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1.  GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of T cell receptor signaling 
Viruses enter into complex interactions within human hosts leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, as expression of E2 inhibited T cell receptor (TCR)-mediated activation of tyrosine kinase (Lck). The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4+ and CD8+ T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.
doi:10.4049/jimmunol.1300589
PMCID: PMC3965330  PMID: 23686495
HIV; GBV-C; T cell activation; TCR; Envelope glycoprotein
2.  Treatment-related changes in serum lipids and inflammation: clinical relevance remains unclear. Analyses from the Women's Interagency HIV Study 
AIDS (London, England)  2013;27(9):1516-1519.
Summary
Among 127 HIV-infected women, the magnitude of HDLc increases after HAART initiation predicted the magnitude of concurrent decreases in inflammation biomarkers. After HAART initiation, changes in LDLc and inflammation were unrelated. In the same population, predicted risk of coronary heart disease based upon levels of standard clinical risk factors was similar before and after HAART treatment. Thus, it remains unknown whether short-term treatment-related changes in standard risk factors may appreciably change risk of CVD.
doi:10.1097/QAD.0b013e32835fd8a9
PMCID: PMC3909663  PMID: 23435295
lipids; HAART; HIV infection; inflammation
3.  Comparison of Lower Genital Tract Microbiota in HIV-Infected and Uninfected Women from Rwanda and the US 
PLoS ONE  2014;9(5):e96844.
Introduction
Previous studies have shown that alterations of the bacterial microbiota in the lower female genital tract influence susceptibility to HIV infection and shedding. We assessed geographic differences in types of genital microbiota between HIV-infected and uninfected women from Rwanda and the United States.
Methods
Genera of lower genital tract bacterial microbiota were identified by high-throughput pyrosequencing of the 16S rRNA gene from 46 US women (36 HIV-infected, 10 HIV-uninfected) and 40 Rwandan women (18 HIV-infected, 22 HIV-uninfected) with similar proportions of low (0–3) Nugent scores. Species of Lactobacillus were identified by assembling sequences along with reference sequences into phylogenetic trees. Prevalence of genera and Lactobacillus species were compared using Fisher's exact tests.
Results
Overall the seven most prevalent genera were Lactobacillus (74%), Prevotella (56%), Gardnerella (55%), Atopobium (42%), Sneathia (37%), Megasphaera (30%), and Parvimonas (26%), observed at similar prevalences comparing Rwandan to US women, except for Megasphaera (20% vs. 39%, p = 0.06). Additionally, Rwandan women had higher frequencies of Mycoplasma (23% vs. 7%, p = 0.06) and Eggerthella (13% vs. 0%, p = 0.02), and lower frequencies of Lachnobacterium (8% vs. 35%, p<0.01) and Allisonella (5% vs. 30%, p<0.01), compared with US women. The prevalence of Mycoplasma was highest (p<0.05) in HIV-infected Rwandan women (39%), compared to HIV-infected US women (6%), HIV-uninfected Rwandan (9%) and US (10%) women. The most prevalent lactobacillus species in both Rwandan and US women was L. iners (58% vs. 76%, p = 0.11), followed by L. crispatus (28% vs. 30%, p = 0.82), L. jensenii (20% vs. 24%, p = 0.80), L. gasseri (20% vs. 11%, p = 0.37) and L. vaginalis (20% vs. 7%, p = 0.10).
Discussion
We found similar prevalence of most major bacterial genera and Lactobacillus species in Rwandan and US women. Further work will be needed to establish whether observed differences differentially impact lower genital tract health or susceptibility to genital infections.
doi:10.1371/journal.pone.0096844
PMCID: PMC4016010  PMID: 24817204
4.  Regulatory B Cells Inhibit Cytotoxic T Lymphocyte (CTL) Activity and Elimination of Infected CD4 T Cells after In Vitro Reactivation of HIV Latent Reservoirs 
PLoS ONE  2014;9(4):e92934.
