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1.  Heterogeneity of CD4+ and CD8+ T-cell Responses to Cytomegalovirus in HIV-Infected and HIV-Uninfected Men Who Have Sex With Men 
The Journal of Infectious Diseases  2014;210(3):400-404.
Studies of T-cell immunity to human cytomegalovirus (CMV) primarily reflect anti-CMV pp65 or immediate early antigen 1 (IE-1) activity. We assessed responses of T cells from human immunodeficiency virus (HIV)–negative and HIV-infected men to peptide pools spanning 19 CMV open reading frames selected because they previously correlated with total CMV-specific T-cell responses in healthy donors. Cells producing cytokines in response to pp65 or IE-1 together composed <12% and <40% of the total CD4+ and CD8+ T-cell responses to CMV, respectively. These proportions were generally similar regardless of HIV serostatus. Thus, analyses of total CMV-specific T-cell responses should extend beyond pp65 and IE-1 regardless of HIV serostatus.
PMCID: PMC4110456  PMID: 24532602
CMV; CD4+ and CD8+ T-cell responses; HIV infection; MACS; pp65; IE-1; intracellular cytokine staining; IFN-γ; TNF-α; IL-2
2.  Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations 
Nature  2015;517(7534):381-385.
Despite antiretroviral therapy (ART), HIV-1 persists in a stable latent reservoir1, 2, primarily in resting memory CD4+ T cells3, 4. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed5 and tested both in vitro and in vivo6–8. A key remaining question is whether virus-specific immune mechanisms including cytolytic T lymphocytes (CTL) can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.
PMCID: PMC4406054  PMID: 25561180
3.  Chronic CMV infection in older women: Longitudinal comparisons of CMV DNA in peripheral monocytes, anti-CMV IgG titers, serum IL-6 levels, and CMV pp65 (NLV)-specific CD8+ T-cell frequencies with twelve year follow-up 
Experimental gerontology  2014;0:84-89.
Chronic cytomegalovirus (CMV) infection may contribute significantly to T-cell immunosenescence, chronic inflammation, and adverse health outcomes in older adults. Recent studies suggest detectable CMV DNA in peripheral monocytes as a better indicator for this persistent viral infection than anti-CMV IgG serology. Here, we conducted longitudinal comparisons of anti-CMV IgG titers, CMV DNA in the peripheral monocytes, serum IL-6 levels, and CMV pp65 (NLV)-specific CD8+ T-cell frequencies in fifteen community-dwelling older women with twelve year follow-up. The results showed that anti-CMV IgG titers did not change over twelve years. Women with detectable CMV DNA had significantly higher IL-6 levels than those without, both at baseline (3.06+0.58 vs 1.19±0.37 pg/ml, respectively, p< .001) and at the follow-up (3.23±0.66 versus 0.98±0.37 pg/ml, respectively, p< .001). In addition, CMV pp65 (NLV)-specific CD8+ T cells were detected only in women who had CMV DNA with similar frequencies at both time points. These findings indicate that anti-CMV IgG serology is not sensitive to change nor useful for monitoring chronic CMV infection over time. They also provide a basis for further investigation into chronic CMV infection as defined by detectable CMV DNA in the peripheral monocytes and its impact on immunity and health in the elderly.
PMCID: PMC3989432  PMID: 24440388
chronic CMV infection; CMV DNA in monocytes; IL-6; CMV pp65 (NLV)-specific CD8+ T cells; longitudinal; aging
4.  Incorrect Identification of Recent HIV Infection in Adults in the United States Using a Limiting-Antigen Avidity Assay 
AIDS (London, England)  2014;28(8):1227-1232.
To evaluate factors associated with misclassification by the limiting-antigen avidity (LAg-avidity) assay among individuals with long-standing HIV infection.
Samples were obtained from the Multicenter AIDS Cohort Study (MACS), and AIDS Linked to the IntraVenous Experience (ALIVE) cohort (1089 samples from 667 individuals, 595 samples collected 2–4 years and 494 samples collected 4–8 years after HIV seroconversion). Paired samples from both time points were available for 422 (63.3%) of the 667 individuals.
Samples were considered to be misclassified if the LAg-Avidity assay result was ≤1.5 normalized optical density (OD-n) units.
