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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Cellular and mitochondrial toxicity of integrase strand transfer inhibitors in human embryonic stem cell models (2024)
Each year, over one million more children are exposed in utero to HIV antiretrovirals (ARVs), yet their safety is often not well characterized during pregnancy. Many ARVs are known to affect mitochondrial function that, if altered during initial stages of embryonic and placental development, could have detrimental consequences for cellular differentiation and early development. Recent studies suggested a concerning association between exposure to the integrase inhibitor (InSTI) dolutegravir (DTG) during early development and increased incidence of neurologic outcomes, such as neural tube defects. As the latter occur within the first four weeks of embryo development, it is crucial to identify the effects of ARVs during the early stages of gestation, on both mitochondrial health and the differentiation of early human embryos.To address this, I sought to characterize and compare the effects of several clinically relevant ARVs in cultured human embryonic stem cell (hESC) models, with respect to cellular and mitochondrial health. I also investigated the effects of ARV exposure on hESC markers of pluripotency, and during their directed germ layer differentiation. I found that even at sub-therapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC cell counts, pluripotency, numerous metrics of mitochondrial health, and dysregulated the expression of genes involved in early differentiation. Notably, first-generation InSTI raltegravir did not induce this hESC toxicity or affect differentiation at any concentration tested. Exposure to some InSTIs at clinically relevant concentrations or even lower can induce adverse effects in hESCs. There are now over 30 ARVs in the HIV therapeutic arsenal but we have little insight into which combination ARV regimens are the most pregnancy-safe. Given the increasingly prevalent use of second-generation InSTIs worldwide, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety following in utero exposure. My study provides crucial pre-clinical information on the relative toxicity of multiple ARVs. This new knowledge can inform future human trials and help guide improved strategies for the treatment of HIV in women of reproductive age.
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Discovery and surveillance of viral spillover threats using probe capture-based targeted genomic sequencing (2023)
Human health and agriculture are constantly threatened by viruses that spillover from wildlife. Genomic sequencing is becoming integral to protecting humanity from these threats. This technology is being used to explore viral biodiversity and identify new potential pathogens. It is also being used to monitor human, livestock, and wildlife populations for potential spillovers.Recovering and analyzing viral genomes in real-world specimens remains a challenge, however. Viral genomic material must be recovered from human, animal, and environmental specimens where it is extremely dilute, fragmented, or incomplete. This makes enrichment of viral genomic material necessary for practical, high-throughput surveillance operations. Hybridization probe capture is a powerful technique used in many fields to enrich target genomic material when preparing specimens for sequencing. This technique uses synthetic nucleotide oligomers (probes) to capture target genomic fragments while background material is washed away. To achieve this, collections of probes must be designed with sequences complementary to the target genome, which becomes problematic for viral targets with extensive genetic diversity and hypervariability. Designing compact probe panels that provide broad coverage of viral taxa presents a substantial computational challenge.A lack of accessible probe design tools has kept this powerful technique out of reach for many public health and agriculture agencies. Consequently, probe capture has not been widely deployed for large-scale discovery and routine surveillance of viral spillover threats. To address this gap, this dissertation presents ProbeTools, a user-friendly software package developed to facilitate probe panel design for diverse and hypervariable viral taxa. In this dissertation,ProbeTools is validated in silico on tens of thousands of reference genome sequences and in vitro on dozens of egg-cultured viral isolates. Next, ProbeTools is applied to two real-world viral discovery and surveillance applications. First, it is used to characterize genomes of novel coronaviruses in bats. Second, it is used to detect avian influenza viruses in environmental specimens from wild bird habitats.The validations and case studies in this dissertation demonstrate the power of probe capture for discovery and surveillance of viral spillover threats. They also demonstrate the suitability and flexibility of ProbeTools when designing probe panels for applied viral genomics.
