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1.  Voriconazole Metabolism, Toxicity, and the Effect of Cytochrome P450 2C19 Genotype 
The Journal of Infectious Diseases  2014;209(12):1941-1948.
Background. Prospective evaluation of the antifungal drug, voriconazole, is needed to determine whether drug toxicity correlates with CYP2C19 genotype or serum concentrations of voriconazole or its metabolites.
Methods. We conducted a prospective study of 95 patients to determine voriconazole toxicity and its relationship to genotype and serum levels of voriconazole and its two metabolites. Efficacy was not evaluated because, in most cases, the drug was given for empirical or prophylactic therapy.
Results. Hallucinations occurred in 16 patients (16.8%), visual changes in 17 (17.9%), photosensitivity in 10 (10.5%), and hepatotoxicity in 6 (6.3%). There was no correlation between photosensitivity or hepatotoxicity and levels of voriconazole or metabolites. Patients with hallucinations had higher average voriconazole levels (4.5 vs 2.5 μg/mL) but with extensive overlap. The recommended oral dose of 200 mg did not provide consistently detectable serum voriconazole levels in adults. CYP2C19 and CYP2C9 genotypes had a minor influence over levels, though the 4 patients homozygous for the 2C19*2 genotype had higher average levels for voriconazole (4.3 vs 2.5 μg/mL) and lower N-oxide levels (1.6 vs 2.5 μg/mL).
Conclusions. CYP2C19 and 2C9 genotypes were not major determinants of voriconazole metabolism. No toxic serum level of voriconazole or its metabolites could be identified.
PMCID: PMC4038142  PMID: 24403552
voriconazole; metabolites; CYP2C19; toxicity
2.  Short Communication: HIV Blips While on Antiretroviral Therapy Can Indicate Consistently Detectable Viral Levels Due to Assay Underreporting 
AIDS Research and Human Retroviruses  2013;29(12):1621-1625.
Viral blips, where HIV RNA plasma viral load (pVL) intermittently increases above the lower limit of assay detection, are a cause for concern. We investigated a number of hypotheses for their cause. We assessed HIV RNA, and total and episomal HIV DNA from 16 individuals commencing antiretroviral therapy (ART) consisting of raltegravir and tenofovir/emtricitabine for 3 years, using two assays: a single-copy assay [SCA; lower limit of quantification (LLOQ), <1 copy/ml] and the Amplicor assay (LLOQ of 50 copies/ml). Two individuals exhibited viral blips. From week 20 onward, the period where ART had achieved its final suppressive levels, pVL ranged from <1 to 330 copies/ml, except for one individual at the final time. Both assays were 98% consistent (108/110) in assessing pVL <50 copies/ml, but the Amplicor assay registered 56% of samples (19/34) as below the LLOQ that were in the 50 to 1000 copy/ml range as quantified by SCA. pVL changes between successive time points did not correlate with changes in cellular infection as measured through either total or episomal HIV DNA. Changes in pVL were correlated (negatively) with changes in total CD4+ T cell numbers (p=0.003), naive (CD45RO–CD62L+CD4+), natural regulatory (CD45RO–CD25+CD127–CD4+), activated effector (CD45RO+CD38++CCR5+CD8+), but not activated (CD38+HLA-DR+) CD4+ T cells. Patients receiving stable, seemingly suppressive ART can have pVL near the 50 copy LLOQ at multiple time points. The high Amplicor assay error rate around this level implies that viral blips underrepresent pVL being more consistently above the LLOQ. Activation of latently infected cells is less likely to contribute to this phenomenon.
PMCID: PMC3848484  PMID: 23844947
3.  Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells 
Journal of virological methods  2013;193(1):184-189.
Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells.
PMCID: PMC3760681  PMID: 23773807
2-LTR; HIV; Episome; Mitochondrial DNA; Retrovirus; PCR
4.  Decreases in Colonic and Systemic Inflammation in Chronic HIV Infection after IL-7 Administration 
PLoS Pathogens  2014;10(1):e1003890.
