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1.  Cycling Memory CD4+ T Cells in HIV Disease Have a Diverse T Cell Receptor Repertoire and a Phenotype Consistent with Bystander Activation 
Journal of Virology  2014;88(10):5369-5380.
The mechanisms of increased memory CD4+ T cell cycling in HIV disease are incompletely understood but have been linked to antigen stimulation, homeostatic signals, or exposure to microbial products and the inflammatory cytokines that they induce. We examined the phenotype and Vβ family distribution in cycling memory CD4+ T cells among 52 healthy and 59 HIV-positive (HIV+) donors. Cycling memory CD4+ T cells were proportionally more frequent in subjects with HIV infection than in controls, more often expressed CD38 and PD-1, and less frequently expressed OX40 and intracellular CD40L. OX40 expression on memory CD4+ T cells was induced in vitro by anti-CD3, interleukin-2 (IL-2), IL-7, or IL-15 but not by Toll-like receptor ligands. In HIV+ donors, memory CD4+ T cell cycling was directly related to plasma lipopolysaccharide (LPS) levels, to plasma HIV RNA levels, and to memory CD8+ T cell cycling and was inversely related to peripheral blood CD4+ T cell counts but not to the levels of IL-2, IL-7, or IL-15, while in HIV-negative donors, memory CD4+ T cell cycling was related to IL-7 levels and negatively related to the plasma levels of LPS. In both controls and HIV+ donors, cycling memory CD4+ T cells had a broad distribution of Vβ families comparable to that of noncycling cells. Increased memory CD4+ T cell cycling in HIV disease is reflective of generalized immune activation and not driven primarily by cognate peptide stimulation or exposure to common gamma-chain cytokines. This cycling may be a consequence of exposure to microbial products, to plasma viremia, or, otherwise, to proinflammatory cytokines.
IMPORTANCE This work provides evidence that the increased memory CD4+ T cell cycling in HIV infection is not a result of cognate peptide recognition but, rather, is more likely related to the inflammatory environment of HIV infection.
PMCID: PMC4019138  PMID: 24522925
2.  Cytomegalovirus-specific responses of CD38+ memory T cells are skewed towards IFN-γ and dissociated from CD154 in HIV-1 infection 
AIDS (London, England)  2014;28(3):311-316.
Despite the strong correlation of T-cell CD38 expression with HIV disease progression, evidence linking CD38 expression and dysfunction at the single cell level is scant. Since CD38+ memory CD4+ T cells, especially those from HIV-infected persons, fail to induce CD154 (CD40L) while responding to a superantigen with interferon (IFN)-γ or interleukin (IL)-2, we aimed to determine if recall responses to cytomegalovirus (CMV) were similarly affected in the CD38+ memory CD4+ T-cell subpopulation.
Design and methods
Peripheral blood mononuclear cells from HIV+ patients and healthy controls were incubated 14 h with CMV antigens, the superantigen Staphylococcus aureus enterotoxin B or medium, and labeled for identification of central memory (TCM) and effector memory (TEM) CD4+ T cells, and for the intracellular detection of induced CD154, IFN-γ and/or IL-2 by flow cytometry.
Compared with CD38− cells, CD38+ TCM cells from patients had less CD40L induction after CMV stimulation, and increased IFN-γ response. Patients’ CD38+ TEM cells showed a lower IL-2 response, and tended to have a greater IFN-γ response, in which CD154 induction frequently failed. CMV-specific responses of patients’ CD38+ TCM and TEM cells were dominated by IFN-γ, and almost all IL-2+ cells co-expressed IFN-γ. IL-2 responses to the polyclonal activator S. aureus enterotoxin B were also significantly less frequent among CD38+ TCM and TEM cells than in CD38− cells.
Patients’ CD38+ memory CD4+ T-cell responses to CMV favor the effector cytokine IFN-γ over IL-2, in the context of deficient CD154 induction, which may limit co-stimulation, proliferation and survival.
PMCID: PMC4109327  PMID: 24594993
AIDS; CD38 antigen; HIV; immune activation; immunologic memory; T cell
3.  Rosuvastatin Treatment Reduces Markers of Monocyte Activation in HIV-Infected Subjects on Antiretroviral Therapy 
Soluble CD14, a marker of monocyte activation and independent predictor of mortality in HIV disease, is reduced by rosuvastatin treatment. Monocyte tissue factor expression in HIV-infected subjects is reduced by rosuvastatin treatment, potentially reducing thrombotic risk.
Background. Statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have anti-inflammatory effects that are independent of their lipid-lowering properties. Despite suppressive antiretroviral therapy (ART), elevated levels of immune activation and inflammation often persist.
Methods. The Stopping Atherosclerosis and Treating Unhealthy Bone With Rosuvastatin in HIV (SATURN-HIV) trial is a randomized, double-blind, placebo-controlled study, designed to investigate the effects of rosuvastatin (10 mg/daily) on markers of cardiovascular disease risk in ART-treated human immunodeficiency virus (HIV)–infected subjects. A preplanned analysis was to assess changes in markers of immune activation at week 24. Subjects with low-density lipoprotein cholesterol <130 mg/dL and heightened immune activation (%CD8+CD38+HLA-DR+ ≥19%, or plasma high-sensitivity C-reactive protein ≥2 mg/L) were randomized to receive rosuvastatin or placebo. We measured plasma (soluble CD14 and CD163) and cellular markers of monocyte activation (proportions of monocyte subsets and tissue factor expression) and T-cell activation (expression of CD38, HLA-DR, and PD1).