Overall, 4.8% (52/1089) of the samples were misclassified, including 1.8% (16/884, 95% confidence intervals [CI]: 1.09%–3.06%) of samples from individuals with viral loads >400 copies/mL and 1.4% (10/705) of samples from individuals with viral loads >400 copies/mL and CD4 cell counts >200 cells/μl (95% CI: 0.68%–2.60%). Age, race, gender, and mode of HIV acquisition were not associated with misclassification. In an adjusted analysis, viral load <400 copies/mL (adjusted odds ratio [aOR]: 3.72, 95% CI: 1.61–8.57), CD4 cell count <50 cells/μl (aOR: 5.41, 95% CI: 1.86–15.74), and low LAg-Avidity result (≤1.5 OD-n) from the earlier time point (aOR: 5.60, 95% CI: 1.55–20.25) were significantly associated with misclassification.
The manufacturer of the LAg-Avidity assay recommends excluding individuals from incidence surveys who are receiving antiretroviral therapy, are elite suppressors, or have AIDS (CD4 cell count <200 cells/μl). The results of this study indicate that those exclusions do not remove all sources of assay misclassification among individuals with long-standing HIV infection.
PMCID: PMC4102639  PMID: 24513567
LAg-Avidity; incidence; MSM; PWID; HIV; misclassification
5.  Hearing Loss among HIV-Seropositive and HIV-Seronegative Men and Women 
Hearing sensitivity among adults has quality of life implications as individuals become older. There are limited data on hearing loss among aging HIV+ adults.
1) To evaluate pure-tone hearing thresholds among HIV+ and HIV- adults with similar demographic characteristics. 2) To determine if HIV disease variables and antiretroviral therapy (ART) are associated with pure-tone threshold levels.
262 men (117 HIV+) from the Baltimore/Washington, DC site of the Multicenter AIDS Cohort Study (MACS) and 134 women (105 HIV+) from the Washington, DC site of the Women's Interagency HIV Study (WIHS) participated. Pure-tone air-conduction thresholds were collected in a sound-treated room at octave frequencies from 250 through 8000 Hz, along with interoctave frequencies of 750, 3000, and 6000 Hz.
In each ear, a low-frequency pure-tone average (LPTA) was calculated using air-conduction thresholds at 250, 500, 1000, and 2000 Hz and a high-frequency PTA (HPTA) was calculated using air-conduction thresholds at 3000, 4000, 6000, and 8000 Hz. Linear mixed regression models tested the effect of HIV on hearing after adjusting for age, sex, race, and noise exposure history. Differential HIV effects for LPTA and HPTA and better/worse ear were also examined. CD4+ and CD8+ T-cell counts, log10 plasma HIV RNA concentrations, ever having had an AIDS-defining condition, and cumulative time on ART were included in the models for HIV+ participants only.
HPTA and LPTA were significantly higher (18% and 12%, respectively), for HIV+ participants compared to HIV- participants for the better ear. The direction of the effect was consistent across both the better and worse ear. There were no significant associations between HIV disease variables or treatment variables and LPTA or HPTA.
HIV+ adults had significantly poorer lower and higher frequency hearing compared to HIV- adults. High frequency hearing loss is consistent with an accelerated aging (presbycusis); hearing loss in the low frequency range among middle-aged individuals is an unexpected finding. Since some vowels and consonants have predominantly low frequency acoustic energy, poorer hearing in lower frequencies may lead to increased communication difficulties in HIV+ individuals.
PMCID: PMC4369193  PMID: 25541676
Hearing loss; Human immunodeficiency virus (HIV); Antiretroviral therapy (ART); Multicenter AIDS Cohort Study (MACS); Women's Interagency HIV Study (WIHS)
6.  Association between Free Testosterone Levels and Anal Human Papillomavirus Types 16/18 Infections in a Cohort of Men Who Have Sex with Men 
PLoS ONE  2015;10(3):e0119447.
Human papillomavirus (HPV) types 16 and 18 cause invasive cervical cancer and most invasive anal cancers (IACs). Overall, IAC rates are highest among men who have sex with men (MSM), especially MSM with HIV infection. Testosterone is prescribed for men showing hypogonadism and HIV-related wasting. While there are direct and indirect physiological effects of testosterone in males, its role in anal HPV16/18 infections in men is unknown.
Free testosterone (FT) was measured in serum from 340 Multicenter AIDS Cohort Study (MACS) participants who were tested for anal HPV16/18-DNA approximately 36 months later. The effect of log10-transformed current FT level on anal HPV16/18 prevalence was modeled using Poisson regression with robust error variance. Multivariate models controlled for other HPV types, cumulative years of exogenous testosterone use, race, age, lifetime number of receptive anal intercourse partnerships, body mass index, tobacco smoking, HIV-infection and CD4+ T-cell counts among HIV-infected, and blood draw timing.
