Daniel E. Nixon

Inflammatory and Coagulation Biomarkers and Mortality in Patients with HIV Infection

Background In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]). We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis. Methods and Findings Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case–control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0–4.1; p = 0.05), 8.3 (95% CI, 3.3–20.8; p < 0.0001), and 12.4 (95% CI, 4.2–37.0; p < 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case–control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1–3.1; p = 0.02) to 1.5 (95% CI, 0.8–2.8) and 1.4 (95% CI, 0.8–2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively. Conclusions IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation. Trial Registration: ClinicalTrials.gov (NCT00027352).

Plasma Levels of Soluble CD14 Independently Predict Mortality in HIV Infection

BACKGROUND Chronic human immunodeficiency virus (HIV) infection is associated with intestinal permeability and microbial translocation that contributes to systemic immune activation, which is an independent predictor of HIV disease progression. The association of microbial translocation with clinical outcome remains unknown. METHODS This nested case-control study included 74 subjects who died, 120 of whom developed cardiovascular disease and 81 of whom developed AIDS during the Strategies for Management of Anti-Retroviral Therapy (SMART) study with matched control subjects. Intestinal fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), soluble CD14 (sCD14), endotoxin core antibody (EndoCAb), and 16S ribosomal DNA (rDNA) were measured in baseline plasma samples. RESULTS Subjects with the highest quartile of sCD14 levels had a 6-fold higher risk of death than did those in the lowest quartile (95% confidence interval, 2.2-16.1; P<.001), with minimal change after adjustment for inflammatory markers, CD4(+) T cell count, and HIV RNA level. No other marker was significantly associated with clinical outcomes. I-FABP, LPS, and sCD14 were increased and EndoCAb was decreased in study subjects, compared with healthy volunteers. sCD14 level correlated with levels of IL-6, C-reactive protein, serum amyloid A and D-dimer. CONCLUSIONS sCD14, a marker of monocyte response to LPS, is an independent predictor of mortality in HIV infection. Therapeutic attenuation of innate immune activation may improve survival in patients with HIV infection.

Inflammation, coagulation and cardiovascular disease in HIV-infected individuals.

Background The SMART study was a trial of intermittent use of antiretroviral therapy (ART) (drug conservation [DC]) versus continuous use of ART (viral suppression [VS]) as a strategy to reduce toxicities, including cardiovascular disease (CVD) risk. We studied the predictive value of high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6) and D-dimer with CVD morbidity and mortality in HIV-infected patients who were enrolled in SMART beyond other measured CVD risk factors. Methods A blood sample was available in 5098 participants who were enrolled in the SMART study for the measurement of IL-6, hsCRP and D-dimer. Hazard ratios (HR) with 95% CI for CVD events were estimated for each quartile (Q) for each biomarker vs the 1st quartile and for 1 SD higher levels. For both treatment groups combined, unadjusted and adjusted HRs were determined using Cox regression models. Results There were 252 participants who had a CVD event over a median follow-up of 29 months. Adjusted HRs (95% CI) for CVD for Q4 vs Q1 were 4.65 (2.61, 8.29), 2.10 (1.40, 3.16), and 2.14 (1.38, 3.33) for IL-6, hsCRP and D-dimer, respectively. Associations were similar for the DC and VS treatment groups (interaction p-values were >0.30). The addition of the three biomarkers to a model that included baseline covariates significantly improved model fit (p<0.001). Area under the curve (AUC) estimates improved with inclusion of the three biomarkers in a model that included baseline covariates corresponding to other CVD risk factors and HIV factors (0.741 to 0.771; p<0.001 for difference). Conclusions In HIV-infected individuals, IL-6, hsCRP and D-dimer are associated with an increased risk of CVD independent of other CVD risk factors. Further research is needed to determine whether these biomarkers can be used to improve CVD risk prediction among HIV positive individuals.

Biomarkers of immune dysfunction in HIV.

Purpose of reviewHIV infection is characterized by chronic immune system activation and inflammatory cytokine production. This review will highlight recent developments using plasma and cellular biomarkers of immune system activation and dysfunction to predict mortality and opportunistic disease in HIV-infected individuals. Recent findingsHIV infection results in features characteristic of early aging of the immune system or ‘immune senescence’, driven by chronic antigen exposure and immune system activation. Microbial translocation of gut bacterial components is associated with chronic immune activation and possibly systemic inflammation. Antiretroviral therapy may not fully normalize this condition. Baseline elevations of certain biomarkers of inflammation or coagulopathy, notably interleukin-6 (IL-6), C-reactive protein (CRP), and D-dimer, have been associated with mortality or opportunistic disease, after adjustment for appropriate variables, in several large randomized clinical trials. It is not known if elevated IL-6 or CRP causes this morbidity and mortality or if they are simply surrogate markers of a global inflammatory state. SummarySeveral inflammatory biomarkers appear to add to our ability to predict mortality or opportunistic disease in HIV-infected individuals. Before biomarkers will be useful, it will be necessary to identify interventions that moderate biomarker levels, and then determine if this moderation attenuates disease outcomes.