Results. After 24 weeks of rosuvastatin, we found significant decreases in plasma levels of soluble CD14 (−13.4% vs 1.2%, P = .002) and in proportions of tissue factor–positive patrolling (CD14DimCD16+) monocytes (−38.8% vs −11.9%, P = .04) in rosuvastatin-treated vs placebo-treated subjects. These findings were independent of the lipid-lowering effect and the use of protease inhibitors. Rosuvastatin did not lead to any changes in levels of T-cell activation.
Conclusions. Rosuvastatin treatment effectively lowered markers of monocyte activation in HIV-infected subjects on antiretroviral therapy.
Clinical Trials Registration NCT01218802.
PMCID: PMC3905756  PMID: 24253250
HIV-1; monocytes; tissue factor; rosuvastatin
4.  HIV-1 is not a major driver of increased plasma IL-6 levels in chronic HIV-1 disease 
Increased plasma IL-6 levels have been associated with HIV-1 disease progression risk, yet the drivers of IL-6 production in HIV-1 infection are not known. This study was designed to explore the relationship between HIV-1 replication and IL-6 induction.
Correlations between plasma levels of IL-6 and HIV-1 RNA were examined in two clinical studies. To more directly assess the induction of IL-6 by HIV-1, several cell and tissue types that support HIV-1 replication in vivo were infected with HIV-1 and expression of IL-6 was measured.
Spearman’s rank correlations were used to examine the relationship between plasma levels of IL-6 and HIV-1 RNA. Macrophages, and colonic and lymph node histocultures were infected with HIV-1 or stimulated with bacterial products, LPS or flagellin, and IL-6 levels in supernatant were measured by ELISA or multiplex bead assay.
In the clinical studies there was weak or no correlation between plasma levels of IL-6 and HIV-1 RNA but IL-6 levels were correlated with plasma levels of the LPS coreceptor CD14. Macrophages stimulated with LPS or flagellin showed robust production of IL-6, but there was no increase in IL-6 production after HIV-1 infection. IL-6 expression was not increased in lymph node histocultures obtained from HIV-1 infected subjects nor after productive HIV-1 infection of colonic or lymph node histocultures ex vivo.
We find no evidence that HIV-1 replication is an important driver of IL-6 expression in vivo or in in vitro systems.
PMCID: PMC3458159  PMID: 22659649
IL-6; HIV-1 RNA; histocultures; macrophages; LPS; flagellin
5.  Perivascular Fat, Inflammation, and Cardiovascular Risk in HIV-infected Patients on Antiretroviral Therapy 
International journal of cardiology  2013;168(4):4039-4045.
HIV-infection is characterized by chronic immune activation that persists despite effective antiretroviral therapy (ART) and is associated with elevated cardiovascular risk. Whether specific perivascular fat depots are associated with inflammation in HIV is unknown.
In a cross-sectional study, epicardial (EAT) and thoracic periaortic (TAT) adipose tissue volume were measured by computed tomography in 100 HIV-infected adults, on stable ART, with LDL-cholesterol ≤130mg/dL and evidence of heightened T-cell activation (CD8+CD38+HLA−DR+ ≥19%) or increased inflammation (high sensitivity C-reactive protein ≥2mg/L).
Overall, 77% were male and 70% African American. Mean (standard deviation) age and body mass index were 47 (10) years and 28 (6.4) kg/m2, respectively. All subjects had HIV-1 RNA <1,000 copies/mL with mean (standard deviation) CD4+ T cell count of 665 (280) cells/μL; 50% were on a protease inhibitor. EAT and TAT were correlated with each other (r=0.766, p<0.0001). Both were associated with metabolic syndrome, atherogenic lipid profile, insulin resistance, total and central body fat, serum biomarkers of inflammation, and soluble CD163, but not with cellular immune activation markers. In multivariable models that adjusted for age, sex, and other measures of adiposity, both perivascular fat depots were independently associated with the presence of coronary calcium.
Perivascular fat is associated with soluble CD163, biomarkers of inflammation, insulin resistance, and subclinical atherosclerosis in this population of virologically suppressed HIV-infected patients on ART. The association of perivascular fat with coronary artery calcification appears to be independent of other measures of adiposity.
PMCID: PMC3805774  PMID: 23886531
Adipose tissue; Atherosclerosis; HIV; Inflammation; Macrophages
Molecular pharmaceutics  2013;10(10):10.1021/mp3007242.
5P12-RANTES is a recently developed chemokine analog that has shown high level protection from SHIV infection in macaques. However, the feasibility of using 5P12-RANTES as a long term HIV prevention agent has not been explored partially due to the lack of available delivery devices that can easily be modified for long-term release profiles. Glycosaminoglycans (GAGs) have been known for their affinity for various cytokines and chemokines, including native RANTES, or CCL5. In this work, we investigated used of GAGs in generating a chemokine drug delivery device. Initial studies used surface plasmon resonance analysis to characterize and compare the affinities of different GAGs to 5P12-RANTES. These different GAGs were then incorporated into drug delivery polymeric hydrogels to engineer sustained release of the chemokines. In vitro release studies of 5P12-RANTES from the resulting polymers were performed and we found that 5P12-RANTES release from these polymers can be controlled by the amount and type of GAG incorporated. Polymer disks containing GAGs with stronger affinity to 5P12-RANTES resulted in more sustained, and longer term release than did polymer disks containing GAGs with weaker 5P12-RANTES affinity. Similar trends were observed by varying the amount of GAGs incorporated into the delivery system. 5P12-RANTES released from these polymers demonstrated good levels of CCR5 blocking, retaining activity even after 30 days of incubation.
