Robust tobacco smoking self-report in two cohorts: pregnant women or men and women living with or without HIV.

Understanding the true burden of tobacco smoking on adverse pregnancy outcomes is critical in generating appropriate interventions to improve outcomes. Self-reporting of human behaviour that is associated with stigma is associated with underreporting in general and may bias the impact of smoking in studies; however, self-reporting is frequently the most practical method of gleaning this information. The objective of this study was to evaluate concordance between self-reported smoking and concentrations of plasma cotinine, a biomarker of smoking, among participants enrolled in two related HIV cohorts. A total of 100 pregnant women (76 living with HIV [LWH] and 24 negative controls) in their third trimester, and 100 men and non-pregnant women (43 LWH and 57 negative controls) were included. Among all participants, 43 pregnant women (49% LWH and 25% negative controls) and 50 men and non-pregnant women (58% LWH and 44% negative controls) were self-reported smokers. The odds of discordance between self-reported smoking and cotinine levels were not significantly different between self-reported smokers and non-smokers, nor between pregnant women and others, but were significantly increased, regardless of self-reported status, among people LWH compared to negative controls. The overall concordance between plasma cotinine and self-reported data among all participants was 94% with a sensitivity and specificity of 90% and 96%, respectively. Taken together, these data demonstrate that participant surveying in a non-judgemental context can lead to accurate and robust self-report smoking data among both persons LWH and not, including in the context of pregnancy.

Mitochondrial DNA somatic mutation burden and heteroplasmy are associated with chronological age, smoking, and HIV infection.

The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low-frequency mutations. We use primer ID-based next-generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D-loop, in 164 women and girls aged 2-72 years, of whom 35% were smokers and 56% were HIV-positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.

Dynamics of leukocyte telomere length in pregnant women living with HIV, and HIV-negative pregnant women: A longitudinal observational study.

BACKGROUND: HIV-mediated inflammation and immune activation can accelerate telomere attrition. In addition, antiretrovirals can inhibit telomerase, possibly shortening telomeres. We examined the longitudinal dynamics of leukocyte telomere length (LTL) during pregnancy in a unique cohort of women living with HIV (WLWH) treated with combination antiretroviral therapy (cART), and HIV-negative control women. METHODS: Blood was collected at three visits during pregnancy, at 13-23, >23-30, and >30-40 weeks of gestation, and for WLWH only, at 6 weeks post-partum. LTL was measured by qPCR and both cross-sectional and longitudinal (MANOVA) models were used to examine possible predictors of LTL among participants who attended all three visits during pregnancy. RESULTS: Among WLWH (n = 64) and HIV-negative women (n = 41), within participant LTL were correlated throughout pregnancy (p<0.001). LTL was shorter among WLWH at first visit, but this difference waned by the second visit. WLWH who discontinued cART post-partum experienced a decrease in LTL. Longitudinally, LTL was similar in both groups and increased as gestation progressed, a change that was more pronounced among women under 35 years. Among WLWH, both smoking throughout pregnancy (p = 0.04) and receiving a ritonavir-boosted protease inhibitor-based regimen (p = 0.03) were independently associated with shorter LTL. CONCLUSIONS: LTL increases as pregnancy progresses; the reasons for this are unknown but may relate to changes in blood volume, hormones, and/or cell subset distribution. While our observations need confirmation in an independent cohort, our data suggest that although some cART regimens may influence LTL, being on cART appears overall protective and that stopping cART post-partum may negatively impact LTL. The effect of smoking on LTL is clearly negative, stressing the importance of smoking cessation.

A mitochondrial DNA D loop insertion detected almost exclusively in non-replicating tissues with maternal inheritance across three generations.

Muscle biopsy identified a possibly pathogenic, mitochondrial DNA D-loop insertion, in each of 5 family members from two generations, that was otherwise undetectable in most other tissues. The tissue specific regulation of heteroplasmy is reflected in an age related increase in muscle heteroplasmy level, across the pedigree. This latter finding is in keeping with previous reports (e.g. T408A, C16327) but differs in having a very high muscle heteroplasmy level, and appears maternally transmitted.

Elevated Cell-Free Mitochondrial DNA in Filtered Plasma Is Associated With HIV Infection and Inflammation.