During HIV infection, IL-10/IL-10 receptor and programmed death-1 (PD-1)/programmed death-1-ligand (PD-L1) interactions have been implicated in the impairment of cytotoxic T lymphocyte (CTL) activity. Despite antiretroviral therapy (ART), attenuated anti-HIV CTL functions present a major hurdle towards curative measures requiring viral eradication. Therefore, deeper understanding of the mechanisms underlying impaired CTL is crucial before HIV viral eradication is viable. The generation of robust CTL activity necessitates interactions between antigen-presenting cells (APC), CD4+ and CD8+ T cells. We have shown that in vitro, IL-10hiPD-L1hi regulatory B cells (Bregs) directly attenuate HIV-specific CD8+-mediated CTL activity. Bregs also modulate APC and CD4+ T cell function; herein we characterize the Breg compartment in uninfected (HIVNEG), HIV-infected “elite controllers” (HIVEC), ART-treated (HIVART), and viremic (HIVvir), subjects, and in vitro, assess the impact of Bregs on anti-HIV CTL generation and activity after reactivation of HIV latent reservoirs using suberoylanilide hydroxamic acid (SAHA). We find that Bregs from HIVEC and HIVART subjects exhibit comparable IL-10 expression levels significantly higher than HIVNEG subjects, but significantly lower than HIVVIR subjects. Bregs from HIVEC and HIVART subjects exhibit comparable PD-L1 expression, significantly higher than in HIVVIR and HIVNEG subjects. SAHA-treated Breg-depleted PBMC from HIVEC and HIVART subjects, displayed enhanced CD4+ T-cell proliferation, significant upregulation of antigen-presentation molecules, increased frequency of CD107a+ and HIV-specific CD8+ T cells, associated with efficient elimination of infected CD4+ T cells, and reduction in integrated viral DNA. Finally, IL-10-R and PD-1 antibody blockade partially reversed Breg-mediated inhibition of CD4+ T-cell proliferation. Our data suggest that, possibly, via an IL-10 and PD-L1 synergistic mechanism; Bregs likely inhibit APC function and CD4+ T-cell proliferation, leading to anti-HIV CTL attenuation, hindering viral eradication.
doi:10.1371/journal.pone.0092934
PMCID: PMC3989168  PMID: 24739950
5.  Association of HIV clinical disease progression with profiles of early immune activation: results from a cluster analysis approach 
AIDS (London, England)  2013;27(9):1473-1481.
Objective
CD4 and CD8 T-cell activation are independent predictors of AIDS. The complete activation profile of both T-cell subtypes and their predictive value for AIDS risk is largely unknown.
Design
A total of 564 AIDS-free women in the Women's Interagency HIV Study were followed over 6.1 years (median) after T-cell activation assessment. A cluster analysis approach was used to evaluate the concurrent activation patterns of CD4 and CD8 T cells at the beginning of follow-up in relation to AIDS progression.
Methods
Percentages of CD4 and CD8 T cells with HLA-DR± and CD38± were assessed by flowcytometry. Eight immunologic variables (four on each CD4+ and CD8+: DR± and CD38±) were assessed to yield a 4-cluster solution on samples obtained before clinical endpoints. Proportional hazards survival regression estimated relative risks for AIDS progression by cluster membership.
Results
Compared with the other three clusters, outstanding activation features of each distinct cluster of women were: Cluster 1: higher CD8+CD38– DR– (average = 41% of total CD8 T-cell pool), CD4+CD38– DR– (average = 53% of total CD4 T-cell pool), and CD8+CD38– DR+ (28%); Cluster 2: higher CD8+CD38+DR– (44%) and CD4+CD38+DR– (58%); Cluster 3: higher CD8+CD38+DR+ (49%) and CD4+ CD38+DR– (48%); Cluster 4: higher CD8+CD38+DR+ (49%), CD4+CD38+DR+ (36%) and CD4+CD38– DR+ (19%). Compared with cluster 1, women in cluster 4 had two-fold increased risk of AIDS progression (Hazard ratio = 2.13; 95% confidence interval = 1.30–3.50) adjusted for CD4 cell count, HIV RNA, and other confounders.
Conclusion
A profile including CD4 and CD8 T-cell activation provided insight into HIV pathogenesis indicating concurrent hyperactivation of CD4 and CD8 T cells is associated with AIDS progression.
doi:10.1097/QAD.0b013e3283601bad
PMCID: PMC3949252  PMID: 23945505
AIDS; cluster analysis; immune activation
6.  Systematic Review of the Use of Dried Blood Spots for Monitoring HIV Viral Load and for Early Infant Diagnosis 
PLoS ONE  2014;9(3):e86461.
Background
Dried blood spots (DBS) have been used as alternative specimens to plasma to increase access to HIV viral load (VL) monitoring and early infant diagnosis (EID) in remote settings. We systematically reviewed evidence on the performance of DBS compared to plasma for VL monitoring and EID.
