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1.  Moderate wine consumption is associated with lower hemostatic and inflammatory risk factors over 8 years: The study of women’s health across the nation (SWAN) 
Moderate wine consumption has been associated with reduced cardiovascular (CV) risk, but most investigations have been conducted in Caucasian populations. To investigate the relationship of wine consumption to CV risk markers, we studied a multi-ethnic sample of middle-aged, healthy women (N = 2900; 48% white, 28% black, 7% Hispanic, 8% Chinese, 9% Japanese) participating in SWAN over 7 years with repeated assessments of CV risk factors. Consumption of wine was stable and common with 20% reporting none, 69% light (<1/day), 7% moderate ( = 1/day), and 4% heavy (>1/day). To guard against underreporting, we took the maximum reported wine consumption over 7 years as the predictor. We used mixed models with a random intercept and random time to assess the relationship between wine consumption and CV risk factors with moderate consumption as the reference. Outcome variables were log-transformed where necessary. Longitudinal models were adjusted for ethnicity, age, and time-varying menopausal status, hormone therapy use, overall alcohol consumption, high density lipoprotein (HDL), statin use, and a healthy lifestyle score based on physical activity, not smoking, and weight maintenance. Interactions of wine consumption with time were not significant. Moderate wine consumers had significantly lower levels of C-reactive protein (CRP, p < .001), fibrinogen (p < .001), factor VII (p < .01), and plasminogen activator inhibitor (PAI-1, p < .05) than women who drank no or little wine. These associations were independent of significant effects of healthy lifestyle and overall alcohol consumption and similar across ethnic groups. Moderate wine consumption may protect against CVD via inflammatory and clotting pathways.
doi:10.3233/NUA-130034
PMCID: PMC4334149
Moderate wine consumption; inflammation; coagulation; longitudinal study; linear mixed models; midlife women
2.  MDSC: a new player in HIV immunopathogenesis 
AIDS (London, England)  2012;26(12):1567-1569.
doi:10.1097/QAD.0b013e328355e682
PMCID: PMC4324169  PMID: 22810370
cellular immunity; myeloid-derived suppressor cell; progressive HIV infection
3.  Cleavage/Alteration of Interleukin-8 by Matrix Metalloproteinase-9 in the Female Lower Genital Tract 
PLoS ONE  2015;10(1):e0116911.
Objective
Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration.
Study Design
Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA.
Results
IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs.
Conclusions
These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.
doi:10.1371/journal.pone.0116911
PMCID: PMC4303271  PMID: 25611319
4.  TLR2 activated B cells are phenotypically similar to the abnormal circulating B cells seen preceding the diagnosis of AIDS related non-Hodgkin lymphoma (NHL) diagnosis 
Background
AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is a common AIDS-defining cancer. Prior studies suggest that chronic B cell activation precedes AIDS-NHL diagnosis. Activation of B cells by multiple factors, including Toll-like receptor (TLR) signaling, leads to the expression of activation-induced cytidine deaminase (AID), a DNA mutating molecule that can contribute to oncogene translocations/mutations, leading to NHL. The goal of this study was to determine whether surface markers expressed on activated and/or germinal center (GC) B cells, and AID expression, were elevated on circulating B cells preceding AIDS-NHL, as well as to determine if TLR signaling contributes to this activated B cell phenotype.
Methods
Stored viable peripheral blood mononuclear cell (PBMC) specimens, obtained prior to AIDS-NHL diagnosis, were assessed by multi-color flow cytometry. Additionally, B cells isolated from PBMC were exposed to TLR ligands in vitro, after which B cell phenotype was assessed by flow cytometry.
Results
An elevated fraction of B cells expressing CD10, CD71, or CD86 was seen in those who went on to develop AIDS-NHL. AID expression was detected in some who developed AIDS-NHL, but not in HIV+ or HIV− controls. TLR2- stimulated purified B cells exhibited the activated B cell phenotype observed in HIV+ subjects prior to AIDS-NHL diagnosis.
Conclusions
These results indicate that an elevated fraction of B cells display an activated/GC phenotype in those HIV+ subjects who go on to develop AIDS-NHL, and suggest that TLR2-mediated activation may play a role in HIV infection-associated B cell activation, potentially contributing to the genesis of AIDS-NHL.
doi:10.1097/QAI.0b013e31829d4d50
PMCID: PMC3778065  PMID: 23722608
Immunophenotype; HIV; AIDS lymphoma; non-Hodgkin lymphoma; activation induced cytidine deaminase (AID); Toll-like receptor (TLR)
5.  A Role for TLR Signaling During B Cell Activation in Antiretroviral-Treated HIV Individuals 
AIDS Research and Human Retroviruses  2013;29(10):1353-1360.