Participants were, on average, 60 (+5.4) years of age, White (86%), and HIV-uninfected (56%); Twenty-four percent tested positive for anal HPV16 and/or 18-DNA (HPV16 prevalence=17.1%, HPV18=9.1%). In adjusted analysis, each half-log10 increase of FT was associated with a 1.9-fold (95% Confidence Interval: 1.11, 3.24) higher HPV16/18 prevalence. Additionally, other Group 1 high-risk HPVs were associated with a 1.56-fold (1.03, 2.37) higher HPV16/18 prevalence. Traditional risk factors for HPV16/18 infection (age, tobacco smoking; lifetime number of sexual partners, including the number of receptive anal intercourse partnerships within 24 months preceding HPV testing) were poorly correlated with one another and not statistically significantly associated with higher prevalence of HPV16/18 infection in unadjusted and adjusted analyses.
Higher free testosterone was associated with increased HPV16/18 prevalence measured approximately three years later, independent of sexual behavior and other potential confounders. The mechanisms underlying this association remain unclear and warrant further study.
PMCID: PMC4368778  PMID: 25794147
7.  The Impact of HAART on the Respiratory Complications of HIV Infection: Longitudinal Trends in the MACS and WIHS Cohorts 
PLoS ONE  2013;8(3):e58812.
To review the incidence of respiratory conditions and their effect on mortality in HIV-infected and uninfected individuals prior to and during the era of highly active antiretroviral therapy (HAART).
Two large observational cohorts of HIV-infected and HIV-uninfected men (Multicenter AIDS Cohort Study [MACS]) and women (Women’s Interagency HIV Study [WIHS]), followed since 1984 and 1994, respectively.
Adjusted odds or hazards ratios for incident respiratory infections or non-infectious respiratory diagnoses, respectively, in HIV-infected compared to HIV-uninfected individuals in both the pre-HAART (MACS only) and HAART eras; and adjusted Cox proportional hazard ratios for mortality in HIV-infected persons with lung disease during the HAART era.
Compared to HIV-uninfected participants, HIV-infected individuals had more incident respiratory infections both pre-HAART (MACS, odds ratio [adjusted-OR], 2.4; 95% confidence interval [CI], 2.2–2.7; p<0.001) and after HAART availability (MACS, adjusted-OR, 1.5; 95%CI 1.3–1.7; p<0.001; WIHS adjusted-OR, 2.2; 95%CI 1.8–2.7; p<0.001). Chronic obstructive pulmonary disease was more common in MACS HIV-infected vs. HIV-uninfected participants pre-HAART (hazard ratio [adjusted-HR] 2.9; 95%CI, 1.02–8.4; p = 0.046). After HAART availability, non-infectious lung diseases were not significantly more common in HIV-infected participants in either MACS or WIHS participants. HIV-infected participants in the HAART era with respiratory infections had an increased risk of death compared to those without infections (MACS adjusted-HR, 1.5; 95%CI, 1.3–1.7; p<0.001; WIHS adjusted-HR, 1.9; 95%CI, 1.5–2.4; p<0.001).
HIV infection remained a significant risk for infectious respiratory diseases after the introduction of HAART, and infectious respiratory diseases were associated with an increased risk of mortality.
PMCID: PMC3595204  PMID: 23554932
8.  p21WAF1/CIP1 RNA Expression in Highly HIV-1 Exposed, Uninfected Individuals 
PLoS ONE  2015;10(3):e0119218.
Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.
PMCID: PMC4352077  PMID: 25746435
9.  Transmission and evolution of Hepatitis C Virus in HCV seroconverters in HIV infected subjects 
Virology  2013;449:339-349.
HIV/HCV co-infection provides a model to determine the role of immunity on HCV transmission and evolution. In this study HCV transmission and evolution were evaluated in 6 HCV seroconverters in HIV-infected subjects with a wide range of CD4 cell count. The HCV envelope E1/E2 sequences were analyzed for transmission bottleneck, viral diversity/divergence, immune pressure, and mutations of HLA class I/II restricted epitopes. HCV infection started with transmission bottleneck in all HIV-infected individuals. During the 1.0–2.0 years of infection there was a shift of viral quasispecies in majority of the subjects from one to next visit. However, HCV diversity, divergence, mutations in HLA class I/II restricted and virus neutralizing epitopes were similar in all subjects regardless of CD4 cell count at the time of HCV infection. Our results suggest that HCV transmission and evolution in HIV-infected subjects may not be influenced by host CD4 cell count at the time of infection.
