T-cell receptor activator of nuclear factor-κB ligand/osteoprotegerin imbalance is associated with HIV-induced bone loss in patients with higher CD4+ T-cell counts.

OBJECTIVE: Higher incidence of osteopenia and osteoporosis underlie increased rates of fragility fracture in HIV infection. B cells are a major source of osteoprotegerin (OPG), an inhibitor of the key osteoclastogenic cytokine receptor activator of nuclear factor-κB ligand (RANKL). We previously showed that higher B-cell RANKL/OPG ratio contributes to HIV-induced bone loss. T-cell OPG production in humans, however, remains undefined and the contribution of T-cell OPG and RANKL to HIV-induced bone loss has not been explored. DESIGN: We investigated T-cell OPG and RANKL production in ART-naive HIV-infected and uninfected individuals in relation to indices of bone loss in a cross-sectional study. METHODS: T-cell RANKL and OPG production was determined by intracellular staining and flow cytometry, and plasma levels of bone resorption markers were determined by ELISA. RESULTS: We demonstrate for the first time in-vivo human T-cell OPG production, which was significantly lower in HIV-infected individuals and was coupled with moderately higher T-cell RANKL production, resulting in a significantly higher T-cell RANKL/OPG ratio. T-cell RANKL/OPG ratio correlated significantly with BMD-derived z-scores at the hip, lumbar spine and femur neck in HIV-infected individuals with CD4 T-cell counts at least 200 cells/µl but not in those with lower counts. CONCLUSION: Our data suggest that T cells may be a physiologically relevant source of OPG and T-cell RANKL/OPG imbalance is associated with HIV-induced bone loss in CD4 T-cell-sufficient patients. Both B and T lymphocytes may thus contribute to HIV-induced bone loss.

Antiretroviral therapy induces a rapid increase in bone resorption that is positively associated with the magnitude of immune reconstitution in HIV infection.

OBJECTIVE: Antiretroviral therapy (ART) paradoxically intensifies bone loss in the setting of HIV infection. Although the extent of bone loss varies, it occurs with virtually all ART types, suggesting a common pathway that may be aligned with HIV disease reversal. Using an animal model of immunodeficiency we recently demonstrated that immune activation associated with CD4 T-cell reconstitution induces increased production of the osteoclastogenic cytokines RANKL and TNFα by immune cells, driving enhanced bone resorption and loss in bone mineral density. DESIGN: To confirm these findings in humans, we investigated the early kinetics of CD4 T-cell recovery in relation to biomarkers of bone turnover and osteoclastogenic regulators in a prospective 24-week cohort study. METHODS: Clinical data and blood sampling for HIV-RNA PCR, CD4 T-cell counts, bone turnover biomarkers, and osteoclastogenic regulators were obtained from ART-naïve HIV-infected study participants initiating standard doses of lopinavir/ritonavir plus tenofovir disoproxil fumarate/emtricitabine at baseline and at weeks 2, 8, 12, and 24 post ART. RESULTS: C-terminal telopeptide of collagen (CTx) a sensitive biomarker of bone resorption rose by 200% above baseline at week 12, remaining elevated through week 24 (α<0.01), and was associated with significant increases in plasma levels of osteoclastogenic regulators [receptor activator of NF-kB ligand (RANKL), tumor necrosis factor alpha, (TNFα)]. Importantly, the magnitude of CD4 T-cell recovery correlated significantly with CTx (rs = 0.387, α=0.01). CONCLUSION: Our data suggest that ART-induced bone loss occurs early, is aligned with early events of immune reconstitution, and these immune changes provide a unifying mechanism to explain in part the skeletal decline common to all ART.

Dysregulated B cell expression of RANKL and OPG correlates with loss of bone mineral density in HIV infection.

HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.

Oral sampling and human papillomavirus genotyping in HIV-infected patients.

BACKGROUND: Oral human papillomavirus (HPV) is associated with several health complications especially in combination with HIV infections. Screening may be useful, but methodologies and results have varied widely in previous studies. We conducted a pilot study in an HIV-positive population to evaluate HPV detection in four different oral sample types. METHODS: Upon enrollment, an oral-rinse (OR) sample was collected in 10 ml saline. Additional samples of the buccal mucosa, tonsils, and oral lesion if present were collected with cytology brushes. DNA was extracted using LC-MagNAPure, and the Linear Array HPV genotyping Assay (Roche) was used for HPV genotyping. RESULTS: In samples from 100 HIV-positive participants, HPV was detected in 39 (%) of the oral rinses, 13 (%) mucosal and 11 (12.9%) tonsil brushings. Of seven lesion brushings collected, four were HPV positive. All participants with HPV detected in mucosal, tonsil, or lesion brushings were also positive in the OR sample. Among the rinse samples, 27 different genotypes were detected with HPV84 (n = 6), HPV55 (n = 5), and HPV83 (n = 5) being the most common. Multiple infections were detected in 17 samples (range 2-9, mean 1.9 types). As potential cofactors, only receptive oral sex was significantly associated with HPV (P = 0.018, odds ratio 2.9, 95% CI 1.2-6.9). CONCLUSION: Sampling is a significant factor for oral prevalence studies. Oral rinse provides the best representation for HPV in the oral cavity. To evaluate associated cofactors other than receptive oral sex, larger studies with case-control design are necessary.