Abstract
The mechanisms underlying B cell activation that persists during antiretroviral therapy (ART) are unknown. Toll-like receptor (TLR) signaling is a critical mediator of innate cell activation and though B cells express TLRs, few studies have investigated a role for TLR signaling in B cell activation during HIV infection. We addressed this question by assessing the activated phenotype and TLR expression/responsiveness of B cells from ART-treated HIV-infected subjects (HIVART+). We evaluated activation markers implicated in B cell-mediated T cell trans infection during HIV pathogenesis. We found no significant difference in TLR expression between B cells of HIVART+ and HIV− subjects. However, B cells of HIVART+ subjects exhibited heightened endogenous expression levels of IL-6 (p=0.0051), T cell cognate ligands CD40 (p=0.0475), CD54 (p=0.0229), and phosphorylated p38 (p<0.0001), a marker of TLR signaling. In vitro, B cells of HIVART+ individuals were less responsive to TLR stimulation compared to B cells of HIV− subjects. The activated phenotype of in vitro TLR-stimulated B cells of HIV− subjects was similar to ex vivo B cells from HIVART+ individuals. TLR2 stimulation was a potent mediator of B cell activation, whereas B cells were least responsive to TLR4 stimulation. Compared to HIV− subjects, the serum level of lipoteichoic acid (TLR2 ligand) in HIVART+ subjects was significantly higher (p=0.0207), correlating positively with viral load (p=0.0127, r=0.6453). Our data suggest that during HIV infection TLR-activated B cells may exert a pathogenic role and B cells from HIVART+ subjects respond to in vitro TLR stimulation, yet exhibit a TLR tolerant phenotype suggesting prior in vivo TLR stimulation.
doi:10.1089/aid.2013.0115
PMCID: PMC3785799  PMID: 23763346
6.  Older Age Is Associated with Peripheral Blood Expansion of Naïve B Cells in HIV-Infected Subjects on Antiretroviral Therapy 
PLoS ONE  2014;9(9):e107064.
Older HIV infected subjects were previously found to have significant B cell expansion during initial antiretroviral therapy in a prospective age-differentiated cohort of older and younger (≥45 vs. ≤30 years) HIV-infected subjects initiating antiretroviral therapy (ART) through the AIDS Clinical Trials Group. Here to further describe this expansion, using a subset of subjects from the same cohort, we characterized B cell phenotypes at baseline and after 192 weeks of ART in both older and younger HIV-infected groups and compared them to uninfected age-matched controls. We also examined whether phenotypes at baseline associated with response to tetanus and hepatitis A vaccine at 12 weeks. Forty six subjects were analyzed in the HIV infected group (21 older, 25 younger) and 30 in the control group (15 per age group). We observed naïve B cells to normalize in younger subjects after 192 weeks of ART, while in older subjects naïve B cells increased to greater levels than those of controls (p = 0.045). Absolute resting memory (RM) cell count was significantly lower in the older HIV infected group at baseline compared to controls and numbers normalized after 192 weeks of ART (p<0.001). Baseline RM cell count positively correlated with week 12 increase in antibody to tetanus vaccine among both younger and older HIV-infected subjects combined (p = 0.01), but not in controls. The age-associated naïve B cell expansion is a novel finding and we discuss several possible explanations for this observation. Relationship between RM cells at baseline and tetanus responses may lead to insights about the effects of HIV infection on B cell memory function and vaccine responses.
doi:10.1371/journal.pone.0107064
PMCID: PMC4160206  PMID: 25207968
7.  Virological and Immunological Characteristics of Human Cytomegalovirus Infection Associated With Alzheimer Disease 
The Journal of Infectious Diseases  2013;208(4):564-572.