Despite antiretroviral therapy (ART), some HIV-infected persons maintain lower than normal CD4+ T-cell counts in peripheral blood and in the gut mucosa. This incomplete immune restoration is associated with higher levels of immune activation manifested by high systemic levels of biomarkers, including sCD14 and D-dimer, that are independent predictors of morbidity and mortality in HIV infection. In this 12-week, single-arm, open-label study, we tested the efficacy of IL-7 adjunctive therapy on T-cell reconstitution in peripheral blood and gut mucosa in 23 ART suppressed HIV-infected patients with incomplete CD4+ T-cell recovery, using one cycle (consisting of three subcutaneous injections) of recombinant human IL-7 (r-hIL-7) at 20 µg/kg. IL-7 administration led to increases of both CD4+ and CD8+ T-cells in peripheral blood, and importantly an expansion of T-cells expressing the gut homing integrin α4β7. Participants who underwent rectosigmoid biopsies at study baseline and after treatment had T-cell increases in the gut mucosa measured by both flow cytometry and immunohistochemistry. IL-7 therapy also resulted in apparent improvement in gut barrier integrity as measured by decreased neutrophil infiltration in the rectosigmoid lamina propria 12 weeks after IL-7 administration. This was also accompanied by decreased TNF and increased FOXP3 expression in the lamina propria. Plasma levels of sCD14 and D-dimer, indicative of systemic inflammation, decreased after r-hIL-7. Increases of colonic mucosal T-cells correlated strongly with the decreased systemic levels of sCD14, the LPS coreceptor - a marker of monocyte activation. Furthermore, the proportion of inflammatory monocytes expressing CCR2 was decreased, as was the basal IL-1β production of peripheral blood monocytes. These data suggest that administration of r-hIL-7 improves the gut mucosal abnormalities of chronic HIV infection and attenuates the systemic inflammatory and coagulation abnormalities that have been linked to it.
Author Summary
HIV infected people who receive antiretroviral therapy (ART) remain at higher risk of non-infectious complications such as cardiovascular disease. This risk is linked to persistent inflammation and immune activation and is higher in those with lower circulating CD4+ T-cell counts. IL-7 therapy can increase CD4+ and CD8+ T-cell counts in peripheral blood, but its effects on gut mucosal T cell restoration was unknown. We gave 3 doses of interleukin (IL)-7 to HIV+ persons receiving ART and performed gut biopsies before and after therapy. IL-7 therapy increased T-cell numbers in the periphery including T-cells that express the α4β7 integrin, a molecule that promotes trafficking to the gut. Gut mucosa studies showed significant increases of T-cells and significant decreases in local inflammation as reflected by expression of myeloperoxidase and tumor necrosis factor. In addition, plasma levels of inflammatory markers that are linked to mortality in HIV infection decreased significantly as did some inflammatory monocyte markers. We conclude that IL-7 administration can improve gut mucosal abnormalities in chronic treated HIV infection and as a result, also may decrease both gut and systemic inflammation.
PMCID: PMC3907377  PMID: 24497828
5.  Negative regulation of human U6 snRNA promoter by p38 kinase through Oct-1 
Gene  2012;497(2):200-207.
Recruitment of Oct-1 protein to the octamer sequence of U6 promoter is critical for optimal transcription by RNA polymerase III. Here we report that p38 kinase inhibitors, SB202190 and SB203580, stimulated U6 promoter activity and this stimulation can be observed only in the presence of octamer sequence. SB202190-treated cell nuclear extract had about 50% increase in Oct-1 binding activity suggesting that the increased U6 promoter activity by p38 kinase inhibitor is mediated through Oct-1. Mutation in octamer sequence significantly reduced the SB202190-stimulated U6 promoter transcription and the distance between octamer and proximal sequence element of U6 promoter is also critical for the p38 kinase inhibitor-stimulated activity. Exogenous Oct-1 expression showed a concentration-dependent activation of U6 promoter that was further stimulated by the p38 kinase inhibitors. When cells were treated with p38 kinase inducer, hydrogen peroxide or phorbol 12-myristate 13-acetate (PMA), U6 promoter activity was down regulated and this inhibition was reversed by p38 kinase inhibitors. Over-expression of p38α kinase down-regulated U6 promoter activity and this inhibition was further enhanced by PMA and p38 kinase inhibitors reversed this inhibition. p38 kinase inhibitor-treated cells had 50% more U6 RNA than the control cells. Taken together, our results show a negative correlation between the p38 kinase levels and Oct-1 binding on U6 promoter, suggesting that U6 promoter is negatively regulated by p38 kinase.
PMCID: PMC3306512  PMID: 22310390
U6 promoter; Oct-1; p38 kinase; SB202190; MAPK
6.  Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T cells by up-regulating ABCA1-mediated cholesterol efflux 
Retinoic acids regulate the reverse cholesterol transport by inducing the ATP binding cassette transporter A1 (ABCA1) dependent cholesterol efflux in macrophages, neuronal as well as intestine cells. In the present study, we aim to test the effect of all trans retinoic acid (ATRA) on ABCA1 expression in human CD4+ T cells and the involvement of cholesterol in ATRA mediated anti-HIV effect.
