We thus believe that the observed changes in GAS5 and miR-21 expression and dysregulations of CD4T cell functions in these virus-controlled PLHIV are likely induced by either immunologic scarring during early active viral infection or, perhaps more likely, by lowgrade inflammation and persistent T cell activation during latent viral infection, or both. Our interpretation of the cumulative data would include the notion that CD4 T cells in PLHIV on ART exhibit an immune aging phenotype induced by a myriad of viral/host factors, including HIV reservoirs, that may secrete undetectable viral components, pro-inflammatory mediators, increased ROS levels, increased gut permeability and gut microbiota, coinfection with other pathogens (such as HBV, HCV, EBV, and CMV), ART regimens, associated malignancies, and social-related stresses (56)(57)(58)(59)(60)(61)(62)(63), all of which may contribute to the failure to restore CD4 T cell homeostasis and/or functionality. These factors can lead to persistent, low-grade inflammation, causing T cell over-activation, exhaustion, senescence, apoptosis, and decreased proliferative potential, as our results suggest. ...
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T cells are critical for the control of viral infections and T cell responses are regulated by a dynamic network of non-coding RNAs, including microRNAs (miR) and long non-coding RNAs (lncRNA). Here we show that an activation-induced decline of lncRNA growth arrest-specific transcript 5 (GAS5) activates DNA damage response (DDR), and regulates cellular functions and apoptosis in CD4 T cells derived from people living with HIV (PLHIV) via upregulation of miR-21. Notably, GAS5-miR21-mediated DDR and T cell dysfunction are observed in PLHIV on antiretroviral therapy (ART), who often exhibit immune activation due to low-grade inflammation despite robust virologic control. We found that GAS5 negatively regulates miR-21 expression, which in turn controls critical signaling pathways involved in DNA damage and cellular response. The sustained stimulation of T cells decreased GAS5, increased miR-21 and, as a result, caused dysfunction and apoptosis in CD4 T cells. Importantly, this inflammation-driven T cell over-activation and aberrant apoptosis in ART-controlled PLHIV and healthy subjects (HS) could be reversed by antagonizing the GAS5-miR-21 axis. Also, mutation of the miR-21 binding site on exon 4 of GAS5 gene to generate a GAS5 mutant abolished its ability to regulate miR-21 expression as well as T cell activation and apoptosis markers compared to the wild-type GAS5 transcript. Our data suggest that GAS5 regulates TCR-mediated activation and apoptosis in CD4 T cells during HIV infection through miR-21-mediated signaling. However, GAS5 effects on T cell exhaustion during HIV infection may be mediated by a mechanism beyond the GAS5-miR-21-mediated signaling. These results indicate that targeting the GAS5-miR-21 axis may improve activity and longevity of CD4 T cells in ART-treated PLHIV. This approach may also be useful for targeting other infectious or inflammatory diseases associated with T cell over-activation, exhaustion, and premature immune aging.
... Regarding antiretroviral therapy, there is a lot of similarity in function between HIV reverse transcriptase and telomerase, which results in telomerase being putatively blocked by NRTIs [5,17,18]. In vivo trials have indicated that TDF is the only NRTI that significantly inhibits telomerase activity and reduces telomere size at therapeutic concentrations [5], although these findings were not confirmed by other studies [19,20]. In this setting, our study is consistent with previous results, according to which TDF treatment had no effect on RTL. ...
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Introduction: Human immunodeficiency virus (HIV) infection and cirrhosis are associated with a senescent phenotype that decreases telomere length. We evaluated the impact of hepatitis C virus (HCV) elimination on telomere length in patients with advanced HCV-related cirrhosis after sustained virological response (SVR), with all-oral direct-acting antiviral agents (DAAs). Methods: Prospective study of 60 HIV/HCV-coinfected and 30 HCV-monoinfected patients with advanced HCV cirrhosis (liver decompensation or liver stiffness measurement (LSM) ≥ 25 kPa, hepatic liver pressure gradient (HVPG) ≥ 10 mmHg, or Child-Pugh-Turcotte (CPT) ≥ 7). The relative telomere length (RTL) was quantified by real-time multiplex PCR (MMqPCR) on peripheral blood mononuclear cells at baseline and 48 weeks after HCV treatment. Generalized linear models (GLMs) adjusted for the most relevant clinical and epidemiological variables and mixed GLMs were used. Results: In comparison with HCV-monoinfected patients, HIV/HCV-coinfected patients were younger (p < 0.001), had lower body mass index (BMI) (p = 0.002), and had been exposed less frequently to interferons (p = 0.011). In addition, they were more frequently men (p = 0.011), smokers (p = 0.005), prior intravenous drug users (IVDUs) (p < 0.001), and alcohol abusers (p = 0.005). RTL was significantly lower in HIV/HCV-coinfected patients than in HCV-monoinfected patients, both at baseline (p < 0.001), and at the end of follow-up (p = 0.032). A significant RTL increase over time was found only for HIV/HCV-coinfected patients (p < 0.001), especially in those patients with compensated cirrhosis (p < 0.001). Conclusion: HCV eradication with all-oral DAAs was associated with an increase in telomere length in HIV/HCV-coinfected patients with advanced cirrhosis, particularly in compensated patients. This finding suggests that HCV clearance may have implications in age-related conditions in this population group.
... Specifically, HIV latency in the era of cART is characterized by the existence of viral reservoirs that prevent HIV-1 eradication and likely drive inflammaging (46,47). Thus, HIV-mediated inflammaging could result from a myriad of insults, such as viral particles or viral RNAs/proteins released from the reservoirs, cell-secreted pro-inflammatory cytokines, HIV-enhanced gut permeability and altered gut microbiota or dysbiosis, frequent cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), and HCV coinfections, the cART regimen itself (NRTIs may take part in telomere damage and T cell senescence, as telomerase has been shown to be inhibited by some NRTIs) (48), as well as other comorbidities including malignancies, personal stresses, or social and environmental factors (49)(50)(51)(52). In addition to central memory CD4 T cells, several groups have reported that T stem cell memory (T SCM ) cells harbor HIV-1 provirus and are relatively "spared" from depletion as compared to other CD4 T cells (53,54). ...