Methods and Findings
Thirteen peer reviewed HIV VL publications and five HIV EID papers were included. Depending on the technology and the viral load distribution in the study population, the percentage of DBS samples that are within 0.5 log of VL in plasma ranged from 52–100%. Because the input sample volume is much smaller in a blood spot, there is a risk of false negatives with DBS. Sensitivity of DBS VL was found to be 78–100% compared to plasma at VL below 1000 copies/ml, but this increased to 100% at a threshold of 5000 copies/ml. Unlike a plasma VL test which measures only cell free HIV RNA, a DBS VL also measures proviral DNA as well as cell-associated RNA, potentially leading to false positive results when using DBS. The systematic review showed that specificity was close to 100% at DBS VL above 5000 copies/ml, and this threshold would be the most reliable for predicting true virologic failure using DBS. For early infant diagnosis, DBS has a sensitivity of 100% compared to fresh whole blood or plasma in all studies.
Conclusions
Although limited data are available for EID, DBS offer a highly sensitive and specific sampling strategy to make viral load monitoring and early infant diagnosis more accessible in remote settings. A standardized approach for sampling, storing, and processing DBS samples would be essential to allow successful implementation.
Trial Registration
PROSPERO Registration #: CRD42013003621.
doi:10.1371/journal.pone.0086461
PMCID: PMC3945725  PMID: 24603442
7.  A Compositional Look at the Human Gastrointestinal Microbiome and Immune Activation Parameters in HIV Infected Subjects 
PLoS Pathogens  2014;10(2):e1003829.
HIV progression is characterized by immune activation and microbial translocation. One factor that may be contributing to HIV progression could be a dysbiotic microbiome. We therefore hypothesized that the GI mucosal microbiome is altered in HIV patients and this alteration correlates with immune activation in HIV. 121 specimens were collected from 21 HIV positive and 22 control human subjects during colonoscopy. The composition of the lower gastrointestinal tract mucosal and luminal bacterial microbiome was characterized using 16S rDNA pyrosequencing and was correlated to clinical parameters as well as immune activation and circulating bacterial products in HIV patients on ART. The composition of the HIV microbiome was significantly different than that of controls; it was less diverse in the right colon and terminal ileum, and was characterized by loss of bacterial taxa that are typically considered commensals. In HIV samples, there was a gain of some pathogenic bacterial taxa. This is the first report characterizing the terminal ileal and colonic mucosal microbiome in HIV patients with next generation sequencing. Limitations include use of HIV-infected subjects on HAART therapy.
Author Summary
Human immunodeficiency virus (HIV) infection related illness progresses despite the control of the virus itself by medications that stop the replication of the virus. This happens because the immune system gets activated. While the causes for such activation of the immune system are not exactly known, immune activation in HIV infection may be occurring as a result of bacteria or their products in the digestive tract. This study looks at the types of bacteria that reside in the lower intestinal tract in patients infected with human immunodeficiency virus, using state of the art sequencing technology, that can simultaneously look at thousands of bacteria. We have found that the bacteria at the end of the small bowel (an area also called the terminal ileum), at the right and left sides of the large intestine and in the stool is different in patients infected with the human immunodeficiency virus. HIV patients harbor more bacteria that have been linked to other human diseases and have been previously described as harmful. This finding is new and could open up a new frontier of study that could now pave the way to gain a deeper understanding of how the HIV causes illness.
doi:10.1371/journal.ppat.1003829
PMCID: PMC3930561  PMID: 24586144
8.  Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples 
PLoS ONE  2014;9(2):e85869.
Background
Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring.
Methods and Findings
A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10).
Conclusions
This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same technology platform to ensure appropriate interpretation of changes in VL.
Prospero registration # CD42013003603.
doi:10.1371/journal.pone.0085869
PMCID: PMC3928047  PMID: 24558359
9.  Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study 
PLoS ONE  2014;9(1):e85675.
Background
Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.
Methods and Findings
We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.
Conclusions
CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.
doi:10.1371/journal.pone.0085675
PMCID: PMC3893217  PMID: 24454917
10.  Regulation of Intestinal Immune Responses through TLR Activation: Implications for Pro- and Prebiotics 
The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs) are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC) and resident immune cells in the lamina propria. Many diseases, including chronic intestinal inflammation (e.g., inflammatory bowel disease), irritable bowel syndrome (IBS), allergic gastroenteritis (e.g., eosinophilic gastroenteritis and allergic IBS), and infections are nowadays associated with a deregulated microbiota. The microbiota may directly interact with TLR. In addition, differences in intestinal TLR expression in health and disease may suggest that TLRs play an essential role in disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is involved in either maintaining intestinal homeostasis or the induction of an inflammatory response. This mini review provides an overview of the current knowledge regarding the contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting intestinal inflammation, with a focus on food allergy. We will also highlight a potential role of the microbiota in regulating gut immune responses, especially through TLR activation.
doi:10.3389/fimmu.2014.00060
PMCID: PMC3927311  PMID: 24600450
toll-like receptors; intestinal epithelial cells; food allergy; microbiota; probiotics; prebiotics; circadian rhythm
11.  Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells 
AIDS (London, England)  2008;22(10):10.1097/QAD.0b013e3282fd6df4.