PMCID: PMC3886841  PMID: 23859720
HIV; drug delivery; prevention; microbicide; chemokine; CCL5; CCR5; glycosaminoglycans; heparin
7.  Dendritic cell recruitment in response to skin antigen tests in HIV-1 infected individuals correlates with the level of T cell infiltration 
AIDS (London, England)  2013;27(7):1071-1080.
To study whether in vivo recruitment of dendritic cells (DCs) in response to antigen administration in the skin is altered during HIV-1 infection.
Skin punch biopsies were collected from HIV-1+ as well as seronegative individuals at 48 hours post intradermal injection of inactivated antigens of mumps virus, Candida albicans or purified protein derivate (PPD) from Mycobacterium tuberculosis.
Cryosections were analyzed by in situ staining and computerized imaging.
Control skin biopsies showed that there was no difference in the number of skin-resident DCs between seronegative and HIV-1+ individuals. Antigen injection resulted in substantial infiltration of DCs compared to the frequencies found in donor-matched control skin. In HIV-1+ individuals, CD123+/CD303+ plasmacytoid DCs and CD11c+ myeloid DCs, including the CD141+ cross-presenting subset, were recruited at lower levels compared to healthy controls in response to PPD and mumps but not C. albicans. The level of DC recruitment correlated with the frequencies of T cells infiltrating the respective antigen sites. Ki67+ cycling T cells at the injection sites were much more frequent in response to each of the antigens in the HIV-1+ individuals, including those with AIDS, compared to healthy controls.
Multiple DC subsets infiltrate the dermis in response to antigen exposure. There was no obvious depletion or deficiency in mobilization of DCs in response to antigen skin tests during chronic HIV-1 infection. Instead, the levels of antigen-specific memory T cells that accumulate at the antigen site may determine the level of DC infiltration.
PMCID: PMC4176731  PMID: 23324660
HIV-1; dendritic cells; plasmacytoid; skin; skin test; delayed-type hypersensitivity reaction; Ki67
8.  HIV Pathogenesis: The Host 
Human immunodeficiency virus (HIV) pathogenesis has proven to be quite complex and dynamic with most of the critical events (e.g., transmission, CD4+ T-cell destruction) occurring in mucosal tissues. In addition, although the resulting disease can progress over years, it is clear that many critical events happen within the first few weeks of infection when most patients are unaware that they are infected. These events occur predominantly in tissues other than the peripheral blood, particularly the gastrointestinal tract, where massive depletion of CD4+ T cells occurs long before adverse consequences of HIV infection are otherwise apparent. Profound insights into these early events have been gained through the use of nonhuman primate models, which offer the opportunity to examine the early stages of infection with the simian immunodeficiency virus (SIV), a close relative of HIV that induces an indistinguishable clinical picture from AIDS in Asian primate species, but importantly, fails to cause disease in its natural African hosts, such as sooty mangabeys and African green monkeys. This article draws from data derived from both human and nonhuman primate studies.
During the first few weeks of HIV infection, massive depletion of CD4+ T cells occurs in the gastrointestinal tract, leading to a leaky gut.
PMCID: PMC3426821  PMID: 22951442
9.  Limited HIV Infection of Central Memory and Stem Cell Memory CD4+ T Cells Is Associated with Lack of Progression in Viremic Individuals 
PLoS Pathogens  2014;10(8):e1004345.
A rare subset of HIV-infected individuals, designated viremic non-progressors (VNP), remain asymptomatic and maintain normal levels of CD4+ T-cells despite persistently high viremia. To identify mechanisms potentially responsible for the VNP phenotype, we compared VNPs (average >9 years of HIV infection) to HIV-infected individuals who have similar CD4+ T-cell counts and viral load, but who are likely to progress if left untreated (“putative progressors”, PP), thus avoiding the confounding effect of differences related to substantial CD4+ T cell depletion. We found that VNPs, compared to PPs, had preserved levels of CD4+ stem cell memory cells (TSCM (p<0.0001), which was associated with decreased HIV infection of these cells in VNPs (r = −0.649, p = 0.019). In addition, VNPs had decreased HIV infection in CD4+ central memory (TCM) cells (p = 0.035), and the total number of TCM cells was associated with increased proliferation of memory CD4+ T cells (r = 0.733, p = 0.01). Our results suggest that, in HIV-infected VNPs, decreased infection of CD4+ TCM and TSCM, cells are involved in preservation of CD4+ T cell homeostasis and lack of disease progression despite high viremia.
Author Summary
Here we assessed correlates of protection from disease progression in a rare subset of HIV-infected individuals, viremic non-progressors (VNP). These individuals have high viral load for several years. In contrast to the majority of infected individuals, however, these individuals do not progress to AIDS. Here we found this lack of progression was associated with selective preservation of two critical subsets of memory CD4+ T cells, central memory (TCM) and stem-cell memory (TSCM) cells. Compared to HIV-infected putative progressors, VNPs had higher proliferation of these indispensable subsets of memory cells. In addition, the long-lived CD4+ TCM and TSCM cells in VNPs had decreased HIV infection compared to the less critical effector memory CD4+ T cells, which indicates a possible mechanism by which VNPs maintain their CD4+ T cell pool after several years of infection, and remain free from AIDS progression.