PMCID: PMC4337793  PMID: 24418568
HIV; HCV Seroconverter; Transmission; Evolution; CD4 cell count
10.  The effect of HAART-induced HIV suppression on circulating markers of inflammation and immune activation 
AIDS (London, England)  2015;29(4):463-471.
To investigate the impact of HAART-induced HIV suppression on levels of 24 serological biomarkers of inflammation and immune activation.
Prospective cohort study.
Biomarkers were measured with multiplex assays in centralized laboratories using stored serum samples contributed by 1,697 men during 8,903 person-visits in the Multicenter AIDS Cohort Study (MACS) from 1984–2009. Using generalized gamma models, we compared biomarker values across three groups, adjusting for possible confounders: HIV-uninfected (NEG); HIV+, HAART-naïve (NAI); and HAART-exposed with HIV RNA suppressed to <50 copies/mL plasma (SUP). We also estimated changes in biomarker levels associated with duration of HIV suppression, using splined generalized gamma regression with a knot at one year.
Most biomarkers were relatively normalized in the SUP group relative to the NAI group; however, 12 biomarkers in the SUP group were distinct (p<0.002) from NEG values: CXCL10, CRP, sCD14, sTNFR2, TNF-α, sCD27, sGP130, IL-8, CCL13, BAFF, GM-CSF, and IL-12p70. Thirteen biomarkers exhibited significant changes in the first year after viral suppression, but none changed significantly after that time.
Biomarkers of inflammation and immune activation moved toward HIV-negative levels within the first year after HAART-induced HIV suppression. Although several markers of T cell activation returned to levels present in HIV-negative men, residual immune activation, particularly monocyte/macrophage activation, was present. This residual immune activation may represent a therapeutic target to improve the prognosis of HIV-infected individuals receiving HAART.
PMCID: PMC4311407  PMID: 25630041
Acquired Immunodeficiency Syndrome; Antiretroviral Therapy, Highly Active; Biological Markers; Inflammation; Prospective Studies; Male
11.  Association between systemic inflammation and obstructive sleep apnea in men with or at risk for HIV infection 
Antiviral therapy  2014;19(8):725-733.
To determine whether markers of systemic inflammation are associated with the presence of moderate-to-severe obstructive sleep apnea (OSA), and whether this association differs based on HIV and HIV treatment status.
HIV-uninfected men (HIV−; n=60), HIV-infected men receiving HAART (HIV+/HAART; n=58), and HIV-infected men not receiving HAART (HIV+/ No HAART; n=41) underwent polysomnograpy and measurement of plasma levels of TNF-alpha, soluble TNF-alpha receptors I and II (sTNFRI and sTNFRII) and IL-6. The relationship between moderate-severe OSA (respiratory disturbance index ≥15 apnea/hypopnea events/hour) and inflammatory markers was assessed with multivariable regression models.
Compared to the HIV− men, HIV+/HAART men and HIV+/No HAART men had higher levels of TNF-alpha, sTNFRI, and sTNFRII, independent of age, race, smoking status, obstructive lung disease (OLD), and BMI. Moderate-to-severe OSA was present in 48% of the sample (HIV−:57%; HIV+/HAART: 41%; HIV+/No HAART: 44%). Among the HIV+/No HAART men, but not in the other groups, TNF-alpha, sTNFRII, and IL-6 levels were higher in those with moderate-severe OSA compared to men with no-to-mild OSA after adjustment for age, race, smoking status, OLD, and BMI. Within this group, the association of high TNF-alpha concentrations with moderate-severe OSA was also independent of CD4 cell count and plasma HIV RNA concentration.
Compared to HIV-infected men on HAART and HIV-uninfected men, markers of systemic inflammation were higher in HIV-infected men not receiving HAART. In these men, TNF-alpha was significantly related to obstructive sleep apnea, independent of HIV-related covariates.
PMCID: PMC4130807  PMID: 24518040
12.  Unique characteristics of histone deacetylase inhibitors in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells 
The latent reservoir for HIV-1 in resting memory CD4+ T cells is a major barrier to eradication. In vitro models involving transformed cell lines have been used to search for small molecules that reactivate latent HIV-1. Histone deacetylase (HDAC) inhibitors can reverse HIV-1 latent infection. Most studies on HDAC inhibitors have been performed in cell line models that differ in important aspects from the resting CD4+ T cells that harbour latent HIV-1 in vivo. Therefore, we evaluated the potency and kinetics of HDAC inhibitors in a primary cell model of HIV-1 latency that involves resting CD4+ T cells.