Treatment with ATRA dramatically up-regulated ABCA1 expression in CD4+ T cells in a time and dose dependent manner. The expression of ABCA1 paralleled with increased ABCA1-dependent cholesterol efflux. This induction was dependent on T cell receptor (TCR) signaling and ATRA failed to induce ABCA1 expression in resting T cells. Moreover, ATRA and liver X receptor (LXR) agonist-TO-901317 together had synergistic effect on ABCA1 expression as well as cholesterol efflux. Increased ABCA1 expression was associated with lower cellular cholesterol staining. Cells treated with either ATRA or TO-901317 were less vulnerable to HIV-1 infection. Combination of retinoic acid and TO-901317 further inhibited HIV-1 entry and their inhibitory effects could be reversed by cholesterol replenishment.
ABCA1 RNA and protein were determined by real-time PCR and immuno blot methods in cells treated with ATRA. Cholesterol efflux rate was measured in cells treated with ATRA and TO-901317.
ATRA up-regulates ABCA1 expression and cholesterol efflux in CD4+ T cells and combination of ATRA and liver X receptor (LXR) agonist further enhanced these effects. Increased cholesterol efflux contributed to reduced HIV-1 entry, suggesting that anti-HIV effect of ATRA is mediated through ABCA1.
PMCID: PMC3391983  PMID: 22676378
ABCA1; ATRA; retinoic acid; TO-901317; RAR; RXR; LXR; cholesterol efflux; HIV-1; CD4+ T cells
8.  LIM kinase 1 - dependent cofilin 1 pathway and actin dynamics mediate nuclear retinoid receptor function in T lymphocytes 
BMC Molecular Biology  2011;12:41.
It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation.
Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1.
We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.
PMCID: PMC3187726  PMID: 21923909
9.  Traditional risk factors and D-dimer predict incident cardiovascular disease events in chronic HIV infection 
AIDS (London, England)  2010;24(10):1509-1517.
Cardiovascular disease (CVD) contributes significantly to HIV-related morbidity and mortality. Chronic immune activation and inflammation are thought to augment the progression of atherosclerotic disease. In this retrospective, case-control study of HIV-infected subjects, we investigated the association of traditional cardiac risk factors, HIV-related disease and inflammation with CVD events.
HIV-infected subjects who experienced an incident CVD event while enrolled in National Institutes of Health clinical protocols from 1995-2009 were matched 2:1 to HIV-infected subjects without known CVD. Markers of inflammation and cell activation were measured in serum or plasma using ELISA-based assays and peripheral mononuclear cells by four-color flow cytometry.
Fifty-two patients experienced an incident CVD event. Events were related to smoking, dyslipidemia, hyperglycemia and family history as well as D-dimer, sVCAM-1, TIMP-1, and soluble tissue factor but not hsCRP. No significant differences in antiviral therapy, CD4+ T-cell count or CD38 and HLA-DR expression were identified between cases and controls. In multivariable analysis, smoking, family history, D-dimer, and glucose were independently related to CVD risk.
In this cohort, CVD risk was related to traditional CVD risk factors and markers of thrombosis and endothelial damage but not to hsCRP or markers of T-cell activation such as CD38+/HLA-DR coexpression. D-dimer may help identify HIV-infected patients at elevated CVD risk.
PMCID: PMC2884071  PMID: 20505494
HIV; cardiovascular disease; myocardial infarction; smoking; D-dimer; tissue factor; VCAM-1
10.  Lack of sex-related differences in saquinavir pharmacokinetics in an HIV-seronegative cohort 
To examine the influence of sex on steady-state saquinavir pharmacokinetics in HIV-seronegative volunteers administered saquinavir without a concomitant protease inhibitor.
Thirty-eight healthy volunteers (14 female) received saquinavir soft-gel capsules 1200 mg three times daily for 3 days to achieve steady-state conditions. Following administration of the 10th dose, blood was collected serially over 8 h for measurement of saquinavir plasma concentrations. Saquinavir pharmacokinetic parameter values were determined using noncompartmental methods and compared between males and females. CYP3A phenotype (using oral midazolam) and MDR-1 genotypes at positions 3435 and 2677 were determined for all subjects in order to characterize possible mechanisms for any observed sex-related differences.