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Dynamics of telomere length and mitochondrial DNA content in a cohort study of HIV-infected and HIV-uninfected pregnant women and cell culture models (2022)
Globally, women constitute around 50% of HIV-infected individuals. While mother-to-child transmission accounts for 90% of new HIV infections among children, combination antiretroviral therapy (cART) reduces the risk from 25% to 2%. Treatment guidelines now promote lifelong cART for all persons living with HIV. This implies that more women will be conceiving on cART and exposing their unborn child over a longer period. Nucleoside reverse transcriptase inhibitors can have off target effects. These drugs can inhibit reverse transcriptase activity of telomerase, which could lead to a shortening of leukocyte telomere length (LTL). LTL has been described as a marker of cellular aging and a predictor of age-related diseases over time. Several antiretrovirals (ARVs) can exert mitochondrial toxicity, leading to mitochondrial dysfunction. The overarching hypothesis of my research was that LTL and mitochondrial DNA (mtDNA) content would be affected by cART in the clinical and cell culture samples.I measured LTL in blood samples collected from 64 HIV-infected and 41 HIV-uninfected women at three visits during pregnancy using monochromatic multiplex quantitative polymerase chain reaction. CART treatment status during pregnancy was not associated with shorter LTL. However, smoking throughout pregnancy and receiving a boosted protease inhibitor regimen were independently associated with shorter LTL among HIV-infected women. Whether these reflect telomere attrition or redistribution of cellular subsets is unclear. In clinical studies, it is challenging to distinguish between the effects of HIV vs. those of cART. I used cultured placental and T-lymphoblast cells to study the changes in mtDNA content following either short term exposure to individual ARVs at increasing concentrations, or longer-term exposure (21 days) to cART regimens at 1×Cmax, (maximum concentration) prior to returning the cells to cART-free medium for ten more days, to allow recovery/repair. Most ARVs and cART studied here induced increased mtDNA content, postulated to reflect mitochondria biogenesis in response to cellular stresses and/or damage, something that could promote the clonal expansion of mtDNA mutations. However, changes in mtDNA content in response to ARV exposure can be both bidirectional and cell-specific; and appear to be reversible. Mitochondria morphological changes were suggestive of increased mitophagy to preserve mitochondrial health.
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HIV infection in the post-combination antiretroviral therapy era: a human model of immune aging (2020)
The deterioration of the immune system is a fundamental characteristic of aging and is accompanied by alterations in immune aging markers including shorter telomere length (TL), decreased mitochondrial DNA (mtDNA) content, and larger differentiated T cell populations. These same markers characterize an accelerated immune aging phenotype in people living with HIV (PLWH). Although HIV is successfully treated with combination antiretroviral therapy (cART), treatment is lifelong and does not fully eliminate the accelerated aging phenotype. Therefore, cART-controlled HIV infection can represent a human model of immune aging compared to the general population. The extent to which longitudinal changes in leukocyte TL (LTL) and blood mtDNA content inter-relate and are together influenced by HIV is unknown. Also unknown is whether slow progressors, a rare population who naturally control HIV without cART, are protected against HIV-mediated accelerated immune aging.The goal of my research was to investigate aging markers in PLWH, considering HIV infection as a human model of immune aging.First, I developed a monochrome multiplex quantitative PCR assay to measure mtDNA content quickly and accurately with low starting material. I measured blood mtDNA content, LTL, and their rates of change among 312 PLWH and 300 HIV-negative controls. I showed that faster rates of LTL attrition and blood mtDNA decline were associated with loss of HIV viral control. Faster LTL attrition was also associated with slower blood mtDNA content decline, regardless of HIV status. I then measured more granular markers of immune aging including lymphocyte subset TL and T cell differentiation in 57 slow progressors with sex- and age-matched cART-controlled PLWH and HIV-negative controls. My analyses revealed that slow progressors had shorter immune subset TL and more differentiated CD8 T cells compared to both cART-controlled PLWH and HIV-negative controls, indicating that they experience even faster immune aging despite naturally controlling the disease.My research confirms that uncontrolled chronic/latent viral infections such as HIV accelerate immune aging but demonstrates that naturally controlling HIV may also age immune cells. This suggests that preventing and controlling chronic/latent viral infections with therapy could extend lifespan and may represent an evolution to the paradigm of aging research.