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Article
HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends—telomeres, leading to accelerated telomere erosion—a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro, recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection.
... NRTIs have been drugs of particular concern as they have been associated with varying levels of mitochondrial dysfunction, which in turn has been associated with cellular senescence (31). However, this is an evolving area of knowledge with inconclusive and conflicting data which is not likely to be sufficiently convincing to clinicians and patients alike to cause a major change in how they approach the treatment of HIV infection (32)(33)(34). ...
... Genomic DNA was isolated from whole blood using QIAamp DNA Blood Mini Kit (Qiagen) according to manufacturer's instructions. Relative telomere length, expressed as ratio of telomere (T) to single-copy gene (S), was determined by monochrome quantitative multiplex polymerase chain reaction (PCR) assay, with minor modifications as described in our prior study [14]. A standard curve was prepared with genomic DNA from a pool of 3 healthy volunteers by serial dilution and was included in triplicate in each run together with a reference sample and negative control. ...
Article
Background: Tenofovir is a potent inhibitor of human telomerase. The clinical relevance of this inhibition is unknown. Methods: NEAT001/ANRS143 is a randomised trial that showed non-inferiority over 96 weeks of ritonavir-boosted darunavir + raltegravir vs tenofovir disoproxil fumarate /emtricitabine in 805 antiretroviral antiretrovrial naïve HIV-infected adults. We compared changes in whole blood telomere length measured with quantitative PCR in 201 randomly selected participants (104 raltegravir and 97 tenofovir disoproxil fumarate /emtricitabine). We performed multivariable estimative and predictive linear regression. Results: At week 96, participants receiving tenofovir disoproxil fumarate/emtricitabine had a statistically significant higher gain in telomere length than participants receiving raltegravir. Difference in mean telomere length change between groups (tenofovir disoproxil fumarate/emtricitabine minus raltegravir) from baseline to week 96 adjusted by baseline telomere length was 0.031 (p=0.009). This difference was not significantly confounded by age, gender, known duration of HIV infection, CD4 (baseline/nadir), CD8 cells, CD4/CD8 ratio, HIV viral load (baseline/week96), tobacco and alcohol consumption, statins or hepatitis C. Discussion: antiretroviral naïve HIV infected adults receiving ritonavir-boosted darunavir and tenofovir disoproxil fumarate/emtricitabine had a significant higher gain in blood telomere length than those receiving ritonavir-boosted darunavir and raltegravir suggesting a better initial recovery from HIV associated immunosenescence.
... no associations have been reported between telomere length and exposure to nucleoside reverse transcriptase inhibitors in vivo 7,8 . In fact, exposure to combination antiretroviral therapy has been associated with longer telomere length in individuals living with HIV. ...
Article
Objectives: To evaluate whether the negative impact of tenofovir on telomere length (TL) is due to immune reconstitution interference or inhibition of telomerase. Methods: One hundred and twenty-eight long-term aviraemic HIV adults treated with tenofovir-containing (n = 79) or tenofovir-sparing regimens (n = 49) were recruited to compare the following: TL in whole blood, PBMCs, CD4+ T cells and CD8+ T cells by quantitative PCR (qPCR); telomerase activity in PBMCs, CD4+ cells and CD8+ T cells using the TRAPeze RT Telomerase Detection Kit; and T cell maturational subset distribution by flow cytometry. Results: In an adjusted analysis, participants treated with tenofovir for at least 4 years had shorter TL in CD8+ T cells (P = 0.04) and lower telomerase activity in CD4+ (P = 0.012) and CD8+ T cells (P = 0.023). Tenofovir treatment was also associated with lower proportions of recent thymic emigrant (RTE) CD4+ cells (P = 0.031) and PD1 marker expression (P = 0.013). Conclusions: In long-term aviraemic HIV adults, the inhibition of telomerase by tenofovir could explain telomere shortening in CD8+ T cells. There is no telomere shortening in the CD4+ compartment and the decrease in telomerase activity could be explained both by the inhibition by tenofovir and by the lower proportion of RTE CD4+cells.
Thesis
L’universalisation des traitements antirétroviraux (ARV) couplée à l’augmentation de la couverture ARV chez les femmes enceintes et allaitantes infectées par le VIH voit l’émergence d’une population grandissante d’enfants non infectés nés de mères séropositives, exposés à la fois au VIH et aux ARV maternels de la conception à la petite enfance. Si l’impact de ces expositions questionne sur la santé de ces enfants et fait l’objet de maintes études, la prophylaxie ARV postnatale qui leur ait administrée dans le cadre de la prévention de la transmission mère-enfant (TME) du VIH nécessite elle aussi être rigoureusement évaluée, ces ARV restant donnés à des enfants non infectés. Dans ce contexte, ces travaux de thèse ont consisté à évaluer la toxicité génomique aigüe et sur le long terme du lopinavir/ritonavir (LPV/r) ou de la lamivudine (3TC) utilisés en prophylaxie infantile pendant un an pour prévenir la TME du VIH par l’allaitement lors de l’essai PROMISE PEP mené dans quatre pays d’Afrique Sub-saharienne (Afrique du Sud, Burkina Faso, Ouganda et Zambie) entre novembre 2009 et mai 2012. Si cette prophylaxie étendue à toute la période de l’allaitement a montré son efficacité, avec des taux de transmission à un an de 1.4% et 1.5% pour le LPV/r et le 3TC respectivement, les recommandations actuelles de l’Organisation Mondiale de la Santé préconisent cependant une prophylaxie à base de névirapine (NVP) ou d’azidothymidine combinée à la NVP plafonnée à 12 semaines. Nos travaux indiquent que les deux prophylaxies se trouvaient associées à une prévalence importante d’enfants exposés non infectés présentant une déplétion de l’ADN mitochondrial (diminution du nombre de copies supérieure ou égale à 50% de la valeur initiale) au cours de la première année de vie. Ils ont également mis en évidence la présence d’ADN mitochondrial délétés chez la quasi-totalité des enfants dès l’initiation de la prophylaxie, démontrant ainsi une forte instabilité génomique. Toutefois, aucune association avec la croissance et le développement neuro-psychomoteur de ces enfants à 6 ans n’a été mise en évidence, alors que la déplétion ne persistait plus à cet âge-là et que les délétions sont irréversibles. Le raccourcissement de la longueur des télomères observé à un an n’a quant à lui montré aucune association avec les deux ARV et n’a eu aucun impact sur la santé des enfants à 6 ans. Ces travaux de thèse contribuent à l’évaluation globale de l’innocuité de la prophylaxie ARV utilisée chez les enfants exposés non infectés mais sont également d’intérêt pour ceux qui sont infectés et dont le traitement de première intention intègre le LPV/r et/ou le 3TC.