Objectives
Valproic acid and intensified antiretroviral therapy may deplete resting CD4+ T-cell HIV infection. We tested the ability of valproic acid to deplete resting CD4+ T-cell infection in patients receiving standard antiretroviral therapy.
Methods
Resting CD4+ T-cell infection was measured in 11 stably aviremic volunteers twice prior to, and twice after Depakote ER 1000 mg was added to standard antiretroviral therapy. Resting CD4+ T-cell infection frequency was measured by outgrowth assay. Low-level viremia was quantitated by single copy plasma HIV RNA assay.
Results
A decrease in resting CD4+ T-cell infection was observed in only four of the 11 patients. Levels of immune activation and HIV-specific T-cell response were low and stable. Valproic acid levels ranged from 26 to 96 μg/ml when measured near trough. Single copy assay was performed in nine patients. In three patients with depletion of resting CD4+ T-cell infection following valproic acid, single copy assay ranged from less than 1–5 copies/ml. Continuous low-level viremia was observed in three patients with stable resting CD4+ T-cell infection (24–87, 8–87, and 1–7 copies/ml respectively) in whom multiple samples were analyzed.
Conclusion
The prospective addition of valproic acid to stable antiretroviral therapy reduced the frequency of resting CD4+ T-cell infection in a minority of volunteers. In patients in whom resting CD4+ T-cell infection depletion was observed, viremia was rarely detectable by single copy assay.
doi:10.1097/QAD.0b013e3282fd6df4
PMCID: PMC3863687  PMID: 18525258
antiretroviral therapy; HIV; latency; resting CD4+ T cells; valproic acid
12.  Association of subclinical atherosclerosis with lipid levels amongst antiretroviral-treated and untreated HIV-infected women in the Women’s Interagency HIV Study 
Atherosclerosis  2012;225(2):408-411.
Objective
We examined serum lipids in association with carotid artery intima-media thickness (CIMT) in HIV-infected and HIV-uninfected women.
Methods
In 2003–4, among 1827 Women’s Interagency HIV Study participants, we measured CIMT and lipids (high-density lipoprotein cholesterol [HDL-c], low-density lipoprotein cholesterol [LDL-c], total cholesterol [TC], non-HDL-c). A subset of 520 treated HIV-infected women had pre-1997 lipid measures. We used multivariable linear regression to examine associations between lipids and CIMT.
Results
In HIV-uninfected women, higher TC, LDL-c and non-HDL-c were associated with increased CIMT. Among HIV-infected women, associations of lipids with CIMT were observed in treated but not untreated women. Among the HIV-infected women treated in 2003–4, CIMT was associated both with lipids measured a decade earlier in infection, and with late lipid measurements.
Conclusion
Among HIV-infected women, hyperlipidemia is most strongly associated with subclinical atherosclerosis in treated women. Among treated women, the association appeared strongest early in the disease course.
doi:10.1016/j.atherosclerosis.2012.09.035
PMCID: PMC3696584  PMID: 23089369
cardiovascular diseases; carotid arteries; HAART; HIV; lipids
13.  Biology of Mucosally Transmitted Sexual Infection—Translating the Basic Science into Novel HIV Intervention: A Workshop Summary 
AIDS Research and Human Retroviruses  2012;28(11):1389-1396.
Abstract
A group of over 200 international scientists came together on April 15 in Sydney, Australia just before the 2012 International Microbicides Conference as a part of a workshop to address the basic concepts and factors that modulate HIV infection at the mucosal surface. The meeting focused on defining the interaction between virus, prevailing host physiology, microbiota, and innate and adaptive immune responses and how they combine to impact the outcome at the moment of potential viral transmission. Speakers examined the biology of HIV entry during transmission, innate and natural antiviral mechanisms at the mucosa, microbicide efficacy, pharmacokinetic, and pharmacodynamics, animal models, and opportunities for combining HIV prevention strategies. Other viral infection models both in vivo and in vitro were considered for the insights they provided into HIV transmission events. The workshop raised important questions that we need to answer to further our basic understanding of host and viral factors influencing HIV transmission to inform the development of novel prevention strategies.
doi:10.1089/aid.2012.0276
PMCID: PMC3484787  PMID: 22966898
14.  Older HIV-infected patients on antiretroviral therapy have B-cell expansion and attenuated CD4 cell increases with immune activation reduction 
AIDS (London, England)  2013;27(10):1563-1571.
Background
The contribution of immune activation to accelerated HIV-disease progression in older individuals has not been delineated.
