Hélène C. F. Côté

Human Immunodeficiency Virus Type 1 Protease Cleavage Site Mutations Associated with Protease Inhibitor Cross-Resistance Selected by Indinavir, Ritonavir, and/or Saquinavir

ABSTRACT We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6 gag ) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A→V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.

Mitochondrial toxicity in the era of HAART: evaluating venous lactate and peripheral blood mitochondrial DNA in HIV-infected patients taking antiretroviral therapy.

Nucleoside analogs can induce mitochondrial toxicity by inhibiting the human DNA polymerase γ. This can lead to a wide range of clinical toxicities, from asymptomatic hyperlactatemia to death. Despite their technical and physiological variability, we propose that random venous lactate measurements can be useful to monitor the development of nucleoside-related mitochondrial toxicity. Recently, we have developed an assay that can measure changes in mitochondrial DNA levels in peripheral blood cells. Using this assay we have characterized changes in mitochondrial DNA (mtDNA) relative to nuclear DNA (nDNA) in peripheral blood cells of patients with symptomatic nucleoside-induced hyperlactatemia. Our results demonstrate that symptomatic hyperlactatemia was associated with markedly low mtDNA/nDNA ratios, which were on average 69% lower than HIV-uninfected controls and 45% lower than HIV-infected asymptomatic/antiretroviral naïve controls. A statistically significant (p = .016) increase in mtDNA/nDNA ratio was observed following discontinuation of antiretroviral therapy. The mtDNA/nDNA ratio remained stable among selected patients who reintroduced antiretroviral therapy with stavudine (d4T)-sparing regimens. Of note, the decline in mtDNA preceded the increase in venous lactate levels. More recently we have evaluated changes in the mtDNA/nDNA ratio in relation to selected antiretroviral drug regimens in a cross-sectional study on a non-random sample of participants within the British Columbia Centre for Excellence in HIV/AIDS Drug Treatment Program. Eligible patients had continuously received saquinavir plus ritonavir with either nevirapine (n = 20), lamivudine (n = 15), d4T (n = 53) or lamivudine + d4T (n = 69), for 4 to 30 months. d4T-sparing regimens were associated with a higher median mtDNA/nDNA ratio than d4T-containing regimens (p = .016), despite the fact that study patients had received d4T-containing regimens for a shorter median time than patients taking d4T-sparing regimens (13 versus 25 months, p = .002). In summary, mtDNA levels are significantly decreased among patients who develop symptomatic, nucleoside-related hyperlactatemia, an effect reversed upon therapy discontinuation. Furthermore, mtDNA/nDNA ratios were statistically significantly lower in patients taking d4T-containing regimens than in those taking selected d4T-sparing regimens in a population setting. These results suggest that measurement of this parameter should be investigated as a potential clinical management tool.

Functional Characterization of Recombinant Human Meizothrombin and Meizothrombin(desF1) THROMBOMODULIN-DEPENDENT ACTIVATION OF PROTEIN C AND THROMBIN-ACTIVATABLE FIBRINOLYSIS INHIBITOR (TAFI), PLATELET AGGREGATION, ANTITHROMBIN-III INHIBITION

Recombinant human prothrombin (rII) and two mutant forms (R155A,R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M−1·min−1) for inhibition by antithrombin III (AT-III) were 2.44 × 105 (rIIa), 6.10 × 104 (rMZa), and 1.05 × 105 (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

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.

Decreased skeletal muscle mitochondrial DNA in patients with statin-induced myopathy