Article
Objective: Myeloid-derived suppressor cells (MDSCs) contribute to HIV progression by impairing antiviral immunity; however, the mechanisms responsible for MDSC development during HIV infection are incompletely understood. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long noncoding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1. The role of HOTAIRM1--HOXA1 in the differentiation and functions of MDSCs during HIV infection remains unclear. Methods: In this study, we measured MDSC induction and suppressive functions by flow cytometry, RT-PCR, and co-culture experiments using CD33 myeloid cells derived from people living with HIV (PLHIV) on antiretroviral therapy (ART). We also manipulated the HOTAIRM1--HOXA1 axis in myeloid cells using knockdown and overexpression approaches. Results: We demonstrate that HOTAIRM1 and HOXA1 expressions are reciprocally upregulated and are responsible for increased levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS), in CD33 myeloid cells derived from PLHIV on ART. We found that overexpression of HOTAIRM1 or HOXA1 in CD33 cells isolated from healthy individuals promoted the differentiation and suppressive functions of MDSCs, whereas silencing of HOTAIRM1 or HOXA1 expression in MDSCs derived from PLHIV significantly inhibited the frequency of MDSCs and expressions of the immunosuppressive molecules and reduced their immunosuppressive effects on T cells. Conclusion: These results indicate that the HOTAIRM1--HOXA1 axis enhances differentiation and suppressive functions of MDSCs and could be a potential therapeutic target for immunomodulation during latent HIV infection.
Article
Objectives: Our aim was to investigate factors associated with baseline blood telomere length in participants enrolled in NEAT 001/ANRS 143, a randomized, open-label trial comparing ritonavir-boosted darunavir (DRV/r) plus raltegravir (RAL) with DRV/r plus tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) in antiretroviral therapy (ART)-naïve HIV-positive adults. Methods: A cross-sectional study of 201 randomly selected participants who had stored samples available was carried out. We measured telomere length (i.e. the relative telomere length, calculated as the telomere to single copy gene ratio) at baseline with monochrome quantitative multiplex polymerase chain reaction (PCR). We used multivariable predictive linear regression to calculate mean differences and 95% confidence intervals (CIs) for the association between baseline telomere length and baseline characteristics. Results: The baseline characteristics of the 201 participants did not differ from those of the 805 participants in the parent trial population: 89% were male, the mean age was 39 years, 83.6% were Caucasian, 93% acquired HIV infection via sexual transmission, the mean estimated time since HIV diagnosis was 2.1 years, the mean HIV-1 RNA load was 4.7 log10 HIV-1 RNA copies/mL, the mean nadir and baseline CD4 counts were 301 and 324 cells/μL, respectively, and the mean CD4:CD8 ratio was 0.4. In the univariate analysis, shorter telomere length was associated with older age (per 10 years) (P < 0.001), HIV-1 RNA ≥ 100 000 copies/mL (P = 0.001), CD4 count < 200 cells/μL (P = 0.037), lower CD4:CD8 ratio (P = 0.018), statin treatment (P = 0.004), and current alcohol consumption (P = 0.035). In the multivariable analysis, older age (P < 0.001) and HIV RNA ≥ 100 000 copies/mL (P = 0.054) were independently associated with shorter telomere length. Conclusions: Both age and HIV RNA viral load correlated with shorter blood telomere length in untreated persons living with HIV. These results suggest that HIV infection and age have synergistic and independent impacts upon immunosenescence.
Article
Background: Although cocaine use may induce/accelerate HIV-associated comorbidities in HIV-infected individuals on antiretroviral therapy (ART), and that HIV itself may accelerate aging, the issue of whether cocaine use plays a role in HIV-associated aging in HIV-infected cocaine users has not been reported. The goals of this study were (1) to explore factor(s) associated with peripheral blood leukocyte telomere length, a marker of cellular replicative history, and telomere shortening in HIV-infected individuals, and (2) to assess whether cocaine use plays a role in accelerating telomere shortening in cocaine users with HIV infection. Methods: Between June 2010 and December 2016, 147 HIV-infected participants in Baltimore, Maryland, were enrolled in a cross-sectional study investigating factor(s) associated with telomere length. Of these 147, 93 participated in a follow-up study to examine factor(s) associated with telomere shortening. Robust regression model was used to analyze cross-sectional data and the generalized estimating equation approach was used to analyze follow-up data. Results: Cross-sectional analyses demonstrated that (1) both daily alcohol consumption and use of non-nucleoside reverse transcriptase inhibitors (NNRTIs) were independently associated with telomere length, and cocaine use modified the associations of daily alcohol use and NNRTI use with telomere length. Longitudinal analyses suggested that both daily alcohol consumption and duration of NNRTI use were independently associated with telomere shortening, and (2) cocaine use induced/accelerated telomere shortening in HIV-infected individuals. Conclusions: Our findings suggest that cocaine use may promote premature aging in HIV-infected individuals who are on ART. Our results emphasize the importance of cocaine abstinence/reduced use, which may retard HIV-associated premature aging.