PMCID: PMC4148445  PMID: 25167059
10.  Plasma proteome analysis reveals overlapping, yet distinct mechanisms of immune activation in chronic HCV and HIV infections 
Human immunodeficiency virus (HIV) infection contributes to accelerated rates of progression of liver fibrosis during hepatitis C virus (HCV) infection, and HCV liver disease contributes to mortality during HIV infection. Although mechanisms underlying these interactions are not well known, soluble and cellular markers of immune activation associate with disease progression during both infections.
We identified proteins varying in expression across the plasma proteomes of subjects with untreated HIV infection, untreated HCV infection with low AST/platelet ratio-index (APRI), untreated HCV infection with high APRI, HIV-HCV co-infection, and controls. We examined correlations between dysregulated proteins and markers of immune activation to uncover biomarkers specific to disease states.
We observed the anticipated higher frequencies of HLADR+CD38+CD4 and CD8 T-cells, higher serum sCD14 levels, and higher serum IL-6 levels for HCV and HIV infected groups compared to controls. Plasma proteome analysis identified 2,297 peptides mapping to 227 proteins, and quantitative analysis of peptide intensity identified significant changes in 85 proteins across the five groups. Abundance for seven of these proteins was validated by ELISA. Forty-three of these proteins correlated with markers of immune activation, including at least two proteins that may directly drive T-cell activation. As a functional validation, we tested the enzymatic pathway product (lysophosphatidic acid, LPA) of one such protein, ENPP2, for ability to activate T-cells in vitro. LPA activated T-cells to express CD38 and HLA-DR.
These data indicate elevated levels of ENPP2 and LPA during advanced HCV disease may play a role in exacerbating immune activation during HCV-HIV co-infection.
PMCID: PMC3762939  PMID: 23507661
11.  Increased Levels of Human Beta-Defensins mRNA in Sexually HIV-1 Exposed But Uninfected Individuals 
Current HIV research  2008;6(6):531-538.
Protection against HIV-1 infection in exposed seronegative (ESN) individuals likely involves natural resistance mechanisms that have not been fully elucidated. Human beta defensins (HBD) are antimicrobial peptides found primarily in mucosae, the main ports of HIV entry. HBD-2 and 3 mRNA are induced by HIV-1 in human oral epithelial cells and exhibit strong anti-HIV-1 activity; in addition, polymorphisms in the DEFB1 gene, which encodes HBD-1, have been associated with resistance/susceptibility to different infections, including HIV-1. Here, we have assessed the association of HBD expression with the ESN phenotype. Peripheral blood and vaginal/endocervical and oral mucosal samples were taken from 47 ESN, 44 seropositive (SP) and 39 healthy controls (HC). HBD-1, 2 and 3 mRNA copy numbers were quantified by real time RT-PCR and A692G/G1654A/A1836G polymorphisms in the DEFB1 gene were detected by restriction fragment length polymorphisms and confirmed by nucleotide sequencing. ESN expressed significantly greater mRNA copy numbers of HBD-2 and 3 in oral mucosa than HC; p=0.0002 and p=0.007, respectively. mRNA copy numbers of HBD-1, 2 and 3 in vaginal/endocervical mucosa from ESN and HC were similar. Homozygosity for the A692G polymorphism was significantly more frequent in ESN (0.39) than in SP (0.05) (p=0.0002). In summary, ESN exhibited enhanced mucosal expression of the innate defense genes HBD-2 and 3; however, additional studies are required to verify these results and the potential association of the A692G polymorphism to the relative resistance of ESN to HIV-1 infection.
PMCID: PMC4126611  PMID: 18991618
HIV-1 (Human immunodeficiency virus type 1); human beta defensins; natural resistance; HIV-1-exposed sero-negatives; polymorphism; mucosa
12.  S-phase entry leads to cell death in circulating T cells from HIV-infected persons 
Journal of leukocyte biology  2008;83(6):1382-1387.
Central memory T cells are thought to play a critical role in memory T cell homoestasis by undergoing self-renewal and by maturating into effector T cells that mediate immunity at tissue sites. Circulating T cells in S phase of the cell cycle are found at increased frequencies during HIV infection and are predominantly composed of cells with a central memory phenotype. Here, we tested the hypothesis that CD4 and CD8 S-phase T cells have different capacities to complete cell cycle and survive. S-phase T cells in peripheral blood from HIV-infected donors were identified by incubating whole blood with BrdU ex vivo. Upon in vitro cultivation, S-phase T cells were more likely to die than to complete mitotic division. Intrinsic differences were observed between CD4 and CD8 S-phase T cells during incubation. Higher frequencies of CD4+ S-phase T cell underwent apoptosis after incubation in medium alone or after TCR stimulation, and CD4+ S-phase T cells were less readily induced to proliferate after incubation with IL-2 than were CD8+ S-phase T cells. CD4+ and CD8+ S-phase T cells expressed low levels of Bcl-2, which could contribute to their heightened susceptibility to cell death. Intrinsic differences in the proliferation and survival of CD4+ and CD8+ S-phase T cells could influence the homeostatic maintenance of these T cell subsets in HIV disease.
PMCID: PMC4126612  PMID: 18372341
T lymphocytes; apoptosis; proliferation
13.  Residual Immune Dysregulation Syndrome in Treated HIV infection 
Advances in immunology  2013;119:51-83.