Serum, cerebrospinal fluid (CSF), and cryopreserved lymphocytes from subjects in the Rush Alzheimer's Disease Center Religious Orders Study were analyzed for associations between cytomegalovirus (CMV) infection and clinical and pathological markers of Alzheimer disease. CMV antibody levels were associated with neurofibrillary tangles (NFTs). CSF interferon γ was only detected in seropositive subjects and was significantly associated with NFTs. The percentage of senescent T cells (CD4+ or CD8+CD28−CD57+) was significantly higher for CMV-seropositive as compared to CMV-seronegative subjects and was marginally associated with the pathologic diagnosis of Alzheimer disease (CD4+) or amyloid-β (CD8+). Immunocytochemical analysis showed induction of amyloid-β in human foreskin fibroblasts (HFFs) infected with each of 3 clinical CMV strains. In the same subjects, there was no association of herpes simplex virus type 1 (HSV-1) antibody levels with CMV antibody levels or clinical or pathological markers of Alzheimer disease. HSV-1 infection of HFFs did not induce amyloid-β. These data support an association between CMV and the development of Alzheimer disease.
doi:10.1093/infdis/jit210
PMCID: PMC3719902  PMID: 23661800
Alzheimer's disease; cytomegalovirus; interferon-gamma; CD28-/CD57+ T cells; amyloid-β
8.  Microbial Translocation and Liver Disease Progression in Women Coinfected With HIV and Hepatitis C Virus 
The Journal of Infectious Diseases  2013;208(4):679-689.
Background. Microbial translocation has been implicated in the pathogenesis of liver fibrosis and cirrhosis. We sought to determine whether markers of microbial translocation are associated with liver disease progression during coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV).
Methods. We measured serial plasma lipopolysaccharide (LPS), endotoxin core antibody, intestinal fatty acid–binding protein (I-FABP), soluble CD14 (sCD14), interleukin 6 (IL-6), interleukin 10, and tumor necrosis factor α (TNF-α) levels over a 5-year period in 44 HIV/HCV-coinfected women, 21 of whom experienced liver disease progression and 23 were nonprogressors.
Results. While LPS levels did not differ significantly over time between progressors and nonprogressors (P = .60), progressors had significantly higher plasma levels of sCD14, a marker of monocyte activation by LPS, at the first time point measured (P = .03) and throughout the study period (P = .001); progressors also had higher IL-6 and I-FABP levels over the 5-year study period (P = .02 and .03, respectively). The associations between progression and sCD14, I-FABP, and IL-6 levels were unchanged in models controlling for HIV RNA and CD4+ T-cell count.
Conclusions. Although LPS levels did not differ between liver disease progressors and nonprogressors, the association of sCD14, I-FABP, and IL-6 levels with liver disease progression suggests that impairment of gut epithelial integrity and consequent microbial translocation may play a role in the complex interaction of HIV and HCV pathogenesis.
doi:10.1093/infdis/jit225
PMCID: PMC3719907  PMID: 23687224
HIV; hepatitis C; microbial translocation; fibrosis; liver disease progression; soluble CD14
9.  Free Glycogen in Vaginal Fluids Is Associated with Lactobacillus Colonization and Low Vaginal pH 
PLoS ONE  2014;9(7):e102467.
Objective
Lactobacillus dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote Lactobacillus remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of Lactobacillus colonization and a low vaginal pH.
Methods
Free glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8–11 years.
Results
Free glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of Lactobacillus was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of L. crispatus and L. jensenii, but not L. iners.
Conclusion
These findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by Lactobacillus, suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact Lactobacillus colonization.
doi:10.1371/journal.pone.0102467
PMCID: PMC4102502  PMID: 25033265
10.  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
11.  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
12.  Early microbial translocation blockade reduces SIV-mediated inflammation and viral replication 
The Journal of Clinical Investigation  2014;124(6):2802-2806.
Damage to the intestinal mucosa results in the translocation of microbes from the intestinal lumen into the circulation. Microbial translocation has been proposed to trigger immune activation, inflammation, and coagulopathy, all of which are key factors that drive HIV disease progression and non-HIV comorbidities; however, direct proof of a causal link is still lacking. Here, we have demonstrated that treatment of acutely SIV-infected pigtailed macaques with the drug sevelamer, which binds microbial lipopolysaccharide in the gut, dramatically reduces immune activation and inflammation and slightly reduces viral replication. Furthermore, sevelamer administration reduced coagulation biomarkers, confirming the contribution of microbial translocation in the development of cardiovascular comorbidities in SIV-infected nonhuman primates. Together, our data suggest that early control of microbial translocation may improve the outcome of HIV infection and limit noninfectious comorbidities associated with AIDS.
doi:10.1172/JCI75090
PMCID: PMC4089442  PMID: 24837437
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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