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Objective: Several pieces of evidence indicate that HIV-infected adults undergo premature aging. The effect of HIV and antiretroviral (ART) exposure on the aging process of HIV-infected children may be more deleterious since their immune system co-evolves from birth with HIV. Design: Seventy-one HIV-infected (HIV+), 65 HIV-exposed-uninfected (HEU) and 56 HIV-unexposed-uninfected (HUU) children, all aged 0-5 years, were studied for biological aging and immune senescence. Methods: Telomere length (TL) and T-cell receptor rearrangement excision circle (TREC) levels were quantified in peripheral blood cells by real-time PCR. CD4 and CD8 cells were analysed for differentiation, senescence and activation/exhaustion markers by flow cytometry. Results: TL were significantly shorter in HIV+ than in HEU and HUU children (overall, p < 0.001 adjusted for age); HIV+ ART-naïve (42%) children had shorter TL compared with children on ART (p = 0.003 adjusted for age). TREC levels and CD8 recent thymic emigrant cells (CD45RACD31) were significantly lower in the HIV+ than in control groups (overall, p = 0.025 and p = 0.005, respectively). Percentages of senescent (CD28CD57), activated (CD38HLA-DR) and exhausted (PD1)CD8 cells were significantly higher in HIV+ than in HEU and HUU children (p = 0.004, p < 0.001 and p < 0.001, respectively). Within the CD4 cell subset, the percentage of senescent cells did not differ between HIV+ and controls, but PD-1 expression was up-regulated in the former. Conclusions: HIV-infected children exhibit premature biological aging with accelerated immune senescence, which particularly affects the CD8 cell subset. HIV infection per se seems to influence the aging process, rather than exposure to ART for prophylaxis or treatment.
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Background: Infection with human immunodeficiency virus type 1 (HIV) is associated with clinical symptoms of accelerated aging, as evidenced by the increased incidence and diversity of age-related illnesses at relatively young ages and supporting findings of organ and cellular pathologic analyses. But it has been difficult to detect an accelerated aging effect at a molecular level. Methods: Here, we used an epigenetic biomarker of aging based on host DNA methylation levels to study accelerated aging effects due to HIV infection. DNA from brain and blood tissue was assayed via the Illumina Infinium Methylation 450 K platform. Results: Using 6 novel DNA methylation data sets, we show that HIV infection leads to an increase in epigenetic age both in brain tissue (7.4 years) and blood (5.2 years). While the observed accelerated aging effects in blood may reflect changes in blood cell composition (notably exhausted cytotoxic T cells), it is less clear what explains the observed accelerated aging effects in brain tissue. Conclusions: Overall, our results demonstrate that the epigenetic clock is a useful biomarker for detecting accelerated aging effects due to HIV infection. This tool can be used to accurately determine the extent of age acceleration in individual tissues and cells.
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Combination antiretroviral therapy (cART) has extended the longevity of human immunodeficiency virus (HIV)-infected individuals. However, this has resulted in greater awareness of age-associated diseases such as chronic obstructive pulmonary disease (COPD). Accelerated cellular senescence may be responsible, but its magnitude as measured by leukocyte telomere length is unknown and its relationship to HIV-associated COPD has not yet been established. We measured absolute telomere length (aTL) in peripheral leukocytes from 231 HIV-infected adults. Comparisons were made to 691 HIV-uninfected individuals from a population-based sample. Subject quartiles of aTL were assessed for relationships with measures of HIV disease severity, airflow obstruction, and emphysema severity on computed tomographic (CT) imaging. Multivariable regression models identified factors associated with shortened aTL. Compared to HIV-uninfected subjects, the mean aTL in HIV-infected patients was markedly shorter by 27 kbp/genome (p<0.001); however, the slopes of aTL vs. age were not different (p=0.469). Patients with longer known durations of HIV infection (p=0.019) and lower nadir CD4 cell counts (p=0.023) had shorter aTL. Shorter aTL were also associated with older age (p=0.026), smoking (p=0.005), reduced forced expiratory volume in one second (p=0.030), and worse CT emphysema severity score (p=0.049). HIV-infected subjects demonstrate advanced cellular aging, yet in a cART-treated cohort, the relationship between aTL and age appears no different from that of HIV-uninfected subjects.
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Objective: To determine whether nucleos(t)ide reverse transcriptase inhibitors (NRTI) contribute to an accelerated loss in telomere length (TL) in HIV-infected patients on antiretroviral therapy (ART). Design: Substudy of randomised controlled trial. Methods: Patients with HIV RNA <50 copies/mL on combination ART (n = 256) were randomised to darunavir/ritonavir (DRV/r) 800/100 mg once daily, either as monotherapy (n = 127) or with 2 NRTIs (n = 129) for up to 144 weeks. TL and telomerase activity was quantified on stored peripheral blood mononuclear cells (PBMC; n = 124) using quantitative real time PCR. Results: Patients in the sub-study had a mean age of 44 years and had received NRTI for a mean of 6.4 years (range 1-20 years). As expected, older patients have significantly shorter TL (p = 0.006), while women had significantly longer TL (p = 0.026). There was no significant association between TL and either the duration of prior NRTI treatment (p = 0.894) or the use of a PI versus NNRTI (p = 0.107). There was no significant difference between patients who continued or ceased NRTI in the mean change/year of TL or telomerase (p = 0.580 and 0.280 respectively). Conclusion: Continuation versus cessation of NRTI treatment was not associated with an accelerated loss in TL or telomerase activity.