Methods
Prospective multicenter cohort of older (≥45 years) and younger (18–30 years) HIV-infected adults initiating 192 weeks of antiretroviral therapy (ART). Longitudinal models of CD4 cell restoration examined associations with age-group, thymic volume, immune activation, and viral load.
Results
Forty-five older and 45 younger adults (median age 50 and 26 years, respectively) were studied. Older patients had fewer naive CD4 cells (P <0.001) and higher HLA-DR/CD38 expression on CD4 (P = 0.05) and CD8 cells (P = 0.07) than younger patients at any time on ART. The rate of naive and total CD4 cell increase was similar between age groups, but older patients had a faster mean rate of B-cell increase (by +0.7 cells/week; P = 0.01), to higher counts than healthy controls after 192 weeks (P = 0.003). Naive CD4 increases from baseline were associated with immune activation reductions (as declines from baseline of %CD8 cells expressing HLA-DR/CD38; P <0.0001), but these increases were attenuated in older patients, or in those with small thymuses. A 15% reduction in activation was associated with naive gains of 29.9 and 6.2 cells/μl in younger, versus older patients, or with gains of 25.7, 23.4, and 2.1 cells/μl in patients with the largest, intermediate, and smallest thymuses, respectively (P <0.01 for interactions between activation reduction and age-group or thymic volume).
Conclusion
Older patients had significant B-cell expansion, higher levels of immune activation markers, and significantly attenuated naive CD4 cell gains associated with activation reduction.
doi:10.1097/QAD.0b013e32835fabc2
PMCID: PMC3805132  PMID: 24047762
aging; immune activation; immunosenescence; thymus
15.  The Role of Bacterial Vaginosis and Trichomonas in HIV Transmission Across The Female Genital Tract 
Current HIV research  2012;10(3):202-210.
Bacterial vaginosis (BV) and Trichomonas vaginalis (TV) infection are both very common and are associated with increased risk of sexual transmission of HIV. There are several mechanisms by which BV and TV could affect susceptibility including inducing pro-inflammatory cytokines and disrupting mucosal barrier function. This review highlights recent advances in our understanding of how these genital conditions lead to an increased risk of HIV infection in women.
PMCID: PMC3788616  PMID: 22384839
Bacterial vaginosis; HIV; Inflammation; Trichomonas vaginalis
16.  Longitudinal Assessment of Pigtailed Macaque Lower Genital Tract Microbiota by Pyrosequencing Reveals Dissimilarity to the Genital Microbiota of Healthy Humans 
AIDS Research and Human Retroviruses  2012;28(10):1244-1249.
Abstract
Vaginal bacterial communities play an important role in human health and have been shown to influence HIV infection. Pigtailed macaques (Macaca nemestrina) are used as an animal model of HIV vaginal infection of women. Since the bacterial microbiota could influence retrovirus infection of pigtailed macaques, the genital microbiota in 10 cycling macaques was determined by pyrosequencing. The microbiota of all macaques was polymicrobial with a median of 13 distinct genera. Strikingly, the genera Sneathia and Fusobacterium, both in the phylum Fusobacteria, accounted for 18.9% and 13.3% of sequences while the next most frequent were Prevotella (5.6%), Porphyromonas (4.1%), Atopobium (3.6%), and Parvimonas (2.6%). Sequences corresponding to Lactobacillus comprised only 2.2% of sequences on average and were essentially all L. amylovorus. Longitudinal sampling of the 10 macaques over an 8-week period, which spanned at least one full ovulatory cycle, showed a generally stable presence of the major types of bacteria with some exceptions. These studies show that the microbiota of the pigtailed macaques is substantially dissimilar to that found in most healthy humans, where the genital microbiota is usually dominated by Lactobacillus sp. The polymicrobial makeup of the macaque bacterial populations, the paucity of lactobacilli, and the specific types of bacteria present suggest that the pigtailed macaque microbiota could influence vaginal retrovirus infection.
doi:10.1089/aid.2011.0382
PMCID: PMC3448102  PMID: 22264029
17.  Baseline Levels of Soluble CD14 and CD16+56− Natural Killer Cells Are Negatively Associated With Response to Interferon/Ribavirin Therapy During HCV-HIV-1 Coinfection 
The Journal of Infectious Diseases  2012;206(6):969-973.