BACKGROUND: Increased cell-free DNA levels are associated with poor health outcomes, and cell-free mitochondrial DNA (cf-mtDNA) has proinflammatory properties. Given that HIV infection is associated with chronic inflammation, we investigated the relationship between cf-mtDNA and proinflammatory cytokine interleukin-6 (IL-6) in the context of HIV infection. We also optimized separation of cell-free plasma from blood. SETTING: In this retrospective cross-sectional study, we collected blood, demographic information, and clinical data from 99 HIV-infected and 103 HIV-uninfected adults and children enrolled in the Children and Women: AntiRetrovirals and Markers of Aging pan-Canadian (CARMA) cohort. METHODS: Plasma was separated from blood by 14,000g centrifugation followed by 0.45-µm filtration to remove cells and platelets. Cf-mtDNA and cell-free nuclear DNA were quantified simultaneously via monochrome, multiplex, quantitative polymerase chain reaction. IL-6 was measured using enzyme-linked immunosorbent assay. RESULTS: Higher speed centrifugation and filtration was necessary to isolate truly cell-free plasma. Higher cf-mtDNA levels were univariately associated with HIV infection, elevated IL-6 levels, younger age, higher white blood cell count, and higher cell-free nuclear DNA levels but not blood mtDNA content or HIV viral load. In a multivariable model, HIV infection (P < 0.001), elevated IL-6 (P = 0.021), younger age (P < 0.001), and higher blood nDNA levels (P = 0.007) were independently associated with higher cf-mtDNA. CONCLUSIONS: People living with HIV have higher levels of circulating cf-mtDNA than their uninfected peers. Increased levels of inflammatory marker IL-6 are associated with elevated cf-mtDNA, independent of the effect of HIV infection. Higher cf-mtDNA levels and white blood cell count in younger people may reflect higher cell turnover in that population.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real-Time Quantitative PCR on the LightCycler 480: Sources of Variability and Quality Control Considerations.

Telomere length (TL) measurement is central to many biomedical research, population, and epidemiology studies, with promising potential as a clinical tool. Various assays are used to determine TL, depending on the type and size of the sample. We describe the detailed optimization of a monochromatic multiplex real-time quantitative PCR (MMqPCR) assay for relative TL using the LightCycler 480. MMqPCR was initially developed using a different instrument with many separate reagents. Differences in instrument performance, reagents, and workflow required substantial optimization for the assay to be compatible with the LightCycler 480. We optimized the chemistry of the assay using a purchased one-component reaction mix and herein describe sources of variability and quality control relevant to the MMqPCR TL assay on any instrument. Finally, the assay was validated against other TL assays, such as terminal restriction fragment, Southern blot, and flow fluorescent in situ hybridization. The correlations obtained between data from MMqPCR and these assays (R(2) = 0.88 and 0.81) were comparable to those seen with the monoplex version (R(2) = 0.85 and 0.82) when the same samples were assayed. The intrarun and interrun CV ranged from 4.2% to 6.2% and 3.2% to 4.9%, respectively. We describe a protocol for measuring TL on the LightCycler platform that provides a robust high-throughput method applicable to clinical diagnostics or large-scale studies of archived specimens.

Association between short leukocyte telomere length and HIV infection in a cohort study: No evidence of a relationship with antiretroviral therapy.

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.

Blood and dried blood spot telomere length measurement by qPCR: assay considerations.

Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.

Leukocyte telomere length in HIV-infected and HIV-exposed uninfected children: shorter telomeres with uncontrolled HIV viremia.