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Telomere length (TL) and immune activation markers were measured in a cohort of HIV-infected (n=102) and age-matched non-HIV-infected (n=41) men. TL was significantly shorter in HIV-infected compared to non-HIV-infected subjects (P = 0.04). Univariate analysis revealed a strong inverse relationship of TL to sCD163, and thus, monocyte/macrophage activation, among the HIV group (ρ = -0.30, P = 0.003). In multivariate modeling among the whole group, HIV positive serostatus (P = 0.06) and sCD163 (P = 0.05) were independent predictors of TL controlling for age and smoking status. Our data demonstrate that increased immune activation relates to shorter TL in HIV.
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Background: Individuals infected with human immunodeficiency virus (HIV) appear to age faster than the general population, possibly related to HIV infection, antiretroviral therapy, and/or social/environmental factors. We evaluated leukocyte telomere length (LTL), a marker of cellular aging, in HIV-infected and uninfected adults. Methods: Clinical data and blood were collected from Children and women: AntiRetrovirals and the Mechanism of Aging (CARMA) cohort study participants. Variables found to be important in univariate analysis were multivariate model candidates. Results: Of the 229 HIV-infected and 166 HIV-uninfected participants, 76% were women, and 71% were current/previous smokers. In a multivariate model of all participants, older age (P < .001), HIV infection (P = .04), active hepatitis C virus (HCV) infection (P = .02), and smoking (P < .003) were associated with shorter LTL. An interaction was detected, whereby smoking was associated with shorter LTL in HIV-uninfected subjects only. Among those, age and smoking (P ≤ .01) were related to shorter LTL. In 2 models of HIV-infected individuals, age (P ≤ .002) and either active HCV infection (P = .05) or peak HIV RNA ≥100 000 copies/mL (P = .04) were associated with shorter LTL, whereas other HIV disease or treatment parameters were unrelated. Conclusions: Our results suggest that acquisition of HIV and viral load are primarily responsible for the association between HIV-positive status and shorter LTL. The lack of association between LTL and time since HIV diagnosis, antiretroviral treatment, or degree of immune suppression would implicate HIV infection-related factors rather than disease progression or treatment. Smoking effects on LTL appear masked by HIV, and HCV infection may accelerate LTL shortening, particularly in coinfected individuals. The effect of early therapeutic intervention on LTL in HIV and HCV infections should be evaluated.
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Objectives: Little is known about the impact of HIV infection on biological ageing in sub-Saharan Africa. The study aimed to assess biological ageing in South African HIV-infected adults and HIV-seronegative individuals using two validated biomarkers, telomere length and CDKN2A expression (a mediator of cellular senescence). Design: A case-control study. Methods: Two hundred and thirty-six HIV-infected adults aged at least 30 years and 250 age and sex frequency matched HIV-seronegative individuals were recruited from clinics in township communities in Cape Town. Biological ageing was evaluated by measurement of telomere length and CDKN2A expression in peripheral blood leukocytes. Results: The median ages of the HIV-infected and HIV-seronegative participants were 39 and 40 years, respectively. Among HIV-infected participants, 87.1% were receiving antiretroviral therapy (ART), their median CD4⁺ cell count was 468 cells/μl and 84.3% had undetectable viral load. Both biomarkers were validated against chronological age in HIV-seronegative individuals. Telomere length was significantly shorter in HIV-infected individuals than in HIV-seronegative individuals (mean relative T/S ratio ±SE:0.91 ± 0.007 vs. 1.07 ± 0.008, P < 0.0001). CD2NKA expression was higher in HIV-infected participants than in HIV-seronegative individuals (mean expression: 0.45 ± 0.02 vs. 0.36 ± 0.03, P = 0.003). Socioeconomic factors were not associated with biological ageing in HIV-infected participants. However, in participants on ART with undetectable viral load, biomarker levels indicated greater biological ageing in those with lower current CD4⁺ cell counts. Conclusion: Telomere length and CDKN2A expression were both consistent with increased biological ageing in HIV-infected individuals. Prospective studies of the impact of HIV on biological ageing in sub-Saharan Africa are warranted.
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Background: HIV-infected patients on combination active antiretroviral therapy (cART) are at increased risk of age-related complications. We hypothesised that nucleos(t)ide reverse transcriptase inhibitors (NRTI) may contribute to accelerated ageing in HIV-infected individuals on cART via inhibition of telomerase activity. Methods: Telomerase activity and telomere length (TL) were measured by quantitative PCR in vitro in activated peripheral blood mononuclear cells (PBMC) cultured with NRTI, and ex vivo in PBMC from uninfected patients exposed to NRTI and from HIV-infected patients on NRTI-containing cART. Results: Lamivudine, abacavir, zidovudine, emtricitabine and tenofovir significantly inhibited telomerase activity in activated PBMC in vitro. Tenofovir was the most potent inhibitor of telomerase activity and caused greatest shortening of TL in vitro at the therapeutic concentration of 0.3μM. PBMC from HIV-infected patients receiving NRTI-containing cART (n=39) had significantly lower telomerase activity than HIV-uninfected patients (n=47; p=0.011) and HIV-infected patients receiving non-NRTI-containing cART (n=11; p<0.001). TL was significantly inversely associated with age (p=0.009) and the total duration on any NRTI (p=0.01). Conclusion: NRTIs, and specifically tenofovir at therapeutic concentrations, inhibit telomerase activity leading to accelerated shortening of TL in activated PBMC. The relationship between NRTI, reduced telomerase activity and accelerated ageing requires further investigation in HIV-infected individuals on cART.