Disease progression of human immunodeficiency virus type 1 (HIV-1) is associated with immune activation. Activation indices are higher during coinfection of hepatitis C virus (HCV) and HIV. The effect of immune activation on interferon α (IFN-α) therapy response is unknown. We evaluated soluble CD14 (sCD14) and natural killer (NK)–cell subsets at baseline, and during pegIFN-α2a/ribavirin therapy in HCV-HIV coinfection. The sCD14 level increased during therapy. Baseline sCD14 positively correlated with baseline HCV level and CD16+56− NK-cell frequency, and both sCD14 and CD16+56− NK cells correlated negatively with magnitude of HCV decline. IL28B genotype was associated with therapy response but not sCD14 or CD16+56− NK frequency. Markers of innate immune activation predict poor host response to IFN-α–based HCV therapy during HCV-HIV coinfection.
doi:10.1093/infdis/jis434
PMCID: PMC3501153  PMID: 22782948
18.  Interleukin 10 Responses Are Associated With Sustained CD4 T-Cell Counts in Treated HIV Infection 
The Journal of Infectious Diseases  2012;206(5):780-789.
Background.Inflammation persists in treated human immunodeficiency virus (HIV) infection and may contribute to an increased risk for non–AIDS-related pathologies. We investigated the correlation of cytokine responses with changes in CD4 T-cell levels and coinfection with hepatitis C virus (HCV) during highly active antiretroviral treatment (HAART).
Methods.A total of 383 participants in the Women's Interagency HIV Study (212 with HIV monoinfection, 56 with HCV monoinfection, and 115 with HIV/HCV coinfection) were studied. HIV-infected women had <1000 HIV RNA copies/mL, 99.7% had >200 CD4 T cells/μL; 98% were receiving HAART at baseline. Changes in CD4 T-cell count between baseline and 2–4 years later were calculated. Peripheral blood mononuclear cells (PBMCs) obtained at baseline were used to measure interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), and tumor necrosis factor α (TNF-α) responses to Toll-like receptor (TLR) 3 and TLR4 stimulation.
Results.Undetectable HIV RNA (<80 copies/mL) at baseline and secretion of IL-10 by PBMCs were positively associated with gains in CD4 T-cell counts at follow-up. Inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α) were also produced in TLR-stimulated cultures, but only IL-10 was significantly associated with sustained increases in CD4 T-cell levels. This association was significant only in women with HIV monoinfection, indicating that HCV coinfection is an important factor limiting gains in CD4 T-cell counts, possibly by contributing to unbalanced persistent inflammation.
Conclusions.Secreted IL-10 from PBMCs may balance the inflammatory environment of HIV, resulting in CD4 T-cell stability.
doi:10.1093/infdis/jis380
PMCID: PMC3491747  PMID: 22693231
19.  A Pilot Trial of Adding Maraviroc to Suppressive Antiretroviral Therapy for Suboptimal CD4+ T-Cell Recovery Despite Sustained Virologic Suppression: ACTG A5256 
The Journal of Infectious Diseases  2012;206(4):534-542.
Background. Despite viral suppression, antiretroviral therapy (ART) does not restore CD4+ T-cell counts in many patients infected with human immunodeficiency virus type 1 (HIV-1).
Methods. In a single-arm pilot trial involving ART recipients with suppressed plasma levels of HIV-1 RNA for at least 48 weeks and stable suboptimal CD4+ T-cell recovery, subjects added maraviroc, a CCR5 antagonist, to their existing ART for 24 weeks. After stopping maraviroc, they were followed for an additional 24 weeks. A Wilcoxon signed-rank test was used to evaluate whether maraviroc was associated with an increase of at least 20 cells/µL in the CD4+ T-cell count.
Results. A total of 34 subjects were enrolled. The median age was 50 years, and the median baseline CD4+ T-cell count was 153 cells/µL. The median increase in CD4+ T-cell count from baseline to week 22/24 was 12 cells/µL (90% confidence interval, 1–22). A CD4+ T-cell count increase of at least 20 cells/µL was not detected (P = .97). Markers of immune activation and apoptosis decreased during maraviroc intensification; this decline partially reversed after discontinuing maraviroc.
Conclusions. Adding maraviroc to suppressive ART for 24 weeks was not associated with an increase in CD4+ T-cell counts of at least 20 cells/µL. Further studies of CCR5 antagonists in the dampening of immune activation associated with HIV infection are warranted.
Clinical Trials Registration. NCT 00709111.
doi:10.1093/infdis/jis376
PMCID: PMC3491731  PMID: 22740718
20.  Entrapment of recent thymic emigrants in lymphoid tissues from HIV-infected patients: association with HIV cellular viral load 
AIDS (London, England)  2002;16(16):2119-2127.
Objective(s)
Depletion of thymus derived naive T-cells is a feature of HIV infection. Here the impact of HIV infection on the compartmentalization of recent thymic emigrants of (RTE) and naive T-cells was examined.