Immune activation mediated change in alpha-1-acid glycoprotein: impact on total and free lopinavir plasma exposure.

BACKGROUND: Immune mediated changes in circulating α-1-acid glycoprotein (AAG), a type 1 acute phase protein, which binds protease inhibitors (PI), may alter protein binding and contribute to PI's pharmacokinetic (PK) variability. METHODS: In a prospective, 2-phase intensive PK study on antiretroviral naive human immunodeficiency virus (HIV)-infected subjects treated with a lopinavir-/ritonavir-based regimen, steady state PK sampling and AAG assays were performed at weeks 2 and 16 of treatment. RESULTS: Median entry age was 43 years (n = 16). Median plasma log(10) HIV-1 RNA, CD4 T-cell counts, and AAG were 5.16 copies/mL, 28 cells/µL, and 143 mg/dL, respectively.The total lopinavir area under the concentration time curve (AUC(12_total)) and maximum concentration (C(max_total)) changed linearly with AAG at mean rates of 16±7 mg*hr/L (slope ± SE); P = .04, and 1.6 ± 0.6 mg/L, P = .02, per 100 mg/dL increase in AAG levels, respectively (n = 15).A 29% drop in AAG levels between week 2 and week 16 was associated with 14% (geometric mean ratio [GMR] = 0.86; 90% confidence interval [CI] = 0.74-0.98) and 13% (GMR = 0.87; 90% CI = 0.79-0.95) reduction in AUC(12_total) and C(max_total), respectively. Neither free lopinavir PK parameters nor antiviral activity (HIV-1 RNA average AUC minus baseline) was affected by change in plasma AAG. CONCLUSIONS: Changes in plasma AAG levels alter total lopinavir concentrations, but not the free lopinavir exposure or antiviral activity. This observation may have implications in therapeutic drug monitoring.

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection.

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8(+) T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8(+) T cells during the acute and chronic stages of infection. Although HCV-specific CD8(+) T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8(+) T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8(+) T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the "death phase" of HCV-specific CD8(+) T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.

Raltegravir with optimized background therapy for resistant HIV-1 infection.

BACKGROUND: Raltegravir (MK-0518) is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase active against HIV-1 susceptible or resistant to older antiretroviral drugs. METHODS: We conducted two identical trials in different geographic regions to evaluate the safety and efficacy of raltegravir, as compared with placebo, in combination with optimized background therapy, in patients infected with HIV-1 that has triple-class drug resistance in whom antiretroviral therapy had failed. Patients were randomly assigned to raltegravir or placebo in a 2:1 ratio. RESULTS: In the combined studies, 699 of 703 randomized patients (462 and 237 in the raltegravir and placebo groups, respectively) received the study drug. Seventeen of the 699 patients (2.4%) discontinued the study before week 16. Discontinuation was related to the study treatment in 13 of these 17 patients: 7 of the 462 raltegravir recipients (1.5%) and 6 of the 237 placebo recipients (2.5%). The results of the two studies were consistent. At week 16, counting noncompletion as treatment failure, 355 of 458 raltegravir recipients (77.5%) had HIV-1 RNA levels below 400 copies per milliliter, as compared with 99 of 236 placebo recipients (41.9%, P<0.001). Suppression of HIV-1 RNA to a level below 50 copies per milliliter was achieved at week 16 in 61.8% of the raltegravir recipients, as compared with 34.7% of placebo recipients, and at week 48 in 62.1% as compared with 32.9% (P<0.001 for both comparisons). Without adjustment for the length of follow-up, cancers were detected in 3.5% of raltegravir recipients and in 1.7% of placebo recipients. The overall frequencies of drug-related adverse events were similar in the raltegravir and placebo groups. CONCLUSIONS: In HIV-infected patients with limited treatment options, raltegravir plus optimized background therapy provided better viral suppression than optimized background therapy alone for at least 48 weeks. (ClinicalTrials.gov numbers, NCT00293267 and NCT00293254.)

Pharmacokinetics of an indinavir-ritonavir-fosamprenavir regimen in patients with human immunodeficiency virus.