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Dynamics of telomere length and mitochondrial health in relation to combination antiretroviral therapy (cART) exposure: a cohort study of HIV/cART-exposed uninfected children and cell culture investigations (2019)
Combination antiretroviral therapy (cART) during pregnancy has considerably reduced the risk of mother-to-child HIV transmission and the number of cART-exposed HIV-exposed uninfected (HEU) children is increasing. With current treatment guidelines recommending the initiation of immediate, lifelong cART at HIV diagnosis, women conceive on therapy and HEU in utero cART exposure spans the entire gestation period. Many antiretrovirals (ARV) cross the placenta and could exert long-term effects on HEUs. Some ARVs inhibit human telomerase reverse transcriptase (hTERT). As hTERT elongates telomeres and protects mitochondrial DNA (mtDNA) from oxidative damage, its inhibition could lead to shorter telomeres and/or increased mitochondrial dysfunction. Leukocyte telomere length (LTL) and mtDNA alterations are biomarkers of cellular aging, and have been implicated in aging and age-related diseases. The objective of my research was to compare HEU and HIV-unexposed uninfected (HUU) children at birth and in early life, with respect to their LTL and blood mtDNA content, and investigate relationships with in utero cART exposure. I measured LTL and blood mtDNA content in 324 HEU and 306 HUU children between 0-3y of age. I found that exposure to maternal cART did not affect LTL at birth, as it was similar in both groups. However, mtDNA content was higher among HEU children, particularly those exposed to boosted-protease inhibitor (PI/r) cART. This increase in mtDNA persisted at least up to age three. Additionally, maternal smoking during pregnancy affected both LTL and mtDNA content at birth. Given these effects of cART on children’s mtDNA, I aimed to further characterize how various cART regimens affect mitochondrial health using an in vitro human cell culture model. I also investigated the potential mitochondrial protection conferred by hTERT. I found that dolutegravir (DTG)-containing regimens negatively affected mitochondria, decreased cell proliferation and increased apoptosis. PI/r-containing regimens also affected certain mitochondrial parameters, but this effect was mitigated by mitochondrial hTERT while that of DTG was not. DTG is increasingly used worldwide, including in pregnancy. These novel findings merit further investigations to evaluate the long-term safety of newer ARV exposure, and the predictive value of these biomarkers on HEU health outcomes. Together, this knowledge could inform treatment guidelines.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Exploration of rare mitochondrial DNA mutations in lymphocyte subsets of people living with human immunodeficiency virus (2024)
Despite advancements in Human Immunodeficiency Virus (HIV) therapy in extending lifespan and preventing AIDS, people living with HIV (PLWH) experience premature aging mainly indicated by higher prevalence and earlier onset of age-related diseases compared to HIV-negative controls. The mechanism of this aging remains unclear; however, some immune system effects observed in HIV are also characteristic of aging. Mitochondrial dysfunction, a hallmark of aging, may be linked to HIV and its treatment, which are known to affect mitochondrial DNA (mtDNA) and function. Further, mtDNA mutations are associated with aging and age-related diseases. In this regard, low frequency (1%) mtDNA mutations have become subject to growing interest. The interplay of mtDNA mutations and immunosenescence is unknown as rare mtDNA mutations have never been characterized in lymphocyte subsets. The goal of my research was to explore rare mtDNA mutations in lymphocyte subsets of PLWH. First, I successfully adapted a unique molecular identifier (UMI)-based sequencing assay to enable sequencing of low concentration mtDNA specimens, necessary for my assaying of sorted lymphocyte specimens. I then sequenced a 264bp segment of mtDNA in the mitochondrial control region of: whole blood, CD4+ T cells, proliferative-competent (CD28+) CD8+ T cells, senescent (CD28-) CD8+ T cells, and B cells (CD19+) in 271 specimens from 48 PLWH and 41 HIV-negative controls. My analyses revealed a pattern of mtDNA variants with specific positions appearing as hotspots in the interrogated region, almost exclusively transition mutations (A↔ G, C↔ T). Total mutation burden differed slightly between lymphocyte subsets, however the distribution of mutations across genome position in each subset agreed closely with each other and with whole blood. No significant associations were observed with HIV status in any of the subsets. My research is the first to characterize rare mtDNA mutations in lymphocyte subsets, offering new insight into the composition of mtDNA mutation burden of whole blood. More research is needed to elucidate the extent of interplay between mtDNA mutations, immunosenescence and aging in HIV. Together, my findings facilitate future studies by validating a modified assay for specimens with low mtDNA concentrations and informing the use of whole blood to study mtDNA mutations.