Antiretroviral therapy has revolutionized the course of HIV infection, improving immune function and decreasing dramatically the mortality and morbidity due to the opportunistic complications of the disease. Nonetheless, even with sustained suppression of HIV replication, many HIV-infected persons experience a syndrome characterized by increased T cell activation and evidence of heightened inflammation and coagulation. This residual immune dysregulation syndrome or RIDS is more common in persons who fail to increase circulating CD4+ T cells to normal levels and in several epidemiologic studies it has been associated with increased morbidity and mortality. These morbid and fatal events are not the typical opportunistic infections and malignancies seen in the early AIDS era but rather comprise a spectrum of cardiovascular events, liver disease, metabolic disorders, kidney disease, bone disease, and a spectrum of malignant complications distinguishable from the opportunistic malignancies that characterized the earlier days of the AIDS epidemic.
While immune activation, inflammation, and coagulopathy are characteristic of untreated HIV infection and improve with drug-induced control of HIV replication, the drivers of RIDS in treated HIV infection are incompletely understood. And while inflammation, immune activation, and coagulopathy are more common in treated persons who fail to restore circulating CD4+ T cells, it is not entirely clear how these two phenomena are linked.
PMCID: PMC4126613  PMID: 23886064
14.  Associations of T cell activation and inflammatory biomarkers with virological response to darunavir/ritonavir plus raltegravir therapy 
One of the goals of antiretroviral therapy (ART) is to attenuate HIV-induced systemic immune activation and inflammation. We determined the dynamics of biomarkers of immune activation, microbial translocation and inflammation during initial ART with a nucleos(t)ide-sparing regimen of darunavir/ritonavir plus raltegravir. We also evaluated associations between these biomarkers and the virological response to the regimen.
We determined baseline and week 24 and 48 levels of CD4+ and CD8+ T cell activation (% HLA-DR+/CD38+), interleukin-6 (IL-6), interferon-γ-inducible protein-10 (IP-10), soluble CD14 (sCD14), D-dimer and lipopolysaccharide. Associations between the biomarkers at baseline were assessed using Spearman's rank correlation. The Wilcoxon signed rank test analysed changes from baseline. Comparisons between groups were made using the Wilcoxon rank sum test, and Cox proportional hazards models assessed predictors of virological failure (VF).
Assays were completed on 107 of 112 subjects after excluding five subjects who had only baseline samples. The subjects included were 94 (88%) men with a median age of 37 years, a median baseline CD4 count of 261.5 cells/mm3 and a median baseline viral load (VL) of 75 876 copies/mL. Subjects with a baseline VL >100 000 copies/mL had higher baseline T cell activation, IL-6, IP-10, sCD14 and D-dimer. These biomarkers declined during treatment (P < 0.05). Although subjects who experienced VF had higher baseline CD4+ T cell activation (P = 0.035), only baseline VL independently predicted VF (hazard ratio for >100 000 versus ≤100 000 copies/mL was 4.5–5.6, P ≤ 0.002).
Darunavir/ritonavir plus raltegravir attenuated immune activation, inflammation and microbial translocation. T cell activation remained higher in subjects with VF than those without. Baseline VL >100 000 copies/mL remained the primary driver of VF.
PMCID: PMC3716396  PMID: 23599363
nucleos(t)ide sparing; soluble CD14; microbial translocation
15.  Soluble CD14 is independently associated with coronary calcification and extent of subclinical vascular disease in treated HIV infection 
AIDS (London, England)  2014;28(7):969-977.
To use multimodality imaging to explore the relationship of biomarkers of inflammation, T-cell activation and monocyte activation with coronary calcification and subclinical vascular disease in a population of HIV-infected patients on antiretroviral therapy (ART).
A panel of soluble and cellular biomarkers of inflammation and immune activation was measured in 147 HIV-infected adults on ART with HIV RNA less than 1000 copies/ml and low-density lipoprotein cholesterol (LDL-C) 130 mg/dl or less. We examined the relationship of biomarkers to coronary calcium (CAC) score and multiple ultrasound measures of subclinical vascular disease.
Overall, median (interquartile range, IQR) age was 46 (40–53) years; three-quarters of participants were male and two-thirds African-American. Median 10-year Framingham risk score was 6%. Participants with CAC more than 0 were older, less likely to be African-American and had higher current and lower nadir CD4+ T-cell counts. Most biomarkers were similar between those with and without CAC; however, soluble CD14 was independently associated with CAC after adjustment for traditional risk factors. Among those with a CAC score of zero, T-cell activation and systemic inflammation correlated with carotid intima–media thickness and brachial hyperemic velocity, respectively. Compared with normal participants and those with CAC only, participants with increasing degrees of subclinical vascular disease had higher levels of sCD14, hs-CRP and fibrinogen (all P<0.05).
Soluble CD14 is independently associated with coronary artery calcification, and, among those with detectable calcium, predicts the extent of subclinical disease in other vascular beds. Future studies should investigate the utility of multimodality imaging to characterize vascular disease phenotypes in this population.
PMCID: PMC4097603  PMID: 24691204
carotid intima–media thickness; coronary artery calcium; endothelial function; HIV; inflammation; microbial translocation; soluble CD14
16.  Immunologic Failure Despite Suppressive Antiretroviral Therapy Is Related to Activation and Turnover of Memory CD4 Cells 
The Journal of Infectious Diseases  2011;204(8):1217-1226.
Background. Failure to normalize CD4+ T-cell numbers despite effective antiretroviral therapy is an important problem in human immunodeficiency virus (HIV) infection.