Methods
Peripheral blood mononuclear cells (PBMC) and lymphoid tissue (LT) from 43 HIV-infected patients and 12 controls were examined for RTE distribution by measuring coding joint T-cell receptor excisional circles (cjTREC) by PCR and naive and memory T-cell subsets and adhesion molecules (L-selection, LFA-1) by flow cytometry.
Results
In HIV-infected patients, the RTE as quantified by cjTRECs in CD4 LT cells were significantly higher than in PBMC. Their values, however, were less than in control subjects, in both the LT and PBMC compartments. This was associated with an increase in L-selectin and LFA-1 expression on LT derived T cells. In PBMC, a significant positive relationship between TREC and naive CD4 cells and an inverse relationship between TREC and cellular viral load (CVL) was observed. Whereas in LT, there was a positive relationship between cjTREC and both naive CD4 cell percentage and CVL.
Conclusions
Collectively, the data suggests that LT is a significant reservoir for RTE. The RTE appeared to be entrapped in LT from HIV-infected subjects. Such entrapment is probably a response to the high viral load in these tissues. These observations may partially explain the decline in RTE observed in the peripheral blood of HIV-infected patients, and the delay in recovery of naive cells in blood after initiation of HAART.
PMCID: PMC3738871  PMID: 12409732
Naive T cells; recent thymic emigrants; TRECs; HIV; AIDS
21.  Early Immune Senescence in HIV Disease 
Current HIV/AIDS reports  2010;7(1):4-10.
Non-AIDS–defining comorbidities that occur despite viral suppression and immune reconstitution using antiretroviral therapy depict early aging process in HIV-infected individuals. During aging, a reduction in T-cell renewal, together with a progressive enrichment of terminally differentiated T cells, translates into a general decline of the immune system, gradually leading to immunosenescence. Inflammation is a hallmark of age-associated comorbidities, and immune activation is a hallmark of HIV disease. Constant stimulation of the immune system by HIV or due to co-infections activates the innate and adaptive immune system, resulting in release of mediators of inflammation. Immune activation coupled with lack of anti-inflammatory responses likely results in accelerated aging in HIV disease. Dysfunctional thymic output, along with HIV-mediated disruption of the gastrointestinal barrier leading to microbial translocation, contributes to the circulating antigenic load driving early senescence in HIV disease.
doi:10.1007/s11904-009-0038-4
PMCID: PMC3739442  PMID: 20425052
22.  Potential cardiovascular disease risk markers among HIV-infected women initiating antiretroviral treatment 
Background
Inflammation and hemostasis perturbation may be involved in vascular complications of HIV infection. We examined atherogenic biomarkers and subclinical atherosclerosis in HIV-infected adults before and after beginning highly-active antiretroviral therapy (HAART).
Methods
In the Women's Interagency HIV Study (WIHS), 127 HIV-infected women studied pre- and post-HAART were matched to HIV-uninfected controls. Six semi-annual measurements of soluble CD14, tumor necrosis factor (TNF)-alpha, soluble interleukin (IL)-2 receptor, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, D-dimer, and fibrinogen were obtained. Carotid artery intima-media thickness (CIMT) was measured by B-mode ultrasound.
Results
Relative to HIV-uninfected controls, HAART-naïve HIV-infected women had elevated levels of soluble CD14 (1945 vs 1662 ng/mL, Wilcoxon signed rank P<0.0001), TNF-alpha (6.3 vs 3.4 pg/mL, P<0.0001), soluble IL-2 receptor (1587 vs 949 pg/mL, P<0.0001), IL-10 (3.3 vs 1.9 pg/mL, P<0.0001), MCP-1 (190 vs 163 pg/mL, P<0.0001) and D-dimer (0.43 vs 0.31 µg/mL, P<0.01). Elevated biomarker levels declined after HAART. While most biomarkers normalized to HIV-uninfected levels, in women on effective HAART, TNF-alpha levels remained elevated compared to HIV-uninfected women (+0.8 pg/mL, P=0.0002). Higher post-HAART levels of soluble IL-2 receptor (P=0.02), IL-6 (P=0.05), and D-dimer (P=0.03) were associated with increased CIMT.
Conclusions
Untreated HIV infection is associated with abnormal hemostasis (e.g., D-dimer), and pro-atherogenic (e.g., TNF-alpha) and anti-atherogenic (e.g., IL-10) inflammatory markers. HAART reduces most inflammatory mediators to HIV-uninfected levels. Increased inflammation and hemostasis are associated with subclinical atherosclerosis in recently treated women. These findings have potential implications for long-term risk of cardiovascular disease in HIV-infected patients, even with effective therapy.
doi:10.1097/QAI.0b013e31825b03be
PMCID: PMC3400505  PMID: 22592585
antiretroviral therapy; cardiovascular diseases; cytokines; hemostasis; HIV; inflammation
23.  Cytomegalovirus Immunoglobulin G Antibody Is Associated With Subclinical Carotid Artery Disease Among HIV-Infected Women 
The Journal of Infectious Diseases  2012;205(12):1788-1796.