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Mitochondrial DNA mutations and interruptions in combination antiretroviral therapy : a retrospective cohort study (2023)
HIV therapy has transformed the lives of people living with HIV (PLWH), turning a once lethal condition into a chronic disease that can be carefully managed over a patient’s lifetime. However, PLWH seem to experience accelerated aging, with a reported decrease in lifespan of up to 10 years, as well as earlier onset and higher prevalence of age-related diseases. Many of these diseases have been linked to interruptions in HIV therapy and/or mutations in the mitochondrial DNA (mtDNA). Current theories of aging implicate the gradual accumulation of mtDNA mutations as a driver of biological aging and may provide a mechanism by which interruptions in HIV therapy could lead to an increased occurrence of age-related diseases. These mutations can be somatic (i.e., de novo) mutations, or higher frequency mtDNA variants (i.e., heteroplasmy). My goal was to determine if interruptions in HIV therapy are associated with an increase in mtDNA somatic or heteroplasmic mutations.Blood mtDNA mutations were characterized among participants living with (n = 219) and without (n = 72) HIV, ranging between 1 and 75 years of age, and enrolled in the Children and Women: AntiRetrovirals and Markers of Aging (CARMA) cohort. Participants living with HIV had between 0 and 17 interruptions in HIV therapy. Mutations were measured in a 264bp region of the mitochondrial D-loop, using a single strand unique molecular identifier-based sequencing to detect ultra-rate substitution mutations. Somatic mtDNA substitution mutations were independently associated with older age (p 0.001), Indigenous ethnicity (p = 0.001), and having a peak HIV viral load ≥100,000 copies/ml among adult participants. Among all participants having exactly two interruptions was associated with a lower somatic mtDNA substitution burden (p = 0.035). The occurrence of heteroplasmy was independently associated with older age (p = 0.002) and smoking (p = 0.05) among all participants, after considering an age-smoking interaction term. Our results are consistent with the current understanding of mitochondrial aging; however, they do not support the hypothesis that interruption in HIV therapy may be driving mutations in the mitochondrial DNA. However, interruptions remain a risk factor for age-related disease and are almost universally discouraged.
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The impact of chronic and latent viral infections on aging in people living with HIV (2023)
Background: Over 38 million people are currently living with HIV. Despite antiretroviral therapies that have increased lifespan, people living with HIV (PLWH) experience faster cellular and immunological aging relative to their HIV-negative peers. Infection with chronic/latent viruses such as herpesviruses and hepatitis viruses may accelerate immunological aging. Individually, these viruses are associated with aging markers and/or age-associated diseases, but their cumulative effect is unknown. We characterized the number and type of seven non-HIV chronic/latent viruses in PLWH and those not living with HIV and investigated associations with leukocyte telomere length (LTL).Methods: A total of 187 PLWH (105F/82M) and 189 HIV-negative controls (105F/84M) were selected and balanced for each decade of age, sex, and HIV group. Past CMV, EBV, HHV-8, HSV-1, and HSV-2 infection was determined serologically; HIV, HCV, and HBV were self-reported. Relative LTL was measured using qPCR. Associations between number of viruses, LTL, and sociodemographic factors were assessed using ordinal logistic and linear regression modelling.Results: PLWH had significantly more non-HIV viruses than HIV-negative controls (p0.0001), as did females compared to males (p0.0001). In multivariable analyses, HIV-positive status, female sex, Indigenous ethnicity, current smoking, and African region of birth were independently associated with a greater frequency of viral infection. Additionally having 3-4 viruses (vs. 0-2) was independently associated with shorter LTL. While no individual non-HIV virus had a significant effect on LTL, some virus combinations were independently associated with shorter or longer LTL. Furthermore, sex-segregated analyses revealed that female and male participants differed with respect to factors associated with both total virus burden and LTL. Conclusions: Our results suggest that carrying a greater number of persistent viruses contributes to immunological aging, with specific viruses and combinations exerting differential effects based on sex. Future studies to better understand the contribution of chronic/latent viral infection on immunological aging and clinical outcomes can shed light on the potential value of viral prevention or treatment initiatives. Such studies when combined with biological and sociocultural considerations between men and women in the context of virus acquisition risk can help us understand how to improve the health lifespan of all populations.