A green fluorescent protein (GFP)-expressing reporter virus NL4-3-Δ6-drGFP was used to generate latent infection in Bcl-2-transduced primary CD4+ T cells. Seventeen HDAC inhibitors were tested in this primary cell model. The effects of these HDAC inhibitors on the reactivation of latent HIV-1 were determined and compared with anti-CD3 and anti-CD28 co-stimulation.
In Bcl-2-transduced primary CD4+ T cells, short-term treatment with HDAC inhibitors resulted in very limited reactivation of latent HIV-1, while prolonged treatment greatly enhanced drug efficacy. The effects of HDAC inhibitors in reactivating latent HIV-1 correlated with their inhibitory effects on class I HDACs. Importantly, HIV-1 reactivated by HDAC inhibitors can quickly re-establish latent infection upon drug removal.
We identified unique features of HDAC inhibitor-induced reactivation of latent HIV-1 in primary CD4+ T cells. Our findings may be useful for the design of eradication trials.
PMCID: PMC3861332  PMID: 23999005
latent reservoir; cytotoxicity; vorinostat
13.  Cardiac fibroblasts mediate IL-17A–driven inflammatory dilated cardiomyopathy 
The Journal of Experimental Medicine  2014;211(7):1449-1464.
IL-17A stimulates cardiac fibroblasts to produce inflammatory mediators critical for the recruitment and differentiation of myeloid cells during inflammatory dilated cardiomyopathy.
Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra−/− mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A–fibroblast–GM-CSF–MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.
PMCID: PMC4076595  PMID: 24935258
14.  Tolerability and Efficacy of PI Versus NNRTI-Based Regimens in Subjects Receiving HAART During Acute or Early HIV Infection 
Little is known about modifications to highly active antiretroviral therapy (HAART) initiated during acute or early HIV infection.
Reasons for first modifications of HAART regimens were recorded using the AIDS Clinical Trials Group form among 363 subjects who initiated HAART within 1 year of seroconversion from 2005 in the Acute Infection and Early Disease Research Program. Modifications recorded as due to “patient choice” or “physician choice” were clarified by query to the recording site. Times to events were analyzed by Kaplan–Meier methods; significance of differences was assessed by the log-rank test.
Two hundred five of 363 (56%) subjects modified therapy, at a median of 425 days after initiation, by changing drugs, discontinuing treatment, or removing or adding drugs. Most modifications were attributed to toxicity (n = 105, 51%), most of which was low grade; regimen simplification (n = 18, 5%); and achievement of viral suppression (n = 15, 7%). Time to first modification was shorter for those with shorter time from infection to initiation (P = 0.005) and those having higher CD4 lymphocyte count at initiation (P = 0.06). Modifications occurred sooner in subjects receiving regimens taken more than once daily (P < 0.001) or with more than 2 pills daily (P < 0.001). Most regimens were nonnucleoside reverse transcriptase inhibitor based or protease inhibitor based, and these did not differ significantly in rate and timing of modification.
HAART initiated early in HIV infection was modified in the majority of cases, usually due to minor toxicities whose incidence was similar for protease inhibitor–based and nonnucleoside reverse transcriptase inhibitor–based regimens. Convenience of regimens (lower pill burden and dosing frequency) was associated with a lower rate of modification.
PMCID: PMC3115738  PMID: 19194317
cohort studies; regimen modification; toxicity
15.  Comparison of Interlaboratory Variation in Absolute T-Cell Counts by Single-Platform and Optimized Dual-Platform Methods 
Previous studies have reported that the adoption of a single-platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual-platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein-Bick et al., Clin Diagn Lab Immunol 2000;7:336–343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344–351). In the present study, we asked whether use of a single-platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited.
Identical study samples were shipped overnight to the MACS laboratories either by the National Institute of Allergy and Infectious Diseases, Division of AIDS Immunology Quality Assessment (NIAID-IQA) proficiency-testing program (n = 14), or by the Los Angeles site of the MACS (n = 10). For each sample, two tubes of blood were received; one was used for an automated complete blood count and differential, and the other for flow cytometry. The latter was performed using both our current dual-platform method (three-color CD45 gating and automated hematology) and the single-platform method (with TruCOUNT® beads to generate the absolute counts).