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Study question: Is the association between paternal age at birth and offspring leukocyte telomere length (LTL) an artifact of early life socioeconomic status (SES)? Summary answer: Indicators of early life SES did not alter the relationship between paternal age at birth and offspring LTL among a population of white female nurses. What is known already: Telomere length is considered a highly heritable trait. Recent studies report a positive correlation between paternal age at birth and offspring LTL. Maternal age at birth has also been positively associated with offspring LTL, but may stem from the strong correlation with paternal age at birth. Study design, size and duration: The Nurses' Health Study (NHS) is an ongoing prospective cohort study of 121 700 female registered nurses who were enrolled in 1976. Great effort goes into maintaining a high degree of follow-up among our cohort participants (>95% of potential person-years). In 1989-1990, a subset of 32 826 women provided blood samples from which we selected participants for several nested case-control studies of telomere length and incident chronic disease. We used existing LTL data on a total of 4250 disease-free women who also reported maternal and paternal age at birth for this study. Participants/materials, setting and methods: Nested case-control studies of stroke, myocardial infarction, cancers of the breast, endometrium, skin, pancreas and colon, as well as colon adenoma, were conducted within the blood sub-cohort. Each study used the following study design: for each case of a disease diagnosed after blood collection, a risk-set sampling scheme was used to select from one to three controls from the remaining participants in the blood sub-cohort who were free of that disease when the case was diagnosed. Controls were matched to cases by age at blood collection (± 1 year), date of blood collection (± 3 months), menopausal status, recent postmenopausal hormone use at blood collection (within 3 months, except for the myocardial infarction case-control study), as well as other factors carefully chosen for each individual study. The current analysis was limited to healthy controls. We also included existing LTL data from a small random sample of women participating in a cognitive sub-study. LTL was measured using the quantitative PCR-based method. Exposure and covariate information are extracted from biennial questionnaires completed by the participants. Main results and the role of chance: We found a strong association between paternal age at birth and participant LTL (P = 1.6 × 10(-5)) that remained robust after controlling for indicators of early life SES. Maternal age at birth showed a weak inverse association with participant LTL after adjusting for age at blood collection and paternal age at birth (P = 0.01). We also noted a stronger association between paternal age at birth and participant LTL among premenopausal than among postmenopausal women (P(interaction) = 0.045). However, this observation may be due to chance as premenopausal women represented only 12.6% (N = 535) of the study population and LTL was not correlated with age at menopause, total or estrogen-only hormone therapy (HT) use suggesting that changes in in vivo estrogen exposure do not influence telomere length regulation. Limitations and reasons for caution: The women in our study are not representative of the general US female population, with an underrepresentation of non-white and low social class groups. Although the interaction was not significant, we noted that the paternal age at birth association with offspring LTL appeared weaker among women whose parents did not own their home at the time of the participant's birth. As telomere dynamics may differ among individuals who are most socioeconomically deprived, SES indicators may have more of an influence on the relationship between paternal age at birth and offspring LTL in such populations. Wider implications of the findings: As of yet, our and prior studies have not identified childhood or adult characteristics that confound the paternal age at birth association with offspring LTL, supporting the hypothesis that offspring may inherit the longer telomeres found in sperm of older men. The biological implications of the paternal age effect are unknown. A recent theory proposed that the inheritance of longer telomere from older men may be an adaptive signal of reproductive lifespan, while another theory links telomere length attrition to female reproductive senescence. However, we are unaware of any data to substantiate a relationship between paternal age at birth and daughter's fertility. Generalizability of our study results to other white female populations is supported by prior reports of paternal age at birth and offspring telomere length. Furthermore, a confounding relationship between paternal or maternal age at birth and SES was not observed in a study of SES and telomere length. Study funding/competing interest(s): This work was supported by the National Institutes of Health (grants numbers: CA87969, CA49449, CA065725, CA132190, CA139586, HL088521, CA140790, CA133914, CA132175, ES01664 to M.D.); and by the American Health Association Foundation. We have no competing interests to declare.
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Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4(+) T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect of in vitro HIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.
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The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).
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Telomere shortening occurs concomitant with organismal aging, and it is accelerated in the context of human diseases associated with mutations in telomerase, such as some cases of dyskeratosis congenita, idiopathic pulmonary fibrosis and aplastic anemia. People with these diseases, as well as Terc-deficient mice, show decreased lifespan coincidental with a premature loss of tissue renewal, which suggests that telomerase is rate-limiting for tissue homeostasis and organismal survival. These findings have gained special relevance as they suggest that telomerase activity and telomere length can directly affect the ability of stem cells to regenerate tissues. If this is true, stem cell dysfunction provoked by telomere shortening may be one of the mechanisms responsible for organismal aging in both humans and mice. Here, we will review the current evidence linking telomere shortening to aging and stem cell dysfunction.
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Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3′-Azido-3′-deoxythymidine 5′-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3′-down azido group: 3′-azido-2′,3′-dideoxyguanosine (AZddG) 5′-triphosphate (AZddGTP), 3′-azido-2′,3′-dideoxy-6-thioguanosine (AZddSG) 5′-triphosphate (AZddSGTP), 3′-azido-2′,3′-dideoxyadenosine (AZddA) 5′-triphosphate (AZddATP) and 3′-azido-2′,3′-dideoxy-2-aminoadenosine (AZddAA) 5′-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3′-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases α and δ, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.
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Telomeres are nucleoprotein structures that cap the ends of chromosomes, protecting them from exonucleases and distinguishing them from double-stranded breaks. Their integrity is maintained by telomerase, an enzyme consisting of a reverse transcriptase, TERT and an RNA template, TERC, and other components, including the pseudouridine synthase, dyskerin, the product of the DKC1 gene. When telomeres become critically short, a p53-dependent pathway causing cell cycle arrest is induced that can lead to senescence, apoptosis, or, rarely to genomic instability and transformation. The same pathway is induced in response to DNA damage. DKC1 mutations in the disease dyskeratosis congenita are thought to act via this mechanism, causing growth defects in proliferative tissues through telomere shortening. Here, we show that pathogenic mutations in mouse Dkc1 cause a growth disadvantage and an enhanced DNA damage response in the context of telomeres of normal length. We show by genetic experiments that the growth disadvantage, detected by disparities in X-inactivation patterns in female heterozygotes, depends on telomerase. Hemizygous male mutant cells showed a strikingly enhanced DNA damage response via the ATM/p53 pathway after treatment with etoposide with a significant number of DNA damage foci colocalizing with telomeres in cytological preparations. We conclude that dyskerin mutations cause slow growth independently of telomere shortening and that this slow growth is the result of the induction of DNA damage. Thus, dyskerin interacts with telomerase and affects telomere maintenance independently of telomere length. • dyskeratosis congenita • heterozygous • mosaic analysis • telomerase • αp53
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In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in PBMCs of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5µM and 1µM was 29% (Interquartile range (IQR) 29%-34%, p=0.042) and 28% (IQR 28%-41%, p=0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, p=0.043) at 3µM and 14% (IQR 10%-29%, p=0.043) at 10µM. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels or mRNA levels of telomerase/shelterin genes.