Background. Cytomegalovirus (CMV) infection has been implicated in immune activation and accelerated progression of immunodeficiency from human immunodeficiency virus (HIV) coinfection. We hypothesized that CMV is associated with vascular disease in HIV-infected adults.
Methods. In the Women's Interagency HIV Study, we studied 601 HIV-infected and 90 HIV-uninfected participants. We assessed the association of CMV immunoglobulin G (IgG) level with carotid artery intima-media thickness, carotid artery distensibility, Young's elastic modulus, and blood pressures. Multivariable models adjusted for age, race/ethnicity, smoking, diabetes, and body mass index.
Results. Mean CMV IgG levels were higher in HIV-infected women compared with HIV-uninfected women (P < .01). Among HIV-infected women, higher CMV IgG level was associated with decreased carotid artery distensibility (P < .01) and increased Young's modulus (P = .02). Higher CMV IgG antibody level was associated with increased prevalence of carotid artery lesions among HIV-infected women who achieved HIV suppression on antiretroviral therapy, but not among viremic or untreated HIV-infected women. Adjustment for Epstein–Barr virus antibody levels and C-reactive protein levels had no effect on the associations between CMV IgG levels and vascular parameters.
Conclusions. Cytomegalovirus antibody titers are increased in HIV-infected women and associated with subclinical cardiovascular disease. Host responses to CMV may be abnormal in HIV infection and associated with clinical disease.
doi:10.1093/infdis/jis276
PMCID: PMC3415890  PMID: 22492856
24.  CD28-Negative CD4+ and CD8+ T Cells in Antiretroviral Therapy–Naive HIV-Infected Adults Enrolled in Adult Clinical Trials Group Studies 
The Journal of Infectious Diseases  2012;205(11):1730-1738.
Background
Individuals infected with human immunodeficiency virus (HIV) have higher risk than HIV-negative individuals for diseases associated with aging. T-cell senescence, characterized by expansion of cells lacking the costimulatory molecule CD28, has been hypothesized to mediate these risks.
Methods
We measured the percentage of CD28−CD4+ and CD8+ T cells from HIV-infected treatment-naive adults from 5 Adult Clinical Trials Group (ACTG) antiretroviral therapy (ART) studies and the ALLRT (ACTG Longitudinal Linked Randomized Trials) cohort, and from 48 HIV-negative adults. Pretreatment and 96-week posttreatment %CD28− cells were assessed using linear regression for associations with age, sex, race/ethnicity, CD4 count, HIV RNA, ART regimen, and hepatitis C virus (HCV) infection.
Results
In total, 1291 chronically HIV-infected adults were studied. Pretreatment, lower CD4 count was associated with higher %CD28−CD4+ and %CD28−CD8+ cells. For CD8+ cells, younger age and HCV infection were associated with a lower %CD28−. ART reduced %CD28− levels at week 96 among virally suppressed individuals. Older age was strongly predictive of higher %CD28−CD8+. Compared to HIV-uninfected individuals, HIV-infected individuals maintained significantly higher %CD28−.
Conclusions
Effective ART reduced the proportion of CD28− T cells. However, levels remained abnormally high and closer to levels in older HIV-uninfected individuals. This finding may inform future research of increased rates of age-associated disease in HIV-infected adults.
doi:10.1093/infdis/jis260
PMCID: PMC3415854  PMID: 22448010
25.  Short Chain Fatty Acids Induce Pro-Inflammatory Cytokine Production Alone And In Combination With Toll-like Receptor Ligands 
Problem
Short chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti-inflammatory. BV, a common alteration of the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro-inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with TLR-ligands affected pro-inflammatory cytokine secretion.
Method of study
Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement.
Results
SCFAs, at 20mM, induced IL-8, IL-6, and IL-1β release while lower levels (0.02–2mM) did not induce cytokine secretion. Levels >20mM were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand- and TLR7 ligand-induced production of IL-8 and TNFα in a time- and dose-dependent manner, but had little effect on LPS-induced cytokine release. SCFAs mediated their effects on pro-inflammatory cytokine production at least in part by inducing generation of reactive oxygen species.
Conclusions
Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro-inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.
doi:10.1111/j.1600-0897.2011.01089.x
PMCID: PMC3288536  PMID: 22059850
Short chain fatty acids; inflammation; bacterial vaginosis

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