The median percent coefficients of variation (%CVs) for the dual-platform and single-platform methods were 6.6 and 9.9, respectively, for CD4 T cell counts, and 5.9 and 8.5, respectively, for CD8 T cell counts (n = 24). These differences were not statistically significant. The differences in absolute T-cell counts between the MACS sites and the median of all laboratories participating in the NIAID-IQA were smaller for the dual-platform than for single-platform absolute count method.
In contrast to previous reports, we did not observe lower interlaboratory variation across the MACS sites for single-platform absolute lymphocyte subset counting relative to dual-platform methods. This result may be at least partly explained by the lower interlaboratory variation with the optimized dual-platform method in this study relative to the previous reports.
PMCID: PMC3086643  PMID: 19813263
HIV; absolute CD4 counts; flow cytometry; single platform; dual platform; interlaboratory variation
16.  The Relationship Between Antibody to R7V and Progression of HIV Type 1 Infection 
The presence of antibody to R7V (anti-R7VAb), a seven-amino acid sequence derived from β2-microglobulin incorporated into HIV-1 virions from the surface of infected cells, has been proposed as an early marker of nonprogressive HIV-1 infection. The present study was undertaken because no prospective studies have tested this hypothesis. Stored samples collected prospectively from 361 HIV-1 seroconverting men in the Multicenter AIDS Cohort Study (0.44–1.53 years after seroconversion) were assayed for the presence or absence of anti-R7VAb, using a standardized ELISA. Using Cox proportional hazards models, crude and adjusted relative hazards (RH) were determined for the following outcomes: (a) clinically defined AIDS, (b) clinically defined AIDS or CD4 T cell count of <200 cells/μl, and (c) death. A total of 143 (39.6%) men had early anti-R7VAb and 218 (60.4%) did not; 192 (53.2%) developed AIDS. At the visit tested, men with anti-R7VAb had significantly lower CD4 T cell counts and higher plasma HIV-1 viral loads than those without antibody. After adjustment for CD4 T cell count, HIV-1 viral load, CCR5 polymorphism, and use of combined antiretroviral therapy, the presence of anti-R7VAb was associated with a higher risk of progression for all outcomes, but not significantly so. Absence of anti-R7VAb was significantly associated with expression of HLA-B*5701 and -B*2705, two alleles associated with slower progression of HIV-1 disease. The early presence of anti-R7VAb in HIV-1 seroconverters was not associated with slower progression of HIV-1 disease.
PMCID: PMC2933163  PMID: 20415637
17.  Lipodystrophy and Inflammation Predict Later Grip Strength in HIV-Infected Men: The MACS Body Composition Substudy 
AIDS Research and Human Retroviruses  2013;29(8):1138-1145.
Body fat changes in HIV-infected persons are associated with increased systemic inflammation and increased mortality. It is unknown whether lipodystrophy is also associated with declines in physical function. Between 2001 and 2003, 33 HIV-infected men with evidence of lipodystrophy (LIPO+), 23 HIV-infected men without lipodystrophy (LIPO−), and 33 seronegative men were recruited from the Multicenter AIDS Cohort Study (MACS) for the Body Composition substudy. Visceral adipose tissue (VAT) was assessed by quantitative computed tomography. Lean body mass (LBM) and extremity fat were measured by dual-energy x-ray absorptiometry. Insulin resistance was estimated by Homeostatic Model Assessment (HOMA). Serum interleukin (IL)-6, soluble tumor necrosis factor (TNF)-α receptors I and II (sTNFRI and sTNFRII), and highly sensitive C-reactive protein (hs-CRP) concentrations were quantified from archived serum samples. These measurements were correlated with grip strength measured in 2007 using linear regression. At the substudy visit, the LIPO+ group had higher HOMA, sTNFRI, sTNFRII, and IL-6 levels than the LIPO− group. In 2007, the LIPO+ group had lower median grip strength than the LIPO− group (34.4 vs. 42.7 kg, p=0.002). Multivariable analysis of HIV+ men showed older age, lower LBM, higher sTNFRII concentrations, and LIPO+ status [adjusted mean difference −4.9 kg (p=0.045)] at the substudy visit were independently associated with lower subsequent grip strength. Inflammation, lower LBM, and lipodystrophy in HIV-infected men were associated with lower subsequent grip strength. These findings suggest that inflammation may contribute to declines in functional performance, independent of age.
PMCID: PMC3715766  PMID: 23550976
18.  Assessing Immunophenotyping Performance: Proficiency-Validation for Adopting Improved Flow Cytometry Methods 
The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform “switch” studies to validate performance before methods for T-cell subset analysis can be changed.
Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program.
Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced.
Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories’ transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.
PMCID: PMC4100219  PMID: 17205569
HIV; immunophenotyping; flow cytometry; quality assessment; multicenter studies
19.  Sleep Disordered Breathing, Fatigue, and Sleepiness in HIV-Infected and -Uninfected Men 
PLoS ONE  2014;9(7):e99258.
Study Objectives
We investigated the association of HIV infection and highly active antiretroviral therapy (HAART) with sleep disordered breathing (SDB), fatigue, and sleepiness.
HIV-uninfected men (HIV−; n = 60), HIV-infected men using HAART (HIV+/HAART+; n = 58), and HIV-infected men not using HAART (HIV+/HAART−; n = 41) recruited from two sites of the Multicenter AIDS cohort study (MACS) underwent a nocturnal sleep study, anthropometric assessment, and questionnaires for fatigue and the Epworth Sleepiness Scale. The prevalence of SDB in HIV- men was compared to that in men matched from the Sleep Heart Health Study (SHHS).
The prevalence of SDB was unexpectedly high in all groups: 86.7% for HIV−, 70.7% for HIV+/HAART+, and 73.2% for HIV+/HAART−, despite lower body-mass indices (BMI) in HIV+ groups. The higher prevalence in the HIV− men was significant in univariate analyses but not after adjustment for BMI and other variables. SDB was significantly more common in HIV− men in this study than those in SHHS, and was common in participants with BMIs <25 kg/m2. HIV+ men reported fatigue more frequently than HIV− men (25.5% vs. 6.7%; p = 0.003), but self-reported sleepiness did not differ among the three groups. Sleepiness, but not fatigue, was significantly associated with SDB.
SDB was highly prevalent in HIV− and HIV+ men, despite a normal or slightly elevated BMI. The high rate of SDB in men who have sex with men deserves further investigation. Sleepiness, but not fatigue, was related to the presence of SDB. Clinicians caring for HIV-infected patients should distinguish between fatigue and sleepiness when considering those at risk for SDB, especially in non-obese men.
PMCID: PMC4084642  PMID: 24991815
20.  HIV Diversity as a Biomarker for HIV Incidence Estimation: Including a High-Resolution Melting Diversity Assay in a Multiassay Algorithm 
Journal of Clinical Microbiology  2014;52(1):115-121.
Multiassay algorithms (MAAs) can be used to estimate cross-sectional HIV incidence. We previously identified a robust MAA that includes the BED capture enzyme immunoassay (BED-CEIA), the Bio-Rad Avidity assay, viral load, and CD4 cell count. In this report, we evaluated MAAs that include a high-resolution melting (HRM) diversity assay that does not require sequencing. HRM scores were determined for eight regions of the HIV genome (2 in gag, 1 in pol, and 5 in env). The MAAs that were evaluated included the BED-CEIA, the Bio-Rad Avidity assay, viral load, and the HRM diversity assay, using HRM scores from different regions and a range of region-specific HRM diversity assay cutoffs. The performance characteristics based on the proportion of samples that were classified as MAA positive by duration of infection were determined for each MAA, including the mean window period. The cross-sectional incidence estimates obtained using optimized MAAs were compared to longitudinal incidence estimates for three cohorts in the United States. The performance of the HRM-based MAA was nearly identical to that of the MAA that included CD4 cell count. The HRM-based MAA had a mean window period of 154 days and provided cross-sectional incidence estimates that were similar to those based on cohort follow-up. HIV diversity is a useful biomarker for estimating HIV incidence. MAAs that include the HRM diversity assay can provide accurate HIV incidence estimates using stored blood plasma or serum samples without a requirement for CD4 cell count data.
PMCID: PMC3911463  PMID: 24153134
22.  A Comparison of Two Measures of HIV Diversity in Multi-Assay Algorithms for HIV Incidence Estimation 
PLoS ONE  2014;9(6):e101043.
Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence.
Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies.
The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion.
MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation.
PMCID: PMC4072769  PMID: 24968135
23.  Emergence and Persistence of CXCR4-Tropic HIV-1 in a Population of Men from the Multicenter AIDS Cohort Study 
The Journal of infectious diseases  2008;198(8):1104-1112.