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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.
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Background: Aging-associated non-communicable comorbidities are more prevalent among HIV-1-infected than HIV-uninfected individuals. Residual HIV-related chronic immune activation and senescence may increase the risk of developing comorbidities. Methods: Immune phenotyping, thymic output, and telomere length were assessed in ART treated HIV-1-infected individuals (cases; n=94) and matched uninfected controls (n=95), over 45 years of age. Results: Cases had lower CD4(+)counts, higher CD8(+)counts, and increased levels of immune activation (sCD14; %CD4(+)CD38(+)HLA-DR(+); %CD8(+)CD38(+)HLA-DR(+)), regulatory T-cells and %PD-1(+)CD4(+)T-cells. Immune senescence levels (%CD27(-)CD28(-)or %CD57(+)) were comparable between cases and controls. PBMC from cases had shorter telomeres, but increased Sj-TREC content and CD31(+)naïve CD4(+)cells. Although CMV-antibody titres were higher in cases, CMV-specific T-cell responses were comparable between cases and controls. T-cell senescence in cases was independently associated with T-cell activation, but not with CMV-specific immune responses. Conclusions: Despite long-term ART, HIV-1-infected adults had higher levels of immune activation, regulatory T-cells, PD-1 expressing CD4(+)cells and shorter telomeres. The increased sCD14 and %CD4(+)CD38(+)HLA-DR(+)cells correlated with shorter telomeres and increased regulatory T-cells. This suggests that HIV-1 impacts immune function irreversibly, with several pathways that are persistently abnormal during effective ART. Therapies aimed at improving immune health during ART are needed.
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Background Whether the reported high risk of age-related diseases in HIV-infected people is caused by biological ageing or HIV-associated risk factors such as chronic immune activation and low-grade inflammation is unknown. We assessed time trends in age-standardised and relative risks of nine serious age-related diseases in a nationwide cohort study of HIV-infected individuals and population controls. Methods We identified all HIV-infected individuals in the Danish HIV Cohort Study who had received HIV care in Denmark between Jan 1, 1995, and June 1, 2014. Population controls were identified from the Danish Civil Registration System and individually matched in a ratio of nine to one to the HIV-infected individuals for year of birth, sex, and date of study inclusion. Individuals were included in the study if they had a Danish personal identification number, were aged 16 years or older, and were living in Denmark at the time of study inclusion. Data for study outcomes were obtained from the Danish National Hospital Registry and the Danish National Registry of Causes of Death and were cardiovascular diseases (myocardial infarction and stroke), cancers (virus associated, smoking related, and other), severe neurocognitive disease, chronic kidney disease, chronic liver disease, and osteoporotic fractures. We calculated excess and age-standardised incidence rates and adjusted incidence rate ratios of outcomes for time after HIV diagnosis, highly active antiretroviral therapy (ART) initiation, and calendar time. The regression analyses were adjusted for age, sex, calendar time, and origin. Findings We identified 5897 HIV-infected individuals and 53 073 population controls; median age was 36·8 years (IQR 30·6–44·4), and 76% were men in both cohorts. Dependent on disease, the HIV cohort had 55 050–57 631 person-years of follow-up and the population controls had 638 204–659 237 person-years of follow-up. Compared with the population controls, people with HIV had high excess and relative risk of all age-related diseases except other cancers. Overall, the age-standardised and relative risks of cardiovascular diseases, cancers, and severe neurocognitive disease did not increase substantially with time after HIV diagnosis or ART initiation. Except for chronic kidney diseases, the age-standardised and relative risks of age-related diseases did not increase with calendar time. Interpretations Severe age-related diseases are highly prevalent in people with HIV, and continued attention and strategies for risk reduction are needed. The findings from our study do not suggest that accelerated ageing is a major problem in the HIV-infected population. Funding Preben og Anna Simonsens Fond, Novo Nordisk Foundation, Danish AIDS Foundation, Augustinus Foundation, and Odense University Hospitals Frie Fonds Midler.
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Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a progressive decline of CD4(+) T cells and by other immune disorders that are similar to those observed during aging and which lead eventually to AIDS. One of the mechanisms involved in HIV-1 induced immunodeficiency may be the lack of telomerase induction and the consequent impairment of the potential required for CD4(+) T cell expansion. Telomerase compensates for the progressive telomere loss during cell division and preserves the replicative potential of T lymphocytes after repeated antigenic stimulation. The enzyme is activated by post-translational modifications, such as phosphorylation and also by the nuclear import of its catalytic subunit hTERT from the cytoplasm. In previous studies we found a reduction of telomerase activity in the nucleus of CD4(+) T cells infected with HIV-1 or non-infected but exposed to Tat protein. However, the mechanism for this loss of activity has not been elucidated yet. In the present study, we found that HIV-1 Tat inhibited telomerase activity in CD4(+) T cells by different mechanisms. First, it reduced nuclear levels of hTERT. Secondly, this protein perturbed the AKT pathway and the molecular interaction with the chaperones required for hTERT phosphorylation, nuclear import and activation. These results suggest that in addition to inducing direct cell death, HIV infection may also reduce the replicative potential of non-infected CD4(+) T cells and this may contribute to the overall immunodeficiency in AIDS patients.