Changes in Inflammatory and Coagulation Biomarkers: A Randomized Comparison of Immediate versus Deferred Antiretroviral Therapy in Patients With Hiv Infection

Objectives:Among a subgroup of participants in the Strategies for Management of Antiretroviral Therapy (SMART) Trial that were naïve to antiretroviral therapy (ART) or off ART (6 months or longer) at study entry, risk of AIDS and serious non-AIDS events were increased for participants who deferred ART compared with those randomized to (re)initiate ART immediately. Our objective was to determine whether ART initiation in this group reduced markers of inflammation and coagulation that have been associated with increased mortality risk in SMART. Changes in these biomarkers have been described after stopping ART, but not after starting ART in SMART. Methods:Stored specimens for 254 participants (126 drug conservation [DC] and 128 viral suppression [VS]) who were naïve to ART or off ART (6 months or longer) were analyzed for interleukin-6, high sensitivity C-reactive protein, and D-dimer at baseline and Months 2 and 6. Results:At Month 6, 62% of the VS group had HIV RNA less than 400 copies/mL and median CD4 count was 190 cells/mm3 higher than for the DC group (590 versus 400 cells/mm3). Compared with DC, the VS group had 32% (95% confidence interval, 19%-43%) lower D-dimer levels at Month 6 (P < 0.001); differences were not significant for high sensitivity C-reactive protein or interleukin-6 levels. Conclusions:In this randomized comparison of immediate versus delayed ART initiation, D-dimer, but not interleukin-6 and high sensitivity C-reactive protein, declined significantly after starting ART. Further studies are needed to determine whether improvements in D-dimer are associated with reduced risk of clinical disease and whether adjunct treatments used in combination with ART can reduce inflammation among individuals with HIV infection.

The Ends on Herpesvirus DNA Replicative Concatemers Contain pac2 cis Cleavage/Packaging Elements and Their Formation Is Controlled by Terminal cis Sequences

ABSTRACT Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 andpac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found thatpac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminalcis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation ofpac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in whichpac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.

Determination of the Underlying Cause of Death in Three Multicenter International HIV Clinical Trials

Abstract Purpose: Describe processes and challenges for an Endpoint Review Committee (ERC) in determining and adjudicating underlying causes of death in HIV clinical trials. Method: Three randomized HIV trials (two evaluating interleukin-2 and one treatment interruption) enrolled 11,593 persons from 36 countries during 1999–2008. Three ERC members independently reviewed each death report and supporting source documentation to assign underlying cause of death; differences of opinion were adjudicated. Results: Of 453 deaths reported through January 14, 2008, underlying causes were as follows: 10% AIDS-defining diseases, 21% non-AIDS malignancies, 9% cardiac diseases, 9% liver disease, 8% non-AIDS-defining infections, 5% suicides, 5% other traumatic events/accidents, 4% drug overdoses/acute intoxications, 11% other causes, and 18% unknown. Major reasons for unknown classification were inadequate clinical information or supporting documentation to determine cause of death. Half (51%) of deaths reviewed by the ERC required follow-up adjudication; consensus was eventually always reached. Conclusion: ERCs can successfully provide blinded, independent, and systematic determinations of underlying cause of death in HIV clinical trials. Committees should include those familiar with AIDS and non-AIDS-defining diseases and have processes for adjudicating differences of opinion. Training for local investigators and procedure manuals should emphasize obtaining maximum possible documentation and follow-up information on all trial deaths.

HIV replication, inflammation, and the effect of starting antiretroviral therapy on plasma asymmetric dimethylarginine, a novel marker of endothelial dysfunction

Background:HIV infection is associated with premature development of cardiovascular disease. Understanding the effects of HIV replication on endothelial dysfunction and platelet activation may identify treatment targets to reduce cardiovascular disease risk. Methods:A subgroup of HIV-infected participants in the Strategies for Management of Antiretroviral Therapy study off antiretroviral therapy (ART) at entry enabled a randomized comparison of immediate versus deferred ART initiation of changes in asymmetric dimethylarginine (ADMA), soluble CD40 ligand (sCD40L), and P-selectin levels. Results:At study entry, median (interquartile range) levels of ADMA, sCD40L, and P-selectin were 0.57 (0.49–0.66) &mgr;g/mL, 251 (135–696) &mgr;mol/L, and 34 (28–44) pg/mL. Compared to those randomized to deferral of ART (n = 114), participants randomized to immediate ART (n = 134) had 10.3% lower ADMA levels (P = 0.003) at 12 months; treatment differences in sCD40L (95% confidence interval: −17% to 44%; P = 0.53) and P-selectin (95% confidence interval: −10% to 10%; P = 0.95) were not significant. The difference in ADMA for those assigned immediate ART compared with those assigned ART deferral was greater among younger patients and those with higher levels of high-sensitivity C-reactive protein and D-dimer (P ⩽ 0.05 for interaction for both) but not HIV RNA level at baseline (P = 0.51). DiscussionART initiation leads to declines in ADMA levels, a marker of nitric oxide–mediated endothelial dysfunction. Improvement in ADMA levels was related to the degree of inflammation and coagulation, suggesting that upregulation of these pathways contributes to premature vascular disease among individuals with HIV infection. Whether declines in ADMA levels impact risk of disease requires further research.

Pulmonary function in an international sample of HIV-positive, treatment-naïve adults with CD4 counts > 500 cells/μL: a substudy of the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial

The aim of the study was to describe the prevalence and correlates of chronic obstructive pulmonary disease (COPD) in a multicentre international cohort of persons living with HIV (PLWH).

Impact of 2-Bromo-5,6-Dichloro-1-β-d-Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation

ABSTRACT Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.