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Placenta mitochondrial DNA mutation burden and the risk of preterm birth in pregnant women living with HIV (2022)
Background: Preterm birth (PTB) (37 weeks of gestation), the leading cause of mortality among children 5 years old, is up to three times more common among women living with HIV (WLWH) compared to the general population. The etiology of PTB remains poorly understood, but oxidative stress, and impaired placenta mitochondrial function are believed to play a role. Both HIV and antiretroviral therapy (ART) can affect mitochondrial DNA (mtDNA), which may contribute to high rates of PTB in WLWH. Maternal smoking and older age, both risk factors inPTB, have been associated with increased mtDNA mutation burden in blood. I herein exploredrare placenta mtDNA mutations and their association with PTB, maternal HIV, smoking, and age.Methods: Placenta mtDNA was sequenced using a molecular barcoding approach to detect rare substitutions within a 264bp region of the mitochondrial D-loop. MtDNA was sequenced from multiple locations within the same placenta, taken at multiple times post-delivery, and extracted by two different methods (silica column and salting-out). A subset of pregnant women (37 HIV+ and 27 HIV-negative), enrolled in two studies, 23 of whom had a PTB, was selected. Mutations were quantified within the 264bp region, within subregions known to be resistant to polymorphisms, and within a region outside hypervariable segments.Results: MtDNA sequencing showed low intra-placenta variability, no significant bias associated with time post-delivery up to 48h, but low correlation between two DNA extraction methods (rho=0.34, p=0.15).Increased placenta mtDNA mutations were associated with maternal smoking (p=0.006), and older age (rho=0.21, p=0.02). WLWH showed increased mtDNA mutations outside of hypervariable regions (p0.001) and at positions typically resistant to polymorphisms (p=0.03). These associations persisted in multivariable models.Conclusions: My results suggest that a single placenta sampling collected within 48h of delivery would be suitable for future studies, but that the method of DNA extraction must be consistent within a study as it influences results.Rare placenta mtDNA mutations increase with maternal HIV, smoking, and age, three factors associated with PTB. However, PTB showed no independent association with placenta mtDNA mutations. The type of mutations differs between HIV and other factors, suggesting different mechanisms.
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Placental Mitochondrial Dysfunction in relation to preterm delivery in HIV pregnancy (2018)
Background: Preterm birth (PTB) (37 weeks of gestation), is the leading cause of mortality and morbidity among children, responsible for >1 million deaths in 2015 . In North America, PTB occurs in 6–10% of births. However, among pregnant women living with HIV, the rates are higher (18-29%). To date, there is no generally accepted mechanism underlying such increased rates. One possible explanation is reduced maternal progesterone production during pregnancy, which may be related to HIV infection and/or antiretroviral (ARV) treatment. Synthesis of progesterone (hormone central to pregnancy maintenance), is dependent on placental mitochondrial function. Given that many ARVs can affect mitochondrial (mt) function, I investigated the possible effects of ARV on placental mtDNA content and progesterone levels.Methods: 136 HIV+ and 60 HIV- pregnant women were enrolled in the Canadian prospective study, the Children and women: Antiretroviral and Markers of Aging (CARMA) cohort. Placenta and blood specimens, as well as clinical and sociodemographic data, were collected. Placental and plasma progesterone levels, as well as placenta mtDNA content, were measured using ELISA and qPCR respectively. We extended these investigations of ARV effects to in vitro models on two human placental cell lines, JEG-3 and BeWo.Results: Within this cohort, HIV-exposed uninfected (HEU) infants were born at an earlier gestational age (p=0.017), with a lower birth weight (p=0.011) compared to controls. PTB showed no association with HIV status, placenta mtDNA or progesterone levels. However, higher mtDNA was associated with preeclampsia (p0.001), which often leads to PTB.