STUDY OBJECTIVE: To evaluate the pharmacokinetic compatibility of a ritonavir-boosted indinavir-fosamprenavir combination among patients with human immunodeficiency virus (HIV). DESIGN: Single-center, nonrandomized, prospective, multiple-dose, two-phase pharmacokinetic study. SETTING: University research center. PATIENTS: Eight adult patients with HIV infection who had been receiving and tolerating indinavir 800 mg-ritonavir 100 mg twice/day for at least 2 weeks. Intervention. After 12-hour pharmacokinetic sampling was performed on all patients (period A), fosamprenavir (a prodrug of amprenavir) 700 mg twice/day was coadministered for 5 days, with a repeat 12-hour pharmacokinetic sampling performed on the fifth day (period B). MEASUREMENTS AND MAIN RESULTS: Pharmacokinetic parameters were determined for indinavir, ritonavir, and amprenavir: area under the concentration-time curve from time 0 to 12 hours after dosing (AUC(0-12)), maximum plasma concentration (C(max)), and 12-hour plasma concentration (C(12)). For each parameter, the geometric mean, as well as the geometric mean ratio (GMR) comparing period B with period A, were calculated. Indinavir C(max) was lowered by 20% (GMR 0.80, 95% confidence interval [CI] 0.67-0.96), AUC(0-12) was lowered by 6% (GMR 0.94, 95% CI 0.74-1.21), and C(12) was increased by 28% (GMR 1.28, 95% CI 0.78-2.10). Ritonavir AUC(0-12) was 20% lower (GMR 0.80, 95% CI 0.54-1.19), C(max) was 15% lower (GMR 0.85, 95% CI 0.55-1.32), and C(12) was 7% lower (GMR 0.93, 95% CI 0.49-1.76). With the exception of indinavir C(max), the changes in indinavir and ritonavir pharmacokinetic parameters observed after fosamprenavir coadministration were not statistically significant. The geometric means of amprenavir AUC(0-12), C(max), and C(12) were 41,517 ng*hour/ml (95% CI 30,317-56,854 ng*hr/ml), 5572 ng/ml (95% CI 4330-7170 ng/ml), and 2421 ng/ml (95% CI 1578-3712 ng/ml), respectively. CONCLUSION: The combination of indinavir 800 mg-ritonavir 100 mg-fosamprenavir 700 mg twice/day appears to be devoid of a clinically significant drug-drug interaction and should be evaluated as an alternative regimen in salvage HIV treatment. This combination may be suitable as part of a background regimen to optimize the therapeutic benefit of newer classes of antiretroviral agents such as the integrase and coreceptor inhibitors in the treatment of multidrug-resistant viruses.

Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status.

Most human immunodeficiency virus type 1 (HIV-1) infections are believed to be the result of exposure to the virus in genital secretions. However, prevention and therapeutic strategies are usually based on characterizations of HIV-1 in blood. To understand better the dynamics between HIV-1 quasispecies in the genital tract and blood, we performed heteroduplex assays on amplified env products from cell-free viral RNA in paired vaginal secretion (VS) and blood plasma (BP) samples of 14 women followed for 1.5 to 3.5 years. Diversity and divergence were less in VS than in BP (P = 0.03 and P < 0.01, respectively), and divergence at both sites was correlated with blood CD4(+) cell levels (VS, P = 0.05; BP, P = 0.01). Evolution of quasispecies was observed in 58% of the women; the loss or gain of quasispecies in VS or BP was always accompanied by such changes at the other site. In addition, sustained compartmentalization of quasispecies in VS was found for four women, even as CD4(+) cell levels decreased to low levels (<50 cells/microl). Quasispecies changes over time were associated with fluctuations in CD4(+) cell levels; concordant increases or decreases in VS and BP divergence had greater CD4(+) cell level changes than intervals with discordant changes (P = 0.05), and women with evolving quasispecies had greater decreases in CD4(+) cell levels compared to that for women who maintained the same quasispecies (P < 0.05). Thus, diversity, divergence, and evolution of cell-free HIV-1 in VS can be different from that in BP, and dynamics between their respective quasispecies are associated with changes in CD4(+) cell levels.

The menstrual cycle does not affect human immunodeficiency virus type 1 levels in vaginal secretions.

To determine whether the menstrual cycle affects human immunodeficiency virus (HIV) type 1 levels in vaginal secretions, vaginal lavage samples were collected at 7, 14, and 21 days after initiation of menses, to compare virus levels during the follicular, ovulatory, and luteal phases. During 33 menstrual cycles in 25 women, HIV-1 RNA levels in vaginal secretions ranged from <1000 to 5.3x10(7) copies per lavage, and weekly changes ranged from <0.5 to 2.5 log(10) copies per lavage. HIV-1 RNA levels in vaginal lavage samples from days 7, 14, and 21 were not significantly different. No discernible pattern was found in changes of vaginal virus loads (VVLs) during the menstrual cycle. VVLs were not correlated with plasma estradiol or progesterone levels (P>.05). These results suggest that hormonal changes during the menstrual cycle do not have a significant effect on HIV-1 RNA levels in vaginal secretions.