Statins are widely used to treat hyperlipidemia and lower cardiovascular disease risk. While statins are generally well tolerated, some patients experience statin-induced myopathy (SIM). Statin treatment has been associated with mitochondrial dysfunction and mitochondrial DNA (mtDNA) depletion. In this retrospective study, skeletal muscle biopsies from patients diagnosed with SIM were studied. These were compared with biopsies from patients clinically assessed as having statin-unrelated myopathy but whose biopsy showed no or negligible pathology. For each biopsy sample, mtDNA was quantified relative to nuclear DNA (mtDNA content) by qPCR, mtDNA deletions were investigated by long-template PCR followed by gel densitometry, and mtDNA oxidative damage was quantified using a qPCR-based assay. For a subset of matched samples, mtDNA heteroplasmy and mutations were investigated by cloning/sequencing. Skeletal muscle mtDNA content was significantly lower in SIM patients (N=23, mean±SD, 2036±1146) than in comparators (N=24, 3220±1594), p=0.006. There was no difference in mtDNA deletion score or oxidative mtDNA damage between the two groups, and no evidence of increased mtDNA heteroplasmy or somatic mutations was detected. The significant difference in skeletal muscle mtDNA suggests that SIM or statin treatments are associated with depletion of skeletal muscle mtDNA or that patients with an underlying predisposition to SIM have lower mtDNA levels. If statins induce mtDNA depletion, this would likely reflect decreased mitochondria biogenesis and/or increased mitochondria autophagy. Further work is necessary to distinguish between the lower mtDNA as a predisposition to SIM or an effect of SIM or statin treatment.

Lopinavir/ritonavir plus nevirapine as a nucleoside-sparing approach in antiretroviral-experienced patients (NEKA study).

Objectives: To compare the efficacy and safety of a nucleoside-sparing approach with a conventional highly active antiretroviral therapy (HAART) regimen in antiretroviral-experienced patients with prolonged viral suppression. Methods: Pilot study including 31 antiretroviral-experienced patients with HIV RNA <80 copies/mL. Subjects were randomly assigned to lopinavir/ritonavir (LPV/rtv) 400/100 mg BID plus nevirapine (NVP) 200 mg BID (NVP group, n = 16) or LPV/rtv plus the 2 previous NRTIs (NRTI group, n = 15). The primary endpoint was the percentage of subjects who maintained viral suppression at week 48. Changes in lipid metabolism, mitochondrial parameters, and LPV trough levels were also assessed. Results: All patients maintained viral suppression after 48 weeks. No subject discontinued therapy because of adverse events. HDL cholesterol increased by 28% at week 24 (P < 0.0001) and 10% after 48 weeks of follow-up (P = 0.319) in the NVP group. In the NRTI group, LDL cholesterol increased by 14% at week 48 (P = 0.076). Mitochondrial DNA/nuclear DNA ratio and mitochondrial respiratory chain complex IV activity showed a trend toward increasing in the NVP group. Mean (SD) LPV trough levels were 6340 (2129) ng/mL in the NRTI group and 5161 (2703) ng/mL in the NVP group (P = 0.140). Conclusions: In antiretroviral-experienced subjects with sustained viral suppression, dual therapy with NVP plus LPV/rtv at standard dosage was as potent and safe as standard-of-care HAART at 48 weeks of follow-up. This approach may reduce mitochondrial toxicity and improve LPV/rtv-associated lipid abnormalities. The results of this pilot study support the study of this approach in a larger, randomized trial.

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.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.

Nucleoside-Related Mitochondrial Toxicity among HIV-Infected Patients Receiving Antiretroviral Therapy: Insights from the Evaluation of Venous Lactic Acid and Peripheral Blood Mitochondrial DNA

Nucleoside analogues inhibit human DNA polymerase gamma. As a result, they can produce mitochondrial toxicity. We evaluated the possible role of random venous lactic-acid determinations as a screening tool for mitochondrial toxicity among patients receiving nucleoside therapy. More recently, we have developed an assay that can detect changes in mitochondrial DNA (mtDNA) levels in peripheral blood cells. Using this assay, we have characterized changes in mtDNA relative to nuclear DNA (nDNA) in peripheral blood cells from patients with symptomatic nucleoside-induced hyperlactatemia. Our results demonstrated that symptomatic hyperlactatemia was associated with markedly low mtDNA : nDNA ratios. A statistically significant increase in the mtDNA : nDNA ratio was observed after the discontinuation of antiretroviral therapy. Full validation of monitoring the mtDNA : nDNA ratio is currently under way.