OBJECTIVES: Nucleoside reverse transcriptase inhibitors (NRTIs) used in HIV antiretroviral therapy can inhibit human telomerase reverse transcriptase. We therefore investigated whether in utero or childhood exposure to NRTIs affects leukocyte telomere length (LTL), a marker of cellular aging. METHODS: In this cross-sectional CARMA cohort study, we investigated factors associated with LTL in HIV-1-infected (HIV(+)) children (n = 94), HIV-1-exposed uninfected (HEU) children who were exposed to antiretroviral therapy (ART) perinatally (n = 177), and HIV-unexposed uninfected (HIV(-)) control children (n = 104) aged 0-19 years. Univariate followed by multivariate linear regression models were used to examine relationships of explanatory variables with LTL for: a) all subjects, b) HIV(+)/HEU children only, and c) HIV(+) children only. RESULTS: After adjusting for age and gender, there was no difference in LTL between the 3 groups, when considering children of all ages together. In multivariate models, older age and male gender were associated with shorter LTL. For the HIV(+) group alone, having a detectable HIV viral load was also strongly associated with shorter LTL (p = 0.007). CONCLUSIONS: In this large study, group rates of LTL attrition were similar for HIV(+), HEU and HIV(-) children. No associations between children's LTL and their perinatal ART exposure or HIV status were seen in linear regression models. However, the association between having a detectable HIV viral load and shorter LTL suggests that uncontrolled HIV viremia rather than duration of ART exposure may be associated with acceleration of blood telomere attrition.

Leukocyte telomere length in HIV-infected pregnant women treated with antiretroviral drugs during pregnancy and their uninfected infants.

OBJECTIVES: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Blood mitochondrial DNA mutations in HIV-infected women and their infants exposed to HAART during pregnancy.

OBJECTIVES: Nucleo(s/t)ide reverse transcriptase inhibitors given to HIV-infected pregnant women to prevent vertical transmission may adversely affect mitochondrial DNA (mtDNA). We hypothesized that HAART-exposed/HIV-uninfected infants may show higher blood mtDNA mutation burden than controls born to HIV-uninfected mothers. METHODS: Blood was collected from in-utero HIV/HAART-exposed infants and controls, as well as from a subset of their mothers. The presence of mtDNA A→C/T→G (AC/TG) mutations was measured by cloning and sequencing D-loop PCR amplicons. Relationships with maternal characteristics were examined. RESULTS: No significant difference was found between the percentage of HIV/HAART-exposed infants with AC/TG mutations (N = 15/57, 26.3%) and controls (N = 10/70, 14.3%) before (P = 0.090) or after controlling for covariates (P = 0.058), although a tendency was observed. However, significantly more HIV/HAART-exposed mothers (N = 18/42, 42.9%) harboured AC/TG mutations compared with controls (N = 7/39, 17.9%) before (P = 0.015) and after (P = 0.012) controlling for covariates. AC/TG mutations were more prevalent in HIV/HAART-exposed mothers than in their infants (N = 42, 42.9 vs. 23.8%, P = 0.033), however, this difference disappeared after controlling for covariates. No difference was seen between control mothers and their infants (N = 39, both 17.9%). In HIV/HAART-exposed mothers, only a detectable HIV plasma viral load near delivery predicted AC/TG mutations. CONCLUSION: Our results suggest that HIV and/or HAART exposure are associated with increased prevalence of AC/TG mtDNA mutations in mothers and show a similar tendency in infants exposed during pregnancy. As accumulation of mtDNA mutations has been linked with aging and age-associated diseases, this may raise concerns in the long term for HIV and HAART-exposed populations.

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes.

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.

HIV-1 drug resistance: degree of underestimation by a cross-sectional versus a longitudinal testing approach.

Genotyping of human immunodeficiency virus type 1 (HIV-1) for antiretroviral drug resistance is routinely used both in clinical practice, to guide the selection of options for an individual's antiretroviral therapy, and in epidemiological studies, to estimate levels of antiretroviral drug resistance in a patient population. However, reliance on results of a single test can result in an underestimation of antiretroviral drug resistance. In the present study, we quantified the prevalence of resistance-associated mutations found in recent genotypic tests of 1734 HIV-1-infected, treatment-experienced subjects who had at least 3 genotypic tests (n = 11,404 genotypic tests total; median, 5 tests/subject) and compared it with that of resistance-associated mutations ever detected in these subjects between 1996 and 2004. Single-point analyses underestimated antiretroviral drug resistance, particularly for nucleoside analogues, in both individuals and patient populations. For example, the prevalence of resistance-associated mutation M184V/I was 25.5% in the most recent genotypes and 58.8% in available historical genotypes. Our results suggest that analysis of a combined historical genotype rather than of a cross-sectional genotype may lead to more accurate estimates of antiretroviral drug resistance in individual patients and in patient populations.