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Exploring mitochondrial DNA abnormalities in HIV-exposed uninfected children diagnosed with autism spectrum disorder: a case control study (2016)
Background: Antiretroviral therapy has reduced mother-to-child HIV transmission from 25-40% to less than 2%. Thus, increasing numbers of HIV-exposed uninfected (HEU) children are being born with perinatal exposure to antiretrovirals. Recently, a Canadian HIV clinic noticed a high prevalence of autism spectrum disorder (ASD) in HEU children. This prompted our analysis of HEU children enrolled in the pan-Canadian Children & Women AntiRetrovirals & Markers of Aging (CARMA) cohort study. Significant differences in mitochondrial DNA to nuclear DNA ratio (mtDNA content) have been observed in ASD children and HEUs as a potential marker for mitochondrial dysfunction, which has been theorized as a possible mechanism underlying abnormal neurodevelopment. We hypothesized that HEU children with ASD would have significantly different leukocyte mtDNA content than HEU children without ASD and/or HUU children with and without ASD.Methods: CARMA HEU children with ASD (n=14) were matched 1:3 on age, sex, and ethnicity to HEU children without ASD (n=42), HUU anonymous controls (n=42), and HUU children with ASD in the BC Autism Spectrum Interdisciplinary Research (ASPIRE) program (n=42). Non-ASD HUU siblings of ASD children (n=9) were also studied and grouped with the HUU controls for the purposes of analyses (n=51 total). MtDNA content was assessed using qPCR.Results: Among 299 HEU children in CARMA, 14 (4.7%) were diagnosed with ASD, substantially (>3-fold) above North American prevalence estimates (1.5%).HEU children with ASD had higher mtDNA content (median[interquartile range]: 163) than non-ASD HEUs (115, p=0.02), HUUs with ASD (110, p=0.0001), and HUU controls (100, p0.0001). Non-autistic HEU children and ASD children with no HIV/cART exposure had higher mtDNA content than controls (p=0.004 and p=0.03, respectively), but did not significantly differ from each other (p=0.2).Conclusions: Our results suggest a possible cumulative association between elevated leukocyte mtDNA content and both HEU and ASD status. This may implicate mitochondrial dysfunction as a contributor to the high ASD prevalence in our cohort. It is unclear if this effect is modulated by exposure to antiretrovirals or maternal HIV but it is consistent with studies suggesting increased mtDNA content as an adaptive mechanism to mitochondrial dysfunction.
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Investigating Markers of Cellular Aging in Human Immunodeficiency Virus Infected and Uninfected Adults (2013)
Background: Despite successful combination antiretroviral therapy (cART), people living with HIV have shorter lifespans than the general population. Leukocyte telomere length (LTL) and mitochondrial DNA (mtDNA) oxidative damage are two frequently studied markers of aging that have recently been linked. Telomerase extends telomeres in highly proliferative tissues and is believed to play a protective role against oxidative stress in mitochondria. Given that both HIV and cART have the potential to accelerate cellular aging processes, we sought to measure LTL and mtDNA apparent oxidative damage (AOD) in the context of HIV infection and treatment.Methods: Demographic and clinical data and whole blood were collected from adults aged 19-75 enrolled in a prospective cohort on HIV therapy and aging (CARMA). LTL were measured by qPCR. Variables statistically correlated with LTL on univariate analysis (p0.15) were candidates for a multivariate model. A subset of subjects with LTL data was selected to explore the relationship between LTL and mtDNA AOD, as measured by a quantitative long PCR based assay. Results: Of the 395 study participants, 58% were HIV infected, 76% were women, and 71% were current or previous smokers. In a multivariate model of all participants, older age, HIV infection, active hepatitis C virus (HCV) infection, and smoking were associated with shorter LTL. Smoking was associated with shorter LTL only in HIV uninfected subjects. Among the latter, age and smoking were independently related to shorter LTL. In contrast, in two models among HIV infected individuals, age and having either active HCV or a peak HIV plasma viral load ≥100,000 copies/ml were associated with shorter LTL. Other HIV disease or cART parameters were unrelated to LTL. In the 115 subjects for whom mtDNA AOD was measured, there was no relationship between AOD and age, HIV status or LTL. Conclusions: HIV infection was independently associated with shorter LTL in this cohort and this was related to peak viremia. Future studies should examine the effects of early HIV and HCV therapy, as well as smoking cessation, on LTL. The mtDNA AOD results suggest that more validation work is needed for use of this assay with clinical samples.
