Biomarkers of Immune Activation and Inflammation in HIV
Biomarkers of Immune Activation and Inflammation in HIV
With their informed consent, all HIV-infected patients receiving care in the Infectious Diseases Department of Pitié-Salpêtrière Hospital (Paris, France) have their clinical, biological and therapeutic data prospectively recorded in standardized electronic medical records (NADIS database). Biological data obtained in Pitié-Salpêtrière Hospital laboratories, including HIV RNA levels and CD4 and CD8 cell counts, are directly imported into the database from the laboratory computer system, thus minimizing collection errors. The quality of the database is ensured by automated checks during data capture, and by regular controls and annual assessments. Routine blood tests are performed at each hospital visit, and residual plasma is stored frozen, and identified by a serial number.
All HIV-1-infected patients who started a first line of cART between January 2006 and December 2009 were screened for eligibility for this study, using the NADIS database. Patients were eligible if they received a first-line combination recommended by contemporary French guidelines (2006 and 2008) [tenofovir/emtricitabine (TDF/FTC) or abacavir/lamivudine (ABC/3TC), combined with either efavirenz or a ritonavir-boosted protease inhibitor (atazanavir (ATV/r), fosamprenavir (FPV/r) or lopinavir (LPV/r))] and had a rapid and sustained virological response, defined by plasma HIV-1 viral load (VL) < 400 HIV-1 RNA copies/mL at 6 months and < 50 copies/mL at 24 months, with no values > 1000 copies/mL between month 6 and month 24, to control for the possible effect of residual viral replication on immune activation and inflammation. Hepatitis C virus (HCV) coinfection (positive HCV antibody) was a noninclusion criterion, as HCV infection can also influence biomarker levels. Eligible patients were enrolled if frozen plasma samples obtained at the time of cART initiation [day 0 (D0)] and at month 24 (M24) were available. Biomarker levels among HIV-infected patients were compared with their levels among 20 HIV-seronegative healthy blood donors. In France, donors can donate blood between 18 and 70 years of age. The study was approved by the Pitié-Salpêtrière institutional review board, and eligible patients were asked to give their written consent to the use of their medical information and plasma samples, as required by French law.
We selected biomarkers of inflammation and immune activation that can be reliably measured in frozen plasma. We evaluated IL-6 as a biomarker of inflammation, IP-10 and MIG as biomarkers of immune activation including T lymphocytes and monocytes, and sCD14 as a biomarker of monocyte activation.
Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IL-6 and sCD14, according to their manufacturer's instructions (HS600B and DC140, respectively; R&D Quantikine, Minneapolis, MN, USA). IP-10 and MIG levels were determined in thawed diluted plasma with Cytometric Bead Array (CBA) kits (BD) on a BD FACS Canto I