Methods. To evaluate potential determinants of immune failure in this setting, we performed a comprehensive immunophenotypic characterization of patients with immune failure despite HIV suppression, persons who experienced CD4+ T-cell restoration with therapy, and healthy controls.
Results. Profound depletion of all CD4+ T-cell maturation subsets and depletion of naive CD8+ T cells was found in immune failure, implying failure of T-cell production/expansion. In immune failure, both CD4+ and CD8+ cells were activated but only memory CD4+ cells were cycling at increased frequency. This may be the consequence of inflammation induced by in vivo exposure to microbial products, as soluble levels of the endotoxin receptor CD14+ and interleukin 6 were elevated in immune failure. In multivariate analyses, naive T-cell depletion, phenotypic activation (CD38+ and HLA-DR expression), cycling of memory CD4+ T cells, and levels of soluble CD14 (sCD14) distinguished immune failure from immune success, even when adjusted for CD4+ T-cell nadir, age at treatment initiation, and other clinical indices.
Conclusions. Immune activation that appears related to exposure to microbial elements distinguishes immune failure from immune success in treated HIV infection.
PMCID: PMC3218674  PMID: 21917895
17.  Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism 
Science translational medicine  2013;5(193):193ra91.
Progressive HIV infection is characterized by dysregulation of the intestinal immune barrier, translocation of immunostimulatory microbial products, and chronic systemic inflammation that is thought to drive progression of disease to AIDS. Elements of this pathologic process persist despite viral suppression during highly active antiretroviral therapy (HAART) and drivers of these phenomena remain poorly understood. Disrupted intestinal immunity can precipitate dysbiosis that induces chronic inflammation in the mucosa and periphery of mice. However, putative microbial drivers of HIV-associated immunopathology versus recovery have not been identified in humans. Using high-resolution bacterial community profiling, we identified a dysbiotic mucosal-adherent community enriched in Proteobacteria and depleted of Bacteroidia members that was associated with markers of mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects. Furthermore, this dysbiosis was evident among HIV-infected subjects undergoing HAART, and the extent of dysbiosis correlated with activity of the kynurenine pathway of tryptophan metabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Gut-resident bacteria with capacity to metabolize tryptophan through the kynurenine pathway were found to be enriched in HIV-infected subjects, strongly correlated with kynurenine levels in HIV-infected subjects, and capable of kynurenine production in vitro. These observations demonstrate a link between mucosal-adherent colonic bacteria and immunopathogenesis during progressive HIV infection, which is apparent even in the setting of viral suppression during HAART. This link suggests that gut-resident microbial populations may influence intestinal homeostasis during HIV disease.
PMCID: PMC4094294  PMID: 23843452
18.  Markers of inflammation and CD8+ T-cell activation, but not monocyte activation are associated with subclinical carotid artery disease in HIV 
HIV medicine  2013;14(6):385-390.
To explore the relationships between lymphocyte and monocyte activation, inflammation, and subclinical vascular disease among HIV-1 infected patients on antiretroviral therapy.
Baseline mean common carotid artery (CCA) intima-media thickness (IMT) and carotid plaque (IMT >1.5cm) were evaluated in the first 60 subjects enrolled in the Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN in HIV (SATURN-HIV) trial. All subjects were adults, on stable ART with evidence of heightened T-cell activation (CD8+CD38+HLA-DR+ ≥19%) or increased inflammation (high sensitivity C-reactive protein ≥2mg/L). All had fasting LDL-cholesterol ≤130mg/dL.
78% were men and 65% African-American. Median (IQR) age and CD4+ count were 47(43,52) years and 648(511, 857) cells/µL, respectively. All had HIV-1 RNA<400 cps/mL. Mean CCA-IMT was correlated with log-transformed CD8+CD38+HLA-DR+% (r=0.326, p=0.043), interleukin-6 (r=0.283, p=0.028), soluble vascular cell adhesion molecule (sVCAM, r=0.434, p=0.004), tumor necrosis factor-α receptor-I (TNFR-I, r=0.591, p=<0.0001) and fibrinogen (r=0.257, p=0.047). After adjustment for traditional CVD risk factors, the association with TNFR-I (p=0.007) and fibrinogen (p=0.033) remained significant. Subjects with plaque (n=22, 37%) were older [51(7.7) vs. 43(9.4) years, mean(SD), p=0.002], had higher CD8+CD38+HLA-DR+% [31(24, 41) vs. 23(20,29)%, median(IQR), p=0.046] and higher sVCAM [737(159) vs. 592(160) ng/mL, p=0.008] compared to those without plaque. Pro-inflammatory monocyte subsets and serum markers of monocyte activation (soluble CD163 and soluble CD14) were not associated with CCA-IMT or plaque.
Participants in SATURN-HIV have a high level of inflammation and immune activation that is associated with subclinical vascular disease despite low serum LDL-C.
PMCID: PMC3640592  PMID: 23332012
T-cell activation; Monocyte activation; Inflammation; Carotid intima-media thickness; Subclinical atherosclerosis
19.  Haemophilia, human immunodeficiency virus and human immunodeficiency virus pathogenesis 
Thrombosis and Haemostasis  2010;104(5):911-914.