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Measuring apparent oxidative damage to mitochondrial DNA to HIV antiretroviral therapy (2012)
Background/objectives: HIV antiretroviral therapy, specifically nucleoside reverse transcriptase inhibitors (NRTIs) have been associated with mitochondrial DNA (mtDNA) alterations, possibly through mtDNA oxidative damage leading to mitochondrial dysfunction, which is associated with degenerative diseases and aging. A published assay exploits that oxidative damage can slow down/inhibit DNA polymerase progression, such that the PCR amplification of damaged mtDNA template yields less product compared to undamaged mtDNA. I sought to optimize this assay using in-house tools and to quantify apparent mtDNA oxidative damage in cultured cells exposed to NRTIs.Methods: Three DNA quantification methods were compared: PicoGreen fluorescence quantification, UV spectrophotometry, and qPCR mtDNA copy number. Human hepatocellular carcinoma cells (HepG2) were exposed to hydrogen peroxide (H₂O₂) followed by recovery time to allow mtDNA repair. To determine whether NRTI exposure induces mtDNA damage, human coronary artery endothelial cells (hCAE) cells and human colorectal adenocarcinoma cells (HT29) cells that had been exposed to various NRTIs were subjected to the assay. To assess the assay’s future applicability to clinical samples, human skeletal muscle DNA samples were also assayed.Results: Quantification of long PCR mtDNA product by UV (CV=6.5%) and qPCR (CV=7.0%) showed lowest variability while PicoGreen quantification was noticeably higher (CV=21%). DNA from H₂O₂-exposed cells showed decreased amplification of long PCR product that increased with repair time. MtDNA depletion occurred in both cultures treated with stavudine. While there was no apparent mtDNA oxidative damage in HT29 cells with any NRTI, both tenofovir and stavudine yielded increased mtDNA oxidative damage in hCAE cells. A wide degree of apparent mtDNA oxidative damage was observed in clinical samples.Conclusions: The preferred method for DNA quantification is qPCR mtDNA copy number. The observed mtDNA depletion indicated that NRTIs were active in both cell lines. The primary hCAE cells incurred greater mtDNA oxidative damage than cancer-derived HT29 cells. Cancer cells may have enhanced anti-oxidant mechanisms, suggesting that primary cells may be better model for studying mtDNA damage. The broad range of mtDNA damage detected in clinical samples bodes well for the assay’s use with diverse samples.
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Therapy-related hepatic mitochondrial dysfunction in patients co-infected with human immunodeficiency virus and hepatitis C virus and in HepG2 cells (2012)
Background: Co-infection with HIV and hepatitis C virus (HCV) worsens liver disease and decreases highly active antiretroviral therapy (HAART) tolerability. HAART usually includes two nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI) or a non-NRTI (NNRTI). NRTIs, particularly D-NRTIs, can induce mitochondrial DNA (mtDNA) depletion, deletions or mutations and lead to mitochondrial dysfunction. Multi-drug resistance protein-1 (MDR1) transports drugs across cellular membranes and may modulate toxicity. This project investigated HAART-related mitochondrial toxicity in liver tissue from HIV/HCV co-infected individuals and in human hepatic (HepG2) cells.Hypotheses: 1) Patients ON-HAART will have altered pathology scores, mtDNA quantity/deletions and mt-mRNA/MDR1-mRNA levels compared to patients OFF-HAART, and these will be influenced by type of HAART.2) Treatment with the d-NRTI didanosine (ddI) and the PI saquinavir (SAQ) will alter HepG2 cell viability, population doubling time (PDT) and mtDNA content.Methods: Double-liver biopsies were collected from HIV/HCV co-infected individuals. One sample was used to score pathology, the other to extract DNA and RNA. mtDNA quantity, mt-mRNA and MDR1-mRNA levels were investigated by quantitative-PCR and mtDNA deletion by long-template PCR. Measurements were compared between individuals ON- versus OFF-HAART, on D-NRTI versus other NRTIs and on PI versus NNRTI. HepG2 cells were exposed to ddI and SAQ. Cell viability, PDT and mtDNA content were investigated.Results: Individuals ON-HAART (N=34) were similar in age, gender and HCV genotype to those OFF-HAART (N=18), and the groups did not differ significantly in pathology score, mtDNA quantity/deletions or mt-mRNA/MDR1-mRNA levels. The same was true for individuals on D-NRTI (N=6) versus other NRTIs (N=28) and on PI (N=17) versus NNRTI (N=8), except that individuals on PI were older (p=0.044) with higher mt-mRNA levels (p=0.015).Treatment of HepG2 cells with ddI lowered mtDNA content while SAQ decreased PDT. Addition of a second drug (SAQ or ddI) exacerbated these effects. ddI transiently decreased viability. Conclusions: The lack of differences between the ON- and OFF-HAART groups supports previous observations that HAART is not associated with increased hepatic mitochondrial toxicity although the cell culture findings suggest complementary toxicity upon co-exposure to ddI/SAQ. This study may inform management of HIV/HCV co-infected individuals.