There was no significant difference in saquinavir AUC0−8 or any other pharmacokinetic parameter value between the sexes. These findings persisted after mathematically correcting for total body weight. The mean weight-normalized AUC0−8 was 29.9 (95% confidence interval 15.5, 44.3) and 29.8 (18.6, 40.9) ng h−1 ml−1 kg−1 for males and females, respectively. No significant difference in CYP3A phenotype was observed between the groups; likewise, the distribution of MDR-1 genotypes was similar for males and females.
In contrast to previous study findings, results from this investigation showed no difference in saquinavir pharmacokinetics between males and females. The discrepancy between our findings and those previously reported may be explained by the fact that we evaluated HIV-seronegative volunteers and administered saquinavir in the absence of concomitant protease inhibitors such as ritonavir. Caution must be exercised when extrapolating pharmacokinetic data from healthy volunteer studies (including sex-based pharmacokinetic differences) to HIV-infected populations or to patients receiving additional concurrent medications.
PMCID: PMC1885032  PMID: 16542198
cytochrome P450; HIV; P-glycoprotein; pharmacokinetics; saquinavir; sex
11.  Naïve T-Cell Dynamics in Human Immunodeficiency Virus Type 1 Infection: Effects of Highly Active Antiretroviral Therapy Provide Insights into the Mechanisms of Naïve T-Cell Depletion 
Journal of Virology  2006;80(6):2665-2674.
Both naïve CD4+ and naïve CD8+ T cells are depleted in individuals with human immunodeficiency virus type 1 (HIV-1) infection by unknown mechanisms. Analysis of their dynamics prior to and after highly active antiretroviral therapy (HAART) could reveal possible mechanisms of depletion. Twenty patients were evaluated with immunophenotyping, intracellular Ki67 staining, T-cell receptor excision circle (TREC) quantitation in sorted CD4 and CD8 cells, and thymic computed tomography scans prior to and ∼6 and ∼18 months after initiation of HAART. Naïve T-cell proliferation decreased significantly during the first 6 months of therapy (P < 0.01) followed by a slower decline. Thymic indices did not change significantly over time. At baseline, naïve CD4+ T-cell numbers were lower than naive CD8+ T-cell numbers; after HAART, a greater increase in naïve CD4+ T cells than naïve CD8+ T cells was observed. A greater relative change (n-fold) in the number of TREC+ T cells/μl than in naïve T-cell counts was observed at 6 months for both CD4+ (median relative change [n-fold] of 2.2 and 1.7, respectively; P < 0.01) and CD8+ T cell pools (1.4 and 1.2; P < 0.01). A more pronounced decrease in the proliferation than the disappearance rate of naïve T cells after HAART was observed in a second group of six HIV-1-infected patients studied by in vivo pulse labeling with bromodeoxyuridine. These observations are consistent with a mathematical model where the HIV-1-induced increase in proliferation of naïve T cells is mostly explained by a faster recruitment into memory cells.
PMCID: PMC1395465  PMID: 16501076
12.  In vivo expansion of CD4+CD45RO–CD25+ T cells expressing foxP3 in IL-2-treated HIV-infected patients 
Journal of Clinical Investigation  2005;115(7):1839-1847.
Administration of IL-2 to HIV-infected patients leads to expansion of a unique subset of CD4+CD45RO–CD25+ cells. In this study, the origin, clonality, and function of these cells were investigated. Analysis of TCR excision circles revealed that the CD4+CD45RO–CD25+ cells were the product of peripheral expansion but remained polyclonal as determined by TCR repertoire analysis. Phenotypically, these cells were distinct from naturally occurring Tregs; they exhibited intermediate features, between those of memory and naive cells, and had lower susceptibility to apoptosis than CD45RO–CD25– or memory T cells. Studies of intracellular cytokine production and proliferation revealed that cytokine-expanded naive CD25+ cells had low IL-2 production and required costimulation for proliferation. Despite elevated expression of forkhead transcription factor P3 (foxP3), they exerted only weak suppression compared with CD45RO+CD25+high cells (Tregs). In summary, in vivo IL-2 administration to HIV-infected patients leads to peripheral expansion of a population of long-lived CD4+CD45RO–CD25+ cells that express high levels of foxP3 but exert weak suppressive function. These CD4+CD25+ cytokine-expanded naive cells, distinct from antigen-triggered cells and Tregs, play a role in the maintenance of a state of low turnover and sustained expansion of the CD4+ T cell pool.
PMCID: PMC1142113  PMID: 15937547

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