Search

Background Biomarkers that provide insight into drivers of aging are needed for people with human immunodeficiency virus (PWH). The study objective was to determine if epigenetic age acceleration (EAA) markers are associated with physiologic frailty measured by the Veterans Aging Cohort Study (VACS) Index and predict all-cause mortality for PWH. Methods Epigenome-wide DNA methylation was profiled in VACS total white blood cell samples collected during 2005–2007 from 531 PWH to generate 6 established markers of EAA. The association of each EAA marker was tested with VACS Index 2.0. All-cause mortality was assessed over 10 years. For each EAA marker, the hazard ratio per increased year was determined using Cox regression. To evaluate mortality discrimination, C-statistics were derived. Results Participants were mostly men (98.5%) and non-Hispanic Black (84.4%), with a mean age of 52.4 years (standard deviation [SD], 7.8 years). Mean VACS Index score was 59.3 (SD, 16.4) and 136 deaths occurred over a median follow-up of 8.7 years. Grim age acceleration (AA), PhenoAA, HannumAA, and extrinsic epigenetic AA were associated with the VACS Index and mortality. HorvathAA and intrinsic epigenetic AA were not associated with either outcome. GrimAA had the greatest mortality discrimination among EAA markers and predicted mortality independently of the VACS Index. One-year increase in GrimAA was associated with a 1-point increase in VACS Index and a 10% increased hazard for mortality. Conclusions The observed associations between EAA markers with physiologic frailty and mortality support future research to provide mechanistic insight into the accelerated aging process and inform interventions tailored to PWH for promoting increased healthspan.

Categorizing clinical risk amidst heterogeneous multimorbidity in older people living with HIV/AIDS (PLWH) may help prioritize and optimize health care engagements.PLWH and their prevalent conditions in 8 health domains diagnosed before January 1, 2015 were identified using 2014-2016 Medicare claims and the Chronic Conditions Data Warehouse. Latent profile analysis identified 4 distinct clinical subgroups based on the likelihood of conditions occurring together [G1: healthy, G2: substance use (SU), G3: pulmonary (PULM), G4: cardiovascular conditions (CV)]. Restricted mean survival time regression estimated the association of each subgroup with the 365 day mean event-free days until death, first hospitalization, and nursing home admission. Zero-inflated Poisson regression estimated hospitalization frequency in 2-year follow-up.Of 11,196 older PLWH, 71% were male, and the average age was 61 (SD 9.2) years. Compared with healthy group, SU group had a mean of 30 [95% confidence interval: (19.0 to 40.5)], PULM group had a mean of 28 (22.1 to 34.5), and CV group had a mean of 22 (15.0 to 22.0) fewer hospitalization-free days over 1 year. Compared with healthy group (2.8 deaths/100 person-years), CV group (8.4) had a mean of 4 (3.8 to 6.8) and PULM group (7.9) had a mean of 3 (0.7 to 5.5) fewer days alive; SU group (6.0) was not different. There was no difference in restricted mean survival time for nursing home admission. Compared with healthy group, SU group had 1.42-fold [95% confidence interval: (1.32 to 1.54)], PULM group had 1.71-fold (1.61 to 1.81), and CV group had 1.28-fold (1.20 to 1.37) higher rates of hospitalization.Identifying clinically distinct subgroups with latent profile analysis may be useful to identify targets for interventions and health care optimization in older PLWH.

BACKGROUND: Age-related changes in immune cell composition and functionality are associated with multimorbidity and mortality. However, many centenarians delay the onset of aging-related disease suggesting the presence of elite immunity that remains highly functional at extreme old age. METHODS: To identify immune-specific patterns of aging and extreme human longevity, we analyzed novel single cell profiles from the peripheral blood mononuclear cells (PBMCs) of a random sample of 7 centenarians (mean age 106) and publicly available single cell RNA-sequencing (scRNA-seq) datasets that included an additional 7 centenarians as well as 52 people at younger ages (20–89 years). FINDINGS: The analysis confirmed known shifts in the ratio of lymphocytes to myeloid cells, and noncytotoxic to cytotoxic cell distributions with aging, but also identified significant shifts from CD4(+) T cell to B cell populations in centenarians suggesting a history of exposure to natural and environmental immunogens. We validated several of these findings using flow cytometry analysis of the same samples. Our transcriptional analysis identified cell type signatures specific to exceptional longevity that included genes with age-related changes (e.g., increased expression of STK17A, a gene known to be involved in DNA damage response) as well as genes expressed uniquely in centenarians’ PBMCs (e.g., S100A4, part of the S100 protein family studied in age-related disease and connected to longevity and metabolic regulation). INTERPRETATION: Collectively, these data suggest that centenarians harbor unique, highly functional immune systems that have successfully adapted to a history of insults allowing for the achievement of exceptional longevity. FUNDING: TK, SM, PS, GM, SA, TP are supported by 10.13039/100000002NIH-10.13039/100000049NIAUH2AG064704 and U19AG023122. MM and PS are supported by 10.13039/100000002NIH10.13039/100000049NIA Pepper center: P30 AG031679-10. This project is supported by the Flow Cytometry Core Facility at BUSM. FCCF is funded by the NIH Instrumentation grant: S10 OD021587.

Although people with HIV are living longer, as they age they remain disproportionately burdened with multimorbidity that is exacerbated in resource-poor settings. The geroscience hypothesis postulates that a discrete set of between five and ten hallmarks of biological ageing drive multimorbidity, but these processes have not been systematically examined in the context of people with HIV. We examine four major hallmarks of ageing (macromolecular damage, senescence, inflammation, and stem-cell dysfunction) as gerodrivers in the context of people with HIV. As a counterbalance, we introduce healthy ageing, physiological reserve, intrinsic capacity, and resilience as promoters of geroprotection that counteract gerodrivers. We discuss emerging geroscience-based diagnostic biomarkers and therapeutic strategies, and provide examples based on recent advances in cellular senescence, and other, non-pharmacological approaches. Finally, we present a conceptual model of biological ageing in the general population and in people with HIV that integrates gerodrivers and geroprotectors as modulators of homoeostatic reserves and organ function over the lifecourse.