In July 1982, the occurrence of three cases of acquired immunodeficiency syndrome (AIDS) in men with haemophilia was an immediate signal to Oscar Ratnoff that AIDS was transmissible through blood products. Work that he led provided important and clear indication that the AIDS agent was transmissible through pooled plasma products and had rapidly infected many men who had haemophilia. Before the blood supply was protected, the risk for infection in haemophilia was related directly to the intensity of therapy with pooled anti-haemophilic factor concentrates. Studies performed among the small proportion of haemophiliacs who remained uninfected despite heavy exposure to these plasma products revealed that the rare protective genotype – homozygosity for the 32 base pair deletion in the CCR5 gene was heavily concentrated in this population. Among those who did not have this protective genotype, a state of diminished immune activation distinguished these high risk uninfected haemophiliacs from haemophiliacs who later acquired human immunodeficiency virus (HIV) infection and from healthy uninfected controls. Immune activation state may not only predict risk for HIV acquisition but also appears to be an important predictor and likely determinant of HIV disease progression. The potential drivers of immune activation in chronic HIV infection include HIV itself, other co-infecting pathogens, homeostatic responses to cytopenia as well as the recently recognised phenomenon of translocation of microbial products across a damaged gut mucosal surface. This latter process is particularly compelling as clinical studies have shown a good relationship between indices of microbial translocation and markers of both immune activation and T cell homeostasis in chronic HIV infection. More recently, we have also found evidence that these microbial products also may drive a heightened tendency to thrombus formation in HIV infection via induction of monocyte tissue factor expression. Thus systemic exposure to microbial elements that are translocated through a gut mucosa damaged in the first few weeks of HIV infection may contribute to the pathogenesis of both immune deficiency and the heightened risk for vascular events that have been noted in persons with HIV infection.
PMCID: PMC3394673  PMID: 20694275
Infectious diseases; immunity; viral infection
20.  Dissociation of CD154 and Cytokine Expression Patterns in CD38+ CD4+ Memory T Cells in Chronic HIV-1 Infection 
Expression of the activation antigen CD38 on T cells is a strong predictor of the risk of HIV disease progression, but it is not known whether CD38 is a marker or mediator of dysfunction. We examined the relationship between CD38 expression and responses to T-cell receptor stimulation in central memory and effector memory CD4+ T cells in HIV-infected persons and in healthy controls. Basal CD38 expression was preserved by blocking golgi transport with brefeldin A. Intracellular expression of interleukin 2, interferon γ, and CD154 was measured after stimulating peripheral blood mononuclear cells with the superantigen staphylococcal enterotoxin B with or without anti-CD28 costimulation. Interferon-γ responses were comparable or increased in stimulated CD38+ memory cells, and the interleukin 2 responses of costimulated CD38+ central memory cells were decreased in HIV infection. In CD38+ cells and especially in CD38+ cells of HIV-infected persons, stimulated memory cells more often failed to express CD154 (CD40 ligand) when induced to express cytokine. A dissociated cytokine and CD154 expression by memory CD4 T cells may impair interactions between T cells and antigen-presenting cells, contribute to impaired immunity and help explain the relationship between CD38 expression and disease progression in chronic HIV infection.
PMCID: PMC3375209  PMID: 20926955
AIDS; CD4+; T cell; immune activation
21.  Quantifying and Predicting the Effect of Exogenous Interleukin-7 on CD4+T Cells in HIV-1 Infection 
PLoS Computational Biology  2014;10(5):e1003630.
Exogenous Interleukin-7 (IL-7), in supplement to antiretroviral therapy, leads to a substantial increase of all CD4+ T cell subsets in HIV-1 infected patients. However, the quantitative contribution of the several potential mechanisms of action of IL-7 is unknown. We have performed a mathematical analysis of repeated measurements of total and naive CD4+ T cells and their Ki67 expression from HIV-1 infected patients involved in three phase I/II studies (N = 53 patients). We show that, besides a transient increase of peripheral proliferation, IL-7 exerts additional effects that play a significant role in CD4+ T cell dynamics up to 52 weeks. A decrease of the loss rate of the total CD4+ T cell is the most probable explanation. If this effect could be maintained during repeated administration of IL-7, our simulation study shows that such a strategy may allow maintaining CD4+ T cell counts above 500 cells/µL with 4 cycles or fewer over a period of two years. This in-depth analysis of clinical data revealed the potential for IL-7 to achieve sustained CD4+ T cell restoration with limited IL-7 exposure in HIV-1 infected patients with immune failure despite antiretroviral therapy.
Author Summary
HIV infection is characterized by a decrease of CD4+ T-lymphocytes in the blood. Whereas antiretroviral treatment succeeds to control viral replication, some patients fail to reconstitute their CD4+ T cell count to normal value. IL-7 is a promising cytokine under evaluation for its use in HIV infection, in supplement to antiretroviral therapy, as it increases cell proliferation and survival. Here, we use data from three clinical trials testing the effect of IL-7 on CD4+ T-cell recovery in treated HIV-infected individuals and use a simple mathematical model to quantify IL-7 effects by estimating the biological parameters of the model. We show that the increase of peripheral proliferation could not explain alone the long-term dynamics of T cells after IL-7 injections underlining other important effects such as the improvement of cell survival. We also investigate the feasibility and the efficiency of repetitions of IL-7 cycles and argue for further evaluation through clinical trials.
PMCID: PMC4031052  PMID: 24853554
22.  HIV-Infected Individuals with Low CD4/CD8 Ratio despite Effective Antiretroviral Therapy Exhibit Altered T Cell Subsets, Heightened CD8+ T Cell Activation, and Increased Risk of Non-AIDS Morbidity and Mortality 
PLoS Pathogens  2014;10(5):e1004078.