Article
HIV disease can lead to accelerated telomere attrition, although certain drugs used as part of antiretroviral therapy (ART) can inhibit telomerase reverse transcriptase activity. This could in turn lead to shorter telomeres. We hypothesized that HIV and ART exposure would be associated with shorter leukocyte telomere length (TL) in exposed mother/infant pairs compared with controls. In these retrospective and prospective observational cohort studies, TL was evaluated in peripheral blood leukocytes obtained from HIV-infected pregnant women treated with ART and their uninfected infants, and compared with HIV untreated (retrospective cohort) or HIV mothers and their infants (prospective cohort). In HIV-infected ART-exposed mothers, leukocyte TL was not significantly shorter than that in HIV untreated mothers or HIV controls, nor was their infants' TL significantly different. Cord blood of ART-exposed infants exhibited TL shorter than that from infants born to HIV-negative mothers. Placenta also showed evidence of shorter TL after adjustment for relevant covariates. Factors associated with shorter maternal and infant TL included smoking and the use of drugs of addiction in pregnancy. These results suggest that maternal HIV infection or exposure to ART has minimal effect on infant leukocyte TL, a reassuring finding. In contrast, tissues that express higher telomerase activity such as umbilical cord blood and placenta appear comparatively more affected by ART. Smoking and the use of drugs of addiction have a negative impact on maternal and infant leukocyte TL, possibly through oxidative telomere damage.
Article
Although antiretroviral therapy for HIV infection prevents AIDS-related complications and prolongs life, it does not fully restore health. Long-term treated patients remain at higher than expected risk for a number of complications typically associated with aging, including cardiovascular disease, cancer, osteoporosis, and other end-organ diseases. The potential effect of HIV on health is perhaps most clearly exhibited by a number of immunologic abnormalities that persist despite effective suppression of HIV replication. These changes are consistent with some of the changes to the adaptive immune system that are seen in the very old ("immunosenescence") and that are likely related in part to persistent inflammation. HIV-associated inflammation and immunosenescence have been implicated as causally related to the premature onset of other end-organ diseases. Novel therapeutic strategies aimed at preventing or reversing these immunologic defects may be necessary if HIV-infected patients are to achieve normal life span.
Article
Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G(2)/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, after Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein and the neuronal marker synaptophysin in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma.
Article
Telomere length has emerged as a marker of exposure to oxidative stress and aging. Race/ethnic differences in telomere length have been infrequently investigated. Leukocyte telomere length (LTL) was assessed 981 white, black and Hispanic men and women aged 45-84 years participating in the Multi-Ethnic Study of Atherosclerosis. Direct measurement and questionnaire were used to assess covariates. Linear regression was used to estimate associations of LTL with race/ethnicity and age after adjustment for sex, income, education, smoking, physical activity, diet and body mass index. On average blacks and Hispanics had shorter telomeres than whites [adjusted mean differences (standard error) in T/S ratio compared to whites: -0.041 (0.018) for blacks and -0.044 (0.018) for Hispanics]. Blacks and Hispanics showed greater differences in telomere length associated with age than whites (adjusted mean differences in T/S ratio per 1 year increase in age -0.0018, -0.0047 and -0.0055 in whites, blacks and Hispanics respectively). Differences in age associations were more pronounced and only statistically significant in women. Race/ethnic differences in LTL may reflect the cumulative burden of differential exposure to oxidative stress (and its predictors) over the lifecourse.
Article
Telomerase is a ribonucleoprotein which has a RNA template to bind and extend telomere ends, so prolonging the life of tumour cells. The aim of this study was to determine whether transcriptase function of telomerase could be inhibited by the reverse transcriptase inhibitors (RTI); azydothymidine (AZT), dideoxyinosine (ddI) and AZT-5' triphosphate (AZT-TP). We examined their effects on the proliferation of cancer cells and the antitumour effects of cisplatin in vitro. The three agents did not cause major changes in telomerase activity or telomere length in MCAS cells. However, in HEC-1 cells changes in telomerase activity and telomere length were observed that were dependent on the RTI concentration and duration of exposure. ddI and AZT-TP reduced telomerase activity and shortened the length of the telomere. In the presence of RTI, the antitumour effects of cisplatin were enhanced. This was particularly evident in HEC-1 cells where there was a marked reduction in cell proliferation, appearance of morphological changes and senescent-like cells in the presence of ddI or AZT-TP. In MCAS cells, TP53 expression was increased by ddI and AZT-TP, while p21 expression was unchanged. In HEC-1 cells the expression of both TP53 and P21 was increased by ddI. Continuous administration of RTI enhanced the cell growth inhibition of cisplatin. RTI also inhibited the proliferation of some cells.
Article
The present study describes the effect of Saquinavir on proliferation, interferon-gamma production and telomerase activity of non-stimulated, or activated non-adherent mononuclear cells (NAMNC), obtained from peripheral blood of healthy donors. Fresh NAMNC, non-stimulated or activated in vitro with PHA or with a mixture of monoclonal antibodies against CD3 and against CD28 membrane antigens (in order to obtain prevalent T cell responses), were exposed to Saquinavir before or at the time of mitogenic stimulation. Control and treated cells were tested for DNA synthesis (3H-thymidine incorporation), interferon-gamma production and telomerase activity (TRAP assay). The results indicate that Saquinavir is able to increase proliferation and interferon-gamma release in PHA-stimulated NAMNC, and telomerase activity either in non-stimulated and in PHA or antibody-activated cells. These results suggest that the activity against HIV infection afforded by Saquinavir, could be corroborated by its effects on the host. These include its adjuvant activity on mitogen-induced responses of lymphocytes, and its possible antagonistic effects against lymphoid cell senescence, through telomerase activation.
Telomeres and human disease: ageing, cancer and beyond
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