Introduction The aim of this proof-of-concept study is to explore sleep health disparity and associations with frailty in middle-aged (i.e., age< 65 years) people living with HIV (PLWH) in an African setting. Methods Data were from 98 participants (age: 56.8±4.3 [mean±SD] years; female: 49 [50%]) enrolled in Botswana, Africa, between March-December 2021. Forty-eight participants were HIV seropositive (i.e., PLWH) and on suppressive antiretroviral therapy regimens; 50 participants were confirmed uninfected (i.e., controls). PLWH and controls were matched for age and sex. Sleep health was assessed subjectively based on answers to two questions related to sleep quality, and was scored 1 (i.e., better sleep quality) if the participant answered “very much” to question “I am sleeping well” and “not at all” to question “I need to sleep during the day” or 0 (i.e., worse sleep quality) if otherwise. To assess frailty, 29 health deficits related to weight loss, obesity, physical activity, emotion, and fatigue were examined to construct a composite frailty index ranged between 0 and 1 (i.e., 0=no deficit present and 1=all deficits present). Logistic regression and linear regression models were conducted respectively to compare sleep quality (dichotomous) and the frailty index (continuous) between PLWH and controls. In addition, linear regression models were used to examine the association between frailty index and sleep quality stratified by HIV serostatus. All models were adjusted for age and sex. Statistical significance was rendered at p< 0.05. Results PLWH displayed 2.5-fold higher odds of having worse sleep quality compared to controls (PLWH: 61.4%, controls: 38.6%; p=0.02). The frailty index did not differ significantly between PLWH (0.15±0.08 [mean±SD]; range: [0.02, 0.36]) and controls (0.14±0.10; range: [0.03, 0.56]; p=0.6). Within PLWH, having worse sleep quality was significantly associated with an increase in the frailty index (0.76 SD of the group) (p< 0.001). No significant association was observed in HIV-seronegative controls (p=0.4). Conclusion Treated PLWH had worse self-reported sleep than controls, and worse sleep quality in PLWH is linked to an increased burden of physical frailty in Africa. Future studies are warranted to elucidate the causal direction and underlying mechanisms. Support (if any) Developmental Award (CFAR; P30AI060354); Pilot Grant (HIV&Aging; R33AG067069-02).

Age-related changes in immune cell composition and functionality are associated with multimorbidity and mortality. However, many centenarians delay the onset of aging-related disease suggesting the presence of elite immunity that remains highly functional at extreme old age. To identify immune-specific patterns of aging and extreme human longevity, we analyzed novel single cell profiles from the peripheral blood mononuclear cells (PBMCs) of 7 centenarians (mean age 106) and publicly available single cell RNA-sequencing (scRNA-seq) datasets that included an additional 7 centenarians as well as 52 people at younger ages (20-89 years). The analysis confirmed known shifts in the ratio of lymphocytes to myeloid cells, and noncytotoxic to cytotoxic cell distributions with aging, but also identified significant shifts from CD4+ T cell to B cell populations in centenarians suggesting a history of exposure to natural and environmental immunogens. Our transcriptional analysis identified cell type signatures specific to exceptional longevity that included genes with age-related changes (e.g., increased expression of STK17A, a gene known to be involved in DNA damage response) as well as genes expressed uniquely in centenarians’ PBMCs (e.g., S100A4, part of the S100 protein family studied in age-related disease and connected to longevity and metabolic regulation). Collectively, these data suggest that centenarians harbor unique, highly functional immune systems that have successfully adapted to a history of insults allowing for the achievement of exceptional longevity.

People living with HIV (PLWH) are disproportionately burdened with multimorbidity and decline in physiologic function compared to their uninfected counterparts, but biological mechanisms that differentially contribute to the decline in muscle function in PLWH compared to uninfected people remains understudied. The study site was Brigham and Women’s Hospital, Harvard Medical School, Boston MA. We evaluated skeletal muscle tissue for levels of total NAD, NAD+ and NADH in middle-aged asymptomatic PLWH, coinfected with hepatitis C virus (HCV) and/or cytomegalovirus (CMV) and compared them to uninfected control participants. Of the 54 persons with muscle biopsy data, the mean age 57 years with 33% women. Total NAD levels declined in skeletal muscle in association with HIV infection, and was exacerbated by HCV and CMV coinfection, with lowest levels of total NAD, NAD+ and NADH among persons that were coinfected with all three viruses (P=0.015, P=0.014, P=0.076; respectively). Levels of total NAD, NAD+ and NADH in skeletal muscle were inversely associated with inflammation (P=0.014, P=0.013, P=0.055; respectively). Coinfections were also associated with measures of inflammation (CD4/CD8 ratio: P< 0.001 and sCD163: P< 0.001), immune activation (CD38 and HLA-DR expression on CD8 T cells: P< 0.001). Additionally, coinfection was associated with increased physiologic frailty, based on the VACS Index 1.0 assessment (P=0.001). Further research is warranted to determine the clinical relevance of preclinical deficits in NAD metabolites in skeletal muscle in association with viral coinfection and inflammation, as well as the observed association between viral coinfection and physiologic frailty.

Poor compliance with medications is a growing concern in geriatric care and is increasingly more relevant among people living with HIV (PLWH) as they age. Our goal was to understand geriatric conditions associated with antiretroviral therapy (ART) nonadherence in a Medicare population of older PLWH. We analyzed Medicare data from PLWH aged 50 years or older who were continuously enrolled in fee-for-service Medicare from January 1, 2014 to June 30, 2015. Prevalent geriatric conditions (dementia, depression, falls, hip fracture, sensory deficits, osteoporosis, orthostatic hypotension, urinary incontinence, frailty) were identified in January 1, 2014–December 31, 2014. ART nonadherence was defined as 70% were males. In adjusted models, age was not associated with nonadherence, frailty status was the only geriatric condition associated with nonadherence [robust: reference, prefrail odds ratio (OR): 0.97, confidence interval (95% CI) 0.86–1.10, frail OR: 1.34 95% CI 1.11–1.61], and odds of nonadherence were lower for polypharmacy [OR: 0.48 (0.43–0.54)]. Our findings suggest that patient-centered care plans aimed at improving ART adherence among older PLWH would benefit from long-term surveillance; a deeper understanding of the role of frailty and polypharmacy, even at chronologically younger ages in PLWH.