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Blood telomere length in infants and their HIV-infected mothers exposed to antiretroviral therapy during pregnancy (2011)
Background/Objectives:Nucleoside reverse transcriptase inhibitors (NRTIs) are part of the antiretroviral therapy (ART) given to HIV-infected pregnant women to prevent vertical HIV transmission. The NRTI zidovudine (AZT) is a known inhibitor of human telomerase, the enzyme responsible for telomere elongation. We hypothesized that average telomere length (ATL) may be shorter in infants born to HIV-infected mothers and exposed to ART in utero, compared to ART-unexposed infants.Methods:Two independent cohorts of pregnant women and their infants were studied, spanning 1990-2000 (SJ) and 2005-2009 (Pregnancy). SJ included 120 HIV⁺ exposed and unexposed pregnancies while Pregnancy included 99 HIV⁺ highly active antiretroviral therapy (HAART)-exposed and HIV⁻ pregnancies. Dried blood spots (SJ) or whole blood (Pregnancy) were collected from the pregnant women and their infants. Relative ATL (rATL) was measured by quantitative PCR. The differences in rATL between HAART/ART-exposed and unexposed maternal, infant and cord blood (CB) were investigated using ANCOVA, adjusting for maternal age, gestational age, smoking (cigarette/marijuana) and illicit drug/methadone use ever in pregnancy. For the HIV/HAART group, additional parameters included CD4+ count, HIV plasma viral load near delivery, length of HAART exposure, HCV infection and ethnicity. Relationships between maternal and infant rATL were also investigated.Results:Infant rATL were significantly longer than maternal rATL for both cohorts (p0.0001). Exposed CB rATL was shorter than controls (p=0.042) but the differences became non-significant after adjusting for covariates. Although a consistent pattern was seen whereby the rATL were 2-6% shorter in the exposed samples compared to the unexposed ones, this difference never reached statistical significance. In the SJ but not the Pregnancy cohort, smoking and illicit drug use in pregnancy were associated with shorter infant (p=0.033) and maternal (p=0.035) blood rATL. In the pregnancy cohort, among the HIV⁺ HAART-exposed, higher CD4+ count (p=0.047) and longer HIV duration (p=0.016) were independently associated with shorter maternal rATL. Maternal and infant rATL were significantly correlated (r=0.43, p=0.002) in the pregnancy but not in the SJ cohort.Conclusion: These results suggest that, if HIV and/or ART/HAART is a risk for telomere attrition in the context of this study, it is less important than other recognized risks.
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Blood mitochondrial DNA mutations in HIV-infected women and their infants exposed to HAART during pregnancy (2010)
Background/Objectives: Nucleoside reverse transcriptase inhibitors (NRTIs) as part of highly active antiretroviral therapy (HAART) are given to human immunodeficiency virus (HIV)-infected pregnant women to prevent HIV vertical transmission. NRTIs can adversely affect mitochondrial DNA (mtDNA) and may induce mtDNA point mutations. We hypothesised 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 infants exposed in utero to HIV/HAART and controls (0-6d), as well as from a subset of their mothers (last visit before delivery). MtDNA mutation burden was measured by an assay involving cloning and sequencing mtDNA D-loop PCR amplicons. The presence of transversion mutations A → C/T → G (AC/TG) was analysed by Chi-squared and Wilcoxon signed-rank tests. Relationships with amount of DNA assayed, maternal age, smoking (marijuana/cigarettes) and illicit drug/methadone use in pregnancy were examined. For the HIV/HAART group, relationships with CD4+ count and HIV plasma viral load (pVL) near delivery, as well as length of HAART exposure were also examined.Results: The Taq error rate from PCR caused a low signal (mutation) to noise (background) ratio. Therefore, only AC/TG mutations, not induced under our assay conditions, were analysed. 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 (p=0.058) controlling for covariates, although a trend was observed. Furthermore, significantly more HIV/HAART-exposed mothers (N=18/42, 42.9%) harboured AC/TG mutations compared to controls (N=7/39, 17.9%) both 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 (p=0.777). No difference was observed between control mothers and their infants (N=39, both 17.9%). In HIV/HAART-exposed group mothers, only a detectable HIV pVL near delivery predicted AC/TG mutations.Conclusion:A subset of mtDNA mutations can be quantified with the developed assay. HIV/HAART exposure in pregnancy may be associated with increased prevalence of maternal mtDNA mutations. Since mtDNA mutations have been linked with aging and age-associated diseases, this raises concerns about the long-term impact of HAART.
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