A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm3) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.
Author Summary
The CD4/CD8 ratio, a hallmark of the collection of T cell defects related to aging –“immunosenescence”- and a predictor of mortality in the general population, often fails to normalize in an important proportion of HIV-infected individuals with adequate CD4+ T cell recovery after ART initiation. However, the immunological and clinical characteristics of this clinical phenotype have not been elucidated. Herein we show that during treated HIV infection, expansion of CD8+ T cells, reflected as a low CD4/CD8 ratio, identifies a subgroup of individuals with a number of immunological abnormalities and a poor prognosis. These subjects exhibit increased innate and adaptive immune activation, an immunosenescent phenotype, CD4+ and CD8+ imbalance in the gut mucosa and higher risk of morbidity and mortality. In contrast, those who normalize the CD4/CD8 ratio have traits of a healthy immune system. We observed that early ART initiation might contribute to more rapid and robust CD4/CD8 ratio normalization compared to later initiation. Hence, the CD4/CD8 ratio might help to further discriminate the risk of disease progression of successfully treated HIV-infected individuals, and a successful response to ART may require both normalization of the peripheral CD4+ T cell count and the ratio of CD4+ to CD8+ T cell counts.
PMCID: PMC4022662  PMID: 24831517
23.  HIV Pathogenesis: The Host 
Human immunodeficiency virus (HIV) pathogenesis has proven to be quite complex and dynamic with most of the critical events (e.g., transmission, CD4+ T-cell destruction) occurring in mucosal tissues. In addition, although the resulting disease can progress over years, it is clear that many critical events happen within the first few weeks of infection when most patients are unaware that they are infected. These events occur predominantly in tissues other than the peripheral blood, particularly the gastrointestinal tract, where massive depletion of CD4+ T cells occurs long before adverse consequences of HIV infection are otherwise apparent. Profound insights into these early events have been gained through the use of nonhuman primate models, which offer the opportunity to examine the early stages of infection with the simian immunodeficiency virus (SIV), a close relative of HIV that induces an indistinguishable clinical picture from AIDS in Asian primate species, but importantly, fails to cause disease in its natural African hosts, such as sooty mangabeys and African green monkeys. This article draws from data derived from both human and nonhuman primate studies.
PMCID: PMC3426821  PMID: 22951442
24.  Oral Mycobiome Analysis of HIV-Infected Patients: Identification of Pichia as an Antagonist of Opportunistic Fungi 
PLoS Pathogens  2014;10(3):e1003996.
Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases. Here, we used pyrosequencing to characterize the oral bacteriome and mycobiome of 12 HIV-infected patients and matched 12 uninfected controls. The number of bacterial and fungal genera in individuals ranged between 8–14 and 1–9, among uninfected and HIV-infected participants, respectively. The core oral bacteriome (COB) comprised 14 genera, of which 13 were common between the two groups. In contrast, the core oral mycobiome (COM) differed between HIV-infected and uninfected individuals, with Candida being the predominant fungus in both groups. Among Candida species, C. albicans was the most common (58% in uninfected and 83% in HIV-infected participants). Furthermore, 15 and 12 bacteria-fungi pairs were correlated significantly within uninfected and HIV-infected groups, respectively. Increase in Candida colonization was associated with a concomitant decrease in the abundance of Pichia, suggesting antagonism. We found that Pichia spent medium (PSM) inhibited growth of Candida, Aspergillus and Fusarium. Moreover, Pichia cells and PSM inhibited Candida biofilms (P = .002 and .02, respectively, compared to untreated controls). The mechanism by which Pichia inhibited Candida involved nutrient limitation, and modulation of growth and virulence factors. Finally, in an experimental murine model of oral candidiasis, we demonstrated that mice treated with PSM exhibited significantly lower infection score (P = .011) and fungal burden (P = .04) compared to untreated mice. Moreover, tongues of PSM-treated mice had few hyphae and intact epithelium, while vehicle- and nystatin-treated mice exhibited extensive fungal invasion of tissue with epithelial disruption. These results showed that PSM was efficacious against oral candidiasis in vitro and in vivo. The inhibitory activity of PSM was associated with secretory protein/s. Our findings provide the first evidence of interaction among members of the oral mycobiota, and identifies a potential novel antifungal.
Author Summary
Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases like oral candidiasis. Here we identify the core oral mycobiome (COM) and core oral bacteriome (COB) in HIV-infected and uninfected individuals, and demonstrate that the COM differs between these two groups. Decrease in abundance of Pichia (a resident oral fungus) in uninfected individuals coincided with increase in abundance of Candida, suggesting an antagonistic relationship. In vitro testing showed that Pichia spent medium (PSM) inhibits growth of pathogenic fungi; these findings were validated in an experimental mouse modal of oral candidiasis. The mechanism by which Pichia antagonizes Candida involves nutrient competition and secretory factor/s that inhibit the latter's ability to adhere, germinate, and form biofilms. This study is the first to characterize the mycobiome and the bacteriome in the oral cavity of HIV infected patients, and provides the first evidence that a fungus present in the same host microenvironment antagonizes Candida and identifies potential novel antifungal approach.
PMCID: PMC3953492  PMID: 24626467
25.  Comparison of Illumina and 454 Deep Sequencing in Participants Failing Raltegravir-Based Antiretroviral Therapy 
PLoS ONE  2014;9(3):e90485.
The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs.
A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser.
Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454.
In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.
PMCID: PMC3946168  PMID: 24603872

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