Testosterone supplementation during energy deficit promotes whole body lean mass accretion, but the mechanisms underlying that effect remain unclear. To elucidate those mechanisms, skeletal muscle molecular adaptations were assessed from muscle biopsies collected before, 1 h, and 6 h after exercise and a mixed meal (40 g protein, 1 h postexercise) following 14 days of weight maintenance (WM) and 28 days of an exercise- and diet-induced 55% energy deficit (ED) in 50 physically active nonobese men treated with 200 mg testosterone enanthate/wk (TEST) or placebo (PLA) during the ED. Participants ( n = 10/group) exhibiting substantial increases in leg lean mass and total testosterone (TEST) were compared with those exhibiting decreases in both of these measures (PLA). Resting androgen receptor (AR) protein content was higher and fibroblast growth factor-inducible 14 (Fn14), IL-6 receptor (IL-6R), and muscle ring-finger protein-1 gene expression was lower in TEST vs. PLA during ED relative to WM ( P < 0.05). Changes in inflammatory, myogenic, and proteolytic gene expression did not differ between groups after exercise and recovery feeding. Mechanistic target of rapamycin signaling (i.e., translational efficiency) was also similar between groups at rest and after exercise and the mixed meal. Muscle total RNA content (i.e., translational capacity) increased more during ED in TEST than PLA ( P < 0.05). These findings indicate that attenuated proteolysis at rest, possibly downstream of AR, Fn14, and IL-6R signaling, and increased translational capacity, not efficiency, may drive lean mass accretion with testosterone administration during energy deficit.

People aging with HIV (PAWH) infection experience greater impairments in physical and cognitive function, in addition to higher rates of peripheral comorbid conditions (e.g., renal failure, diabetes, bone fracture, hypertension, cardiovascular disease, polypharmacy, and multimorbidity). While multifactorial drivers, including HIV infection itself, antiretroviral therapy-related toxicities, disparities in care, and biobehavioral factors, likely contribute, there remains an overarching question as to what are the relevant age-related mechanisms and models that could inform interventions that promote health span and life span in PAWH? This workshop was convened to hear from experts on the biology of aging and HIV researchers studying PAWH to focus on advancing investigations at the interface of HIV and Aging. In this study, we summarize the discussions from the Harvard Center for AIDS Research and Boston Claude D. Pepper cosponsored workshop on HIV and Aging, which took place in October 2018.

Background: People living with HIV (PLWH) are disproportionately burdened with multimorbidity and decline in physiologic function compared with their uninfected counterparts, but biological mechanisms that differentially contribute to the decline in muscle function in PLWH compared with uninfected people remain understudied. Setting: The study site was Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Methods: We evaluated skeletal muscle tissue for levels of total nicotinamide adenine dinucleotide (NAD), NAD+, and nicotinamide adenine dinucleotide (NADH) in middle-aged asymptomatic PLWH, coinfected with hepatitis C virus and/or cytomegalovirus and compared them with uninfected control participants. Results: Of the 54 persons with muscle biopsy data, the mean age was 57 years with 33% women. Total NAD levels declined in skeletal muscle in association with HIV infection and was exacerbated by hepatitis C virus and cytomegalovirus coinfection, with lowest levels of total NAD, NAD+, and NADH among persons who were coinfected with all 3 viruses (P = 0.015, P = 0.014, and P = 0.076, respectively). Levels of total NAD, NAD+, and NADH in skeletal muscle were inversely associated with inflammation (P = 0.014, P = 0.013, and P = 0.055, respectively). Coinfections were also associated with measures of inflammation (CD4/CD8 ratio: P < 0.001 and sCD163: P < 0.001) and immune activation (CD38 and human leukocyte antigen-DR expression on CD8 T cells: P < 0.001). In addition, coinfection was associated with increased physiologic frailty based on the Veteran Aging Cohort Study 1.0 index assessment (P = 0.001). Conclusions: Further research is warranted to determine the clinical relevance of preclinical deficits in NAD metabolites in skeletal muscle in association with viral coinfection and inflammation, as well as the observed association between viral coinfection and physiologic frailty.

Since emerging into the human population in late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached across the globe to infect >80 million people. The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 can range in severity from mild and asymptomatic to severe and fatal. Identifying risk factors for adverse outcomes in COVID-19 is a major challenge. In the context of the existing HIV-1 pandemic, whether COVID-19 disproportionately burdens people living with HIV-1 infection (PLWH) is unclear. The following discussion highlights pressing questions and challenges in the HIV-1 and SARS-CoV-2 syndemic, including (i) age, sex, and race as drivers of COVID-19 severity; (ii) whether chronic inflammation common in PLWH influences immune response; (iii) whether disease severity and trajectory models for COVID-19 ought to be calibrated for PLWH; (iv) vaccine considerations, and finally, (v) long-term health outcomes in PLWH that are further burdened by coinfection with SARS-CoV-2.

In Greek mythology, Tithonus was granted eternal life but not eternal youth. As time passes he withers, slowly losing his health and all that he knew, lamenting a cruel immortality.(1)

Background: Severe energy deficits during military operations, produced by significant increases in exercise and limited dietary intake, result in conditions that degrade lean body mass and lower-body muscle function, which may be mediated by concomitant reductions in circulating testosterone. Methods: We conducted a three-phase, proof-of-concept, single centre, randomised, double-blind, placebo-controlled trial (CinicalTrials.gov, NCT02734238) of non-obese men: 14-d run-in, free-living, eucaloric diet phase; 28-d live-in, 55% exercise- and diet-induced energy deficit phase with (200 mg testosterone enanthate per week, Testosterone, n = 24) or without (Placebo, n = 26) exogenous testosterone; and 14-d recovery, free-living, ad libitum diet phase. Body composition was the primary end point; secondary endpoints included lower-body muscle function and health-related biomarkers. Findings: Following energy deficit, lean body mass increased in Testosterone and remained stable in Placebo, such that lean body mass significantly differed between groups [mean difference between groups (95% CI), 2.5 kg (3.3, 1.6); P