We examined the emergence of CXCR4 (i.e., X4) tropism in 67 male human immunodeficiency virus type 1 (HIV-1) seroconverters from the Multicenter AIDS Cohort Study (MACS) who were selected to reflect the full spectrum of rates of HIV-1 disease progression. A mean of 10 serial samples per donor were evaluated by a laboratory-validated, commercially available assay to determine phenotypic coreceptor use. A total of 52% of men had dual- or mixed-tropic HIV-1 detected at 1 or more of the time points tested. Use of X4 by HIV-1 was detected more frequently among men who developed AIDS (defined as a CD4+ T cell count of < 200 cells/μL and/or an AIDS-defining illness)≤11 years after seroconversion than among those who did not (P = .005), as well as among men who exhibited a total T cell count decline (i.e., a CD3+ inflection point), compared with those who did not (P = .03). For men in whom both X4 virus and an inflection point were detected, emergence of X4 virus preceded the inflection point by a median of 0.83 years. The median CD4+ T cell count at first detection of X4 viruses before the onset of AIDS was 475 cells/μL. We conclude that HIV-1 variants that used X4 frequently emerged at high CD4+ T cell counts and may contribute to the decrease in T cell numbers during late HIV-1 infection.
PMCID: PMC2753263  PMID: 18783316
24.  Incidence and epidemiology of anal cancer in the Multicenter AIDS Cohort Study (MACS) 
To examine the incidence and risk factors for anal cancer in a multicenter cohort of HIV-positive and negative men who have sex with men followed between 1984 and 2006 (MACS).
Prospective analysis using Poisson regression and Cox proportional hazard models, and a nested case-control study using conditional logistic regression.
There were 28 cases of anal cancer among the 6,972 men who were evaluated. The incidence rate was significantly higher in HIV-positive men than in HIV-negative men (IR= 69 vs. 14 per 100,000 person-years). Among HIV-positive men, anal cancer incidence was higher in the HAART era than the pre-HAART era (IR=137 vs. 30 per 100,000 person-years). In multivariate analysis restricted to the HAART era, anal cancer risk increased significantly with HIV infection (RH=4.7, 95%CI=1.3–17), and increasing number of unprotected receptive anal sex partners at the first three study visits (p-trend=0.03). Among HIV-positive men, current HAART use did not decrease anal cancer risk.
HIV-positive men had increased risk of anal cancer. Improved survival of HIV-positive individuals following HAART initiation may allow for sufficient time for human papillomavirus (HPV) associated anal dysplasias to develop into malignancies, thus explaining the increased incidence of anal cancer in the HAART era.
PMCID: PMC3991563  PMID: 18614927
anal; rectal; cancer; incidence; MACS; sexual risk; HAART
25.  Lower Adiponectin is Associated with Subclinical Cardiovascular Disease among HIV-infected Men 
AIDS (London, England)  2014;28(6):901-909.
To examine if altered levels of adipokines, adipose-derived peptides associated with myocardial infarction in the general population, may contribute to subclinical coronary atherosclerosis in HIV-infected persons.
Nested cohort study.
We studied HIV-infected(HIV+) and HIV-uninfected(HIV−) men in the Multicenter AIDS Cohort Study with noncontrast CT to measure coronary artery calcium and regional adiposity; 75% additionally underwent coronary CT angiography to measure plaque composition and stenosis. Adiponectin and leptin levels were assessed. Multiple regression models were used to assess associations between adipokine levels and HIV disease parameters, regional adiposity, and plaque adjusted for age, race, HIV serostatus and CVD risk factors (RFs).
Significant findings were limited to adiponectin. HIV+ men (n=493) had lower adiponectin levels than HIV− men (n=250) after adjusting for CVD RFs (p<0.0001), which became non-significant after adjustment for abdominal visceral and thigh subcutaneous adipose tissue. Among HIV+ men, lower adiponectin levels were associated with higher CD4+ T cell counts (p= 0.004), longer duration of antiretroviral therapy (p= 0.006) and undetectable HIV RNA levels (p = 0.04) after adjusting for age, race and CVD RFs; only CD4+ cell count remained significant after further adjustment for adipose tissue. In both groups, lower adiponectin levels were associated with increased odds of coronary stenosis > 50% (p <0.007). Lower adiponectin levels were associated with increased extent of plaque in HIV+ and of mixed plaque in HIV− men.
Adiponectin levels were lower in HIV-infected men and related to the severity of subclinical atherosclerosis, independent of traditional CVD risk factors.
PMCID: PMC3967406  PMID: 24401646
Adipokines; adiponectin; leptin; heart; subclinical coronary atherosclerosis; metabolic side effects of HIV infection; coronary CT angiography; cardiac CT

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