The prevalence of age-standardized comorbidities is significantly elevated for PLWH across an array of cohorts. However, healthcare needs of older people living with (PLWH) and without (PWOH) HIV may be similar if they have similar geriatric conditions. PLWH and PWOH aged 65+ and eligible for Medicare from 7/1/2014-1/1/2015 were matched 1:1 on age, sex, race, and census region (n=7654). Cox regression assessed count of prevalent geriatric conditions (dementia, depression, falls, hip fracture, sensory deficits, osteoporosis, orthostatic hypotension, urinary incontinence, frailty, and polypharmacy), and risk for clinical or utilization outcomes (cancer, kidney disease, muscle wasting, hepatitis C, liver disease, myocardial infarction, stroke; hospitalization, nursing home and home health admission) during follow-up between 1/1/2015-12/31/2016. PLWH and PWOH are similar in count of geriatric conditions. Compared to those with none, those having 2+ geriatric conditions were similar across PLWH and PWOH in their risk of ≥1 clinical outcome (PLWH: HR 1.57 95% CI [1.29-1.90]; PWOH: HR 1.31 [1.02-1.67]), hospitalization (PLWH: HR 2.35 [1.96-2.83]; PWOH: HR 2.07 [1.65-2.60]), and home health admission (PLWH: HR 2.09 [1.58-2.76]; PWOH: HR 2.20 [1.55-3.12]). Having 2+ geriatric conditions, PWOH had 4.45 times the risk (95% CI 3.16-6.26) and PLWH had 2.88 times the risk (95% CI 2.18-3.81) of NH admission compared to no geriatric conditions. In this study, PLWH use nursing homes less than PWOH despite having a similar number of geriatric conditions and clinical outcomes. Further research to understand this apparent discrepancy will be critical to achieve equity in nursing home access.

OBJECTIVES: Short-term energy deficit reduces acute measures of mixed muscle protein synthesis (MPS) and suppresses the hypothalamic-pituitary axis and endogenous testosterone synthesis. We hypothesized that testosterone supplementation could mitigate the effects of energy deficit on MPS. We conducted a randomized, double-blind, placebo-controlled trial to determine the effects of 28 days of tightly-controlled severe energy deficit (deficit 55% of total energy requirements) on measures of mixed-MPS and proteome-wide protein dynamics in non-obese men either given 200 mg testosterone enanthate (Testosterone, n = 24) or placebo (Placebo, n = 26) injections per week. METHODS: Participants received daily aliquots of deuterated water ((2)H(2)O) for 42 consecutive days (14-d weight maintenance period followed by 28-d energy deficit). Muscle biopsies were collected at rest in a fasted state at the end of the weight maintenance phase (PRE) and at the middle (MID) and end (POST) of the 28-d energy deficit. Mixed-MPS and proteome-wide protein fractional synthesis rates (FSR) were quantified. Changes over time and differences between Testosterone and Placebo were determined for mixed-MPS, and cross-sectional comparisons between Testosterone and Placebo were performed at MID and POST for proteome dynamics. RESULTS: In both Testosterone and Placebo, mixed-MPS were 40% and 33% lower (P

In the era of effective antiretroviral therapy, the number of older people with HIV (PWH) is increasing, and those aging with HIV are experiencing an increasing burden of age-associated comorbidities. Life expectancy among older PWH is approaching that of demographically comparable HIV-uninfected (HIV-) adults. With this changing demographic of PWH come new challenges for researchers and clinicians in how to identify, address, and manage the complex interplay of treated HIV infection and aging-associated factors. In response to these challenges, the annual International Workshop on HIV and Aging was initiated in 2009 as a multidisciplinary platform for scientific discourse on the research and clinical complications arising from the aging population of PWH. The multidisciplinary nature of the workshop has resulted in a wide range of topics addressed over the past 9 years, from basic mechanisms in aging and HIV pathogenesis, to epidemiology of aging within large cohorts, interventions, and implementation of clinical programs. Herein, we summarize the key topics discussed at the 9th Annual International Workshop on HIV and Aging 2018, including "inflammaging," mitochondrial dysfunction, exercise interventions, HIV-associated neurocognitive impairment, metabolic dysfunction, menopause, and polypharmacy. In addition to recent developments in research and clinical care, we discuss open questions and future research directions required to better understand the interaction of HIV and aging.

Background The use of circulating clinically routine biomarkers and volitional physical activity using wristband accelerometry in preclinical middle-aged adults may provide sensitive measures of physical function and predict sooner the onset of age- and HIV-related physical decline. Methods Nested cross-sectional cohort study of adult men 50–65 years old with HIV infection on potent antiretroviral therapy and uninfected control participants within the Boston metropolitan area. Gait speed derived from wristband accelerometry, gait speed derived from a standardized 6-minute walk test, cellular immune biomarker levels (CD4 T cell, CD8 T cell), and serum anabolic biomarker levels (total and free testosterone, and sex-hormone-binding globulin) were measured. Results Of the five measured biomarkers, four were significantly associated with volitional gait speed based on accelerometry, whereas only one was associated with gait speed based on the 6-minute walk test collected in a laboratory environment. Conclusion Levels of selected immune and anabolic biomarkers were associated with volitional physical activity in middle-aged individuals. Digital and circulating biomarkers may be useful in future studies designed to identify presymptomatic individuals at increased risk for age- and HIV-associated functional decline.

HIV-infected individuals are at an increased risk of osteoporosis despite effective viral suppression. Observations that myostatin null mice have increased bone mass led us to hypothesize that simian immunodeficiency virus (SIV)-associated bone loss may be attenuated by blocking myostatin/TGFβ signaling. In this proof-of-concept study, pair-housed juvenile male rhesus macaques were inoculated with SIVmac239. Four weeks later, animals were treated with vehicle or Fc-conjugated soluble activin receptor IIB (ActR2B·Fc, iv. 10 mg ∗ kg−1 ∗ week−1) – an antagonist of myostatin and related members of TGFβ superfamily. Limb and trunk bone mineral content (BMC) and density (BMD) using dual-energy X-Ray absorptiometry, circulating markers of bone growth and turnover, and serum testosterone levels were measured at baseline and during the 12-week intervention period. The increase in BMC was significantly greater in the ActRIIB.Fc-treated group (+8 g) than in the placebo group (−4 g) (p < 0.05). BMD also increased significantly more in the ActRIIB.Fc-treated macaques (+0.03 g/cm2) than in the placebo-treated animals (+0 g/cm2) (p < 0.005). Serum osteocalcin was about two-fold higher in the ActRIIB.Fc-treated group than in the placebo group (p < 0.05), but serum C-terminal telopeptide and testosterone levels did not differ significantly between groups. The expression levels of TNFalpha (p < 0.05), GADD45 (p

Summary Because people age differently, age is not a sufficient marker of susceptibility to disabilities, morbidities, and mortality. We measured nineteen blood biomarkers that include constituents of standard hematological measures, lipid biomarkers, and markers of inflammation and frailty in 4704 participants of the Long Life Family Study (LLFS), age range 30–110 years, and used an agglomerative algorithm to group LLFS participants into clusters thus yielding 26 different biomarker signatures. To test whether these signatures were associated with differences in biological aging, we correlated them with longitudinal changes in physiological functions and incident risk of cancer, cardiovascular disease, type 2 diabetes, and mortality using longitudinal data collected in the LLFS. Signature 2 was associated with significantly lower mortality, morbidity, and better physical function relative to the most common biomarker signature in LLFS, while nine other signatures were associated with less successful aging, characterized by higher risks for frailty, morbidity, and mortality. The predictive values of seven signatures were replicated in an independent data set from the Framingham Heart Study with comparable significant effects, and an additional three signatures showed consistent effects. This analysis shows that various biomarker signatures exist, and their significant associations with physical function, morbidity, and mortality suggest that these patterns represent differences in biological aging. The signatures show that dysregulation of a single biomarker can change with patterns of other biomarkers, and age-related changes of individual biomarkers alone do not necessarily indicate disease or functional decline.

Sustained periods of severe energy deficit during military operations, produced by significant increases in aerobic-type physical work and limited dietary intake, result in conditions that degrade lean body mass (LBM) and lower-body muscle function, which may be mediated by concomitant reductions in circulating testosterone. Therefore, we studied the effects of restoring testosterone by supplementation (200 mg testosterone enanthate/wk, TEST), compared to placebo (1 mL sesame seed oil/wk, PLA), on body composition and lower-body muscular strength and endurance during 28 d of severe exercise- and diet-induced energy deficit in non-obese, young men. Following energy deficit, concentrations of free testosterone decreased, total testosterone remained unchanged, and sex-hormone binding globulin (SHBG) increased in PLA; whereas, total and free testosterone increased and SHBG was unchanged in TEST, such that, total and free testosterone were greater and SHBG concentrations were lower in TEST than PLA. During energy deficit, TEST attenuated weight loss and increased DXA-derived LBM compared to PLA, but both groups had a similar increase in type I slow twitch myosin heavy chain cross-sectional area (CSA) and decrease in type II fast twitch myosin heavy chain CSA. Lower-body muscle function declines and adverse event rates did not differ by group during energy deficit. Supplemental testosterone may be an effective pharmacological LBM enhancement strategy during short-term periods of exercise- and diet-induced energy deficit in non-obese, young men, but does not appear to mitigate lower-body functional decline. Funding:The research reported in this publication is supported by the Collaborative Research to Optimize Warfighter Nutrition II and III projects and the Joint Program Committee-5, funded by the US Department of Defense. Declaration of Interest: J.C.W., O.T.C., and K.M.G. reported that their institution received funding from the US Department of Defense for work associated with this publication. H.R.L. reported receiving personal fees from Pfizer, Inc., for work outside this publication. All remaining authors declare no competing interests. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. Ethical Approval: This study was approved by the Pennington Biomedical Research Center (PBRC) Institutional Review Board and the Human Research Protection Office of the US Army Medical Research and Materiel Command.

BackgroundPeople aging with HIV are living with increased risk for functional decline compared with uninfected adults of the same age. Early preclinical changes in biomarkers in middle-aged individuals at risk for mobility and functional decline are needed. ObjectiveThis pilot study aims to compare measures of free-living activity with lab-based measures. In addition, we aim to examine differences in the activity level and patterns by HIV status. MethodsForty-six men (23 HIV+, 23 HIV−) currently in the MATCH (Muscle and Aging Treated Chronic HIV) cohort study wore a consumer-grade wristband accelerometer continuously for 3 weeks. We used free-living activity to calculate the gait speed and time spent at different activity intensities. Accelerometer data were compared with lab-based gait speed using the 6-minute walk test (6-MWT). Plasma biomarkers were measured and biobehavioral questionnaires were administered. ResultsHIV+ men more often lived alone (P=.02), reported more pain (P=.02), and fatigue (P=.048). In addition, HIV+ men had lower blood CD4/CD8 ratios (P

BACKGROUND People aging with HIV are living with increased risk for functional decline compared with uninfected adults of the same age. Early preclinical changes in biomarkers in middle-aged individuals at risk for mobility and functional decline are needed. OBJECTIVE This pilot study aims to compare measures of free-living activity with lab-based measures. In addition, we aim to examine differences in the activity level and patterns by HIV status. METHODS Forty-six men (23 HIV+, 23 HIV−) currently in the MATCH (Muscle and Aging Treated Chronic HIV) cohort study wore a consumer-grade wristband accelerometer continuously for 3 weeks. We used free-living activity to calculate the gait speed and time spent at different activity intensities. Accelerometer data were compared with lab-based gait speed using the 6-minute walk test (6-MWT). Plasma biomarkers were measured and biobehavioral questionnaires were administered. RESULTS HIV+ men more often lived alone (P=.02), reported more pain (P=.02), and fatigue (P=.048). In addition, HIV+ men had lower blood CD4/CD8 ratios (P CONCLUSIONS Accelerometer-based assessment of gait speed and activity patterns are lower for asymptomatic men living with HIV compared with uninfected controls and may be useful as preclinical digital biomarkers that precede differences captured in lab-based measures.

BACKGROUND: Human immunodeficiency virus (HIV)–infected individuals are at increased risk of age-associated functional impairment, even with effective antiretroviral therapy (ART). A concurrent characterization of skeletal muscle, physical function, and immune phenotype in aviremic middle-aged HIV-infected adults represents a knowledge gap in prognostic biomarker discovery. METHODS: We undertook a prospective observational study of 170 middle-aged, HIV-infected ambulatory men and women with CD4(+) T-cell counts of at least 350/µL and undetectable plasma viremia while on effective ART, and uninfected control participants. We measured biomarkers for inflammation and immune activation, fatigue, the Veterans Aging Cohort Study mortality index, and physical function. A subset also received a skeletal muscle biopsy and computed tomography scan. RESULTS: Compared to the uninfected participants, HIV-infected participants displayed increased immune activation (P < .001), inflammation (P = .001), and fatigue (P = .010), and in a regression model adjusting for age and sex displayed deficits in stair-climb power (P < .001), gait speed (P = .036), and predicted metabolic equivalents (P = .019). Skeletal muscle displayed reduced nuclear peroxisome proliferator-activated receptor-γ coactivator 1α–positive myonuclei (P = .006), and increased internalized myonuclei (P < .001) that correlated with immune activation (P = .003) and leukocyte infiltration (P < .001). Internalized myonuclei improved a model for HIV discrimination, increasing the C-statistic from 0.84 to 0.90. CONCLUSIONS: Asymptomatic HIV-infected middle-aged adults display atypical skeletal muscle profiles, subclinical deficits in physical function, and persistent inflammation and immune activation. Identifying biomarker profiles for muscle dysregulation and risk for future functional decline in the HIV-infected population will be key to developing and monitoring preventive interventions. CLINICAL TRIALS REGISTRATION: NCT03011957.

Human immunodeficiency virus (HIV)-infected individuals are at increased risk of age-associated functional impairment, even with effective antiretroviral therapy (ART). Whether accelerated aging occurs in chronic HIV infection and the biological mechanisms that underlay aging with chronic HIV infection remains unclear. We undertook a prospective observational study of 170 middle-aged, HIV-infected ambulatory men and women with CD4+ T-cell counts of at least 350 per ml and undetectable plasma viremia while on effective antiretroviral therapy, and uninfected control participants. We measured biomarkers for inflammation and immune activation, fatigue, the Veterans Aging Cohort Study (VACS) mortality index, and conducted physical function assessment and accelerometer tracking. A subset of participants also received a skeletal muscle biopsy and computerized tomography scan. Compared to the uninfected, HIV+ participants displayed increased immune activation (P

Slower gait speed is a well-established indicator for increased risk of morbidity and mortality. However, gold standard measurement methods like the six-minute walk test (6MWT), are difficult to use in a clinical setting and may not capture important differences in free-living, volitional activity. We enrolled 46 men (23 HIV+, 23 HIV-) from a prospective observational study (MATCH) of men and women, 50–65 years old. Participants wore an activity tracker (i.e., Nokia Pulse Ox) for three weeks. Tracker data was used to create average gait speed and a measure of percent time at quartiles of maximum observed gait speed. Lab-based, 6MWT gait speed did not differ significantly by HIV status. Free-living, tracker assessed gait speed was significantly slower among HIV+ compared to uninfected participants. HIV+ participants also spent more time at lower activity levels. Free-living gait speed may provide an important digital biomarker of risks not detected by traditional lab-based measures.

Background The physiological consequences of severe energy deficit include hypogonadism and the loss of fat-free mass. Prolonged energy deficit also impacts physical performance, mood, attentiveness, and decision-making capabilities. This study will determine whether maintaining a eugonadal state during severe, sustained energy deficit attenuates physiological decrements and maintains mental performance. This study will also assess the effects of normalizing testosterone levels during severe energy deficit and recovery on gut health and appetite regulation. Methods Fifty physically active men will participate in a 3-phase, randomized, placebo-controlled study. After completing a 14-d, energy-adequate, diet acclimation phase (protein: 1.6 g ∙ kg− 1 ∙ d− 1; fat: 30% total energy intake), participants will be randomized to undergo a 28-d, 55% energy deficit phase with (DEF + TEST: 200 mg testosterone enanthate per week) or without (DEF) exogenous testosterone. Diet and physical activity will be rigorously controlled. Recovery from the energy deficit (ad libitum diet, no testosterone) will be assessed until body mass has been recovered within ± 2.5% of initial body mass. Body composition, stable isotope methodologies, proteomics, muscle biopsies, whole-room calorimetry, molecular biology, activity/sleep monitoring, personality and cognitive function assessments, functional MRI, and comprehensive biochemistries will be used to assess physiological and psychological responses to energy restriction and recovery feeding while volunteers are in an expected hypogonadal versus eugonadal state. Discussion The Optimizing Performance for Soldiers (OPS) study aims to determine whether preventing hypogonadism will mitigate declines in physical and mental function that typically occur during prolonged energy deficit, and the efficacy of testosterone replacement on recovery from severe underfeeding. Trial Registration: NCT02734238 .

HIV-infected individuals may be at increased risk of poor physical function. Chronic inflammation has been associated with decreased physical function in the elderly and may also influence physical function in HIV-infected individuals.This cross-sectional study assessed physical function in 65 HIV-infected women aged 40 and older on stable antiretroviral treatment using the Short Physical Performance Battery (SPPB): a standardized test of balance, walking speed, and lower- extremity strength developed for elderly populations. The relationship between low SPPB score, selected demographic and medical characteristics, and high inflammatory biomarker profile was analyzed using Fisher's exact test and Wilcoxon rank sum test.The median age of subjects was 49 years (interquartile range [IQR] 45-55), and the median CD4 T-cell count was 675 cells/mm(3) (IQR 436-828). Thirteen subjects (20%) had a low SPPB score. Subjects with a low SPPB score were more likely to be cigarette smokers (p=0.03), had more medical comorbidities (p=0.01), and had higher levels of interleukin-6 (IL-6) (p0.05). They also tended to be older (median age 55 vs. 48, p=0.06), more likely to have diabetes (p=0.07), and have higher levels of soluble tumor necrosis factor-1 (p=0.09).Twenty percent of women aged 40 and older with well-treated HIV had poor physical-function performance, which was associated with the high burden of comorbidities in this population and with increased IL-6. However, it is unclear from this cross-sectional study whether increased inflammation was related to poor physical function or to other factors, such as age and medical comorbidities.

Objectives To determine reference values for laboratory tests in individuals aged 85 and older. Design Cross-sectional cohort study. Setting International. Participants Long Life Family Study (LLFS) participants (N~5,000, age: range 25–110, median 67, 45% male). Measurements Serum biomarkers were selected based on association with aging-related diseases and included complete blood count, lipids (triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total cholesterol), 25-hydroxyvitamin D2 and D3, vitamin D epi-isomer, diabetes mellitus–related biomarkers (adiponectin, insulin, insulin-like growth factor 1, glucose, glycosylated hemoglobin, soluble receptor for advanced glycation endproduct), kidney disease–related biomarkers (albumin, creatinine, cystatin), endocrine biomarkers (dehydroepiandrosterone, sex-hormone binding globulin, testosterone), markers of inflammation (interleukin 6, high-sensitivity C-reactive protein, N-terminal pro b-type natriuretic peptide), ferritin, and transferrin. Results Of 38 measured biomarkers, 34 were significantly correlated with age. Summary statistics were generated for all biomarkers according to sex and 5-year age increments from 50 and up after excluding participants with diseases and treatments that were associated with biomarkers. A biomarker data set was also generated that will be useful for other investigators seeking to compare biomarker levels between studies. Conclusion Levels of several biomarkers change with older age in healthy individuals. The descriptive statistics identified herein will be useful in future studies and, if replicated in additional studies, might also become useful in clinical practice. The availability of the reference data set will facilitate appropriate calibration of biomarkers measured in different laboratories.

Bayesian networks are probabilistic models that represent complex distributions in a modular way and have become very popular in many fields. There are many methods to build Bayesian networks from a random sample of independent and identically distributed observations. However, many observational studies are designed using some form of clustered sampling that introduces correlations between observations within the same cluster and ignoring this correlation typically inflates the rate of false positive associations. We describe a novel parameterization of Bayesian networks that uses random effects to model the correlation within sample units and can be used for structure and parameter learning from correlated data without inflating the Type I error rate. We compare different learning metrics using simulations and illustrate the method in two real examples: an analysis of genetic and non-genetic factors associated with human longevity from a family-based study and an example of risk factors for complications of sickle cell anemia from a longitudinal study with repeated measures.

The review articles in this issue provide an improved appreciation for microRNA (miRNA) as an essential feature of lineage commitment and regulatory guidance during tissue development that, when absent or hampered, often lead to disease states. In the coming years, there is much to be learned about adaptive (and maladaptive) states by examining how the expression of miRNAs is influenced by the genetic architecture of miR genes, clusters, and mirtrons, as well as miRNA polymorphism and polymorphism in their mRNA targets. We are also introduced to several modes of miRNA regulation (negative feedback, positive feedback, and cross regulatory) that monitor, modulate, or resolve signaling pathways in a variety of biologic processes that include sepsis response, fibrosis, acute exercise, and steroid biology. Perhaps the homeostasis or micromanagement of these miRNA regulatory systems, when perturbed, arrive at new stable networked interactions that have an undesired effect of promoting or antagonizing disease severity and cancer progression. Clearly, a better understanding of these miRNA regulatory networks, as well as improved therapeutic tools for guiding miRNA expression and their targets toward desired outcomes, will be the subject of many advances in miRNA biology over the coming years.

Efficient muscle regeneration requires the clearance of dead and dying tissue via phagocytosis before remodeling. We have previously shown that mice lacking stem cell antigen-1 (Sca-1) display a defect in skeletal muscle regeneration characterized by increased fibrosis and decreased turnover of the extracellular matrix. In the present study we demonstrate that Sca-1−/− mice have a defect in their capacity to recruit soluble IgM, and subsequently C3 complement, to damaged muscle. We hypothesize that this defect in recruitment delays or decreases phagocytosis by macrophages, contributing to the previously observed fibrotic phenotype of these mice. As the primary source of soluble IgM is peritoneal B-1a cells, which are a subset of self-renewing B cells, we analyzed this cell population and observed a significant reduction in B-1a cells in Sca-1−/− animals. Interestingly, these mice are protected from ischemia-reperfusion injury, an acute inflammatory reaction also mediated by IgM and C3 complement that has been linked to a deficit in B-1a cells in previous studies. Collectively, these data reveal a novel role for Sca-1 in innate immunity during muscle regeneration and indicate that further elucidation of immuno-myogenic processes will help to better understand and promote muscle regeneration.

Sca-1 (stem cell antigen-1) is a member of the Ly-6 family of proteins and regulates cell proliferation, differentiation, and self-renewal in multiple tissues. In skeletal muscle, Sca-1 inhibits both proliferation and differentiation of myogenic cells. Sca-1 expression is dynamically regulated during muscle regeneration, and mice lacking Sca-1 display increased fibrosis following muscle injury. Here, we show that Sca-1 expression is negatively regulated by TGF-β1 and that this inhibition is dependent on Smad3. We demonstrate that levels of TGF-β1 in skeletal muscle rapidly increase on injury and that the majority of this TGFβ1 is produced by infiltrating macrophages. Sca-1 is expressed in multiple cell types, and we demonstrate that TGF-β1 represses Sca-1 expression in T cells and other immune cell populations derived from the spleen, indicating that regulation by TGF-β1 is a general feature of Sca-1 expression in multiple cell types. Elucidation of the mechanisms by which Sca-1 expression is regulated may aid in the understanding of muscle homeostasis, potentially identifying novel therapeutic targets for muscle diseases.—Long, K., Montano, M., Pavlath, G. K. Sca-1 is negatively regulated by TGF-β1 in myogenic cells.

Muscle atrophy remains a significant concern in multiple inflammatory conditions, including injury, sepsis, cachexia, and HIV‐associated wasting. Herein, we show that inflammatory stressors, including TNF‐α, IFN‐γ, or lipopolysaccharide, potently induced the novel expression of the RNA editor ADAR1, an observation not previously described in muscle cells. We also observed that cytokine stimulation suppressed muscle‐associated microRNAs, an observation also not previously demonstrated. To map potential effects of ADAR1 induction in the muscle program, we conducted knockdown and overexpression studies in the mouse C2C12 muscle precursor cell (MPC) line and in primary human MPCs. We show that knockdown of stress‐induced ADAR1 increased inflammation‐mediated declines in the muscle differentiation markers Myogenin and myosin heavy chain, and knockdown reduced levels of active phosphorylated Akt (phospho‐Akt), but had no effect on microRNA transcript levels, suggesting a role for ADAR1 in buffering inflammatory stress effects on myogenic transcription and protein synthesis pathways. In addition, overexpression of recombinant ADAR1 suppressed active phosphorylated double‐stranded RNA (dsRNA)‐dependent protein kinase (phospho‐PKR), consistent with a role for ADAR1 in limiting inflammation‐driven catabolic atrophy pathways. Collectively, these data identify a novel regulatory role for ADAR1 activation under inflammatory stress to both promote muscle protein synthesis pathways and limit atrophy pathways. Clin Trans Sci 2010; Volume 3: 73–80

BACKGROUND: Infants of human immunodeficiency virus (HIV)-infected women have high mortality, but the immunologic integrity and protection afforded by the breast milk of HIV-infected women is unknown. METHODS: We determined morbidity and mortality by 24 months among breast-fed infants of 588 HIV-infected and 137 HIV-uninfected women followed-up in a clinical trial in Botswana. A matched case-control study compared clinical, behavioral, and breast milk immunologic parameters among 120 HIV-infected women by infant outcome. Breast milk factors were also compared between HIV-infected and HIV-uninfected women. RESULTS: Twenty-four-month mortality was 29.5% among HIV-infected infants, 6.7% among HIV-exposed uninfected infants, and 1.6% among HIV-unexposed infants. No differences were detected in breast milk immunologic profiles of HIV-infected women whose infants were either ill or well. Discontinuation of breast-feeding was the strongest predictor of illness (P

Testosterone treatment induces erythrocytosis that could potentially affect blood viscosity and cardiovascular risk. We thus investigated the effects of testosterone administration on blood viscosity and erythrocyte deformability using mouse models. Blood viscosity, erythrocyte deformability, and hematocrits were measured in normal male and female mice, as well as in females and castrated males after short-term (2 wk) and long-term (5–7 mo) testosterone intervention (50 mg/kg, weekly). Castrated males for long-term intervention were studied in parallel with the normal males to assess the effect of long-term testosterone deprivation. An additional short-term intervention study was conducted in females with a lower testosterone dose (5 mg/kg). Our results indicate no rheological difference among normal males, females, and castrated males at steady-state. Short-term high-dose testosterone increased hematocrit and whole-blood viscosity in both females and castrated males. This effect diminished after long-term treatment, in association with increased erythrocyte deformability in the testosterone-treated mice, suggesting the presence of adaptive mechanism. Considering that cardiovascular events in human trials are seen early after intervention, rheological changes as potential mediator of vascular events warrant further investigation.

Neisseria gonorrhoeae is an obligate human pathogen that represents a significant health concern, particularly in the developing world. Although generally associated with an acute inflammatory infection of urogenital epithelia cells, infections have been noted in multiple tissues and many infected individuals can become asymptomatic carriers. Few studies of immune response to N. gonorrhoeae infection in peripheral blood have evaluated the production of T helper cytokines (TH1/TH2) induced early after infection. We developed a quantitative realtime PCR assay based on the gonococcal rmpIII gene to monitor dose-response effects of infection on cytokine release from peripheral blood mononuclear cells (PBMCs). We observed upregulation of CD69 transcription and surface CD25 expression on lymphocytes, consistent with early T-cell activation. We observed dosage-dependent transcription of the chemotactic factor IL-8 and previously unreported activation of the chemoattractant MCP-2. Multiplex analysis of broad cytokine protein production revealed a differential increase in the TH1 and TH2 associated cytokines: IL-2, IL-4, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha and MCP-1. Markov models of protein accumulation implicated a cross-regulated response to infection, notably for IL-8, IL-10 and IL-12. Taken together, the cytokine profile we observed early in response to whole gonococcal bacteria was broader than has been previously described and may have relevance for the contribution of antagonistic signaling events early in infection and in understanding peripheral immune mechanisms engaged to control infection.

Perinatal transmission of human immunodeficiency virus (HIV)-1 represents a major problem in many regions of the world, especially Southern Africa. With the exception of viral and proviral load, the role for maternal cofactors in perinatal transmission outcome is largely unknown. In this study, an assessment was made of peripheral blood mononuclear cells (PBMC) gene-expression profiles to better understand transcriptional changes associated with HIV-1 infection and perinatal transmission among young adult mothers with infants in Botswana. Peripheral blood mononuclear cells specimens were used from 25 HIV+ drug naive and 20 HIV− healthy mothers, similar in age and location, collected in 1999–2000 and 2003, and processed with the exact same methods, as previously described. Expression profiling of 22 277 microarray gene probes implicated a broad initiation of innate response gene-sets, including toll-like receptor, interferon-stimulated and antiviral RNA response pathways in association with maternal HIV-1 infection. Maternal transmission status was further associated with host genes that influence RNA processing and splicing patterns. In addition to real-time polymerase chain reaction validation of specific genes, enriched category validation of PBMC profiles was conducted using two independent data sets for either HIV-1 infection or an unrelated RNA virus, severe acute respiratory virus infection. HIV-1 pathogen-specific host profiles should prove a useful tool in infection and transmission intervention efforts worldwide. Supplementary information The online version of this article (doi:10.1038/sj.gene.6364297) contains supplementary material, which is available to authorized users.

The association between sexual activity and human herpes virus-8 (HHV-8) infection has been established, but the mode of acquisition is still unclear. Blood samples from 238 individuals from Northern Cameroon were tested to evaluate the incidence of herpes simplex virus-2 (HSV-2), human immunodeficiency virus (HIV), Treponema pallidum, and HHV-8 infections and to identify their possible association. The presence of HSV-2 antibodies was associated significantly with gender, age, and HIV, HHV-8 antilatent, and T. pallidum antibodies, but not with HHV-8 antilytic antibodies. In a multivariate model older age, female gender, seropositivity for HIV, for HHV-8 latent antigens and for T. pallidum were associated independently with seropositivity for HSV-2. HSV-2-seropositive individuals had significantly higher titers of antibodies to both lytic (P = 0.019) and latent (P = 0.021) HHV-8 antigens. These results suggest that HSV-2 infection can contribute to sexual transmission of HHV-8 infection. J. Med. Virol. 74:467–472, 2004. © 2004 Wiley-Liss, Inc.

The major group of human immunodeficiency viruses (HIV-1) that comprise the current global pandemic have diversified during their worldwide spread and may be divided into at least 10 distinct subtypes or clades, A through J. Subtype B predominates in North America and Europe, subtype E predominates in Southeast Asia, and subtype C predominates in sub-Saharan Africa. Functional distinctions in long terminal repeat (LTR) architecture among HIV subtypes have been identified, thus raising the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. In addition to the transcriptional specificity of the HIV-1 LTR, productive HIV-1 replication is also dependent upon the viral Tat protein. Therefore, we sought to investigate whether interactions between host signaling pathways and the NF-kappaB regions of different HIV-1 subtypes, together with subtype-specific interactions between Tat, TAR, and cellular proteins, modulate the efficiency of HIV-1 clade-specific gene transcription. We demonstrate that the NF-kappaB sites of subtypes B and E both bind NF-kappaB-related complexes. However, the duplicated kappaB sites of the C subtype do not compete for NF-kappaB binding. Also, clade E Tat protein possesses the highest transactivation capacity, regardless of the LTR context. Furthermore, preliminary evidence suggests that the acetylation of subtype-specific Tat proteins may correlate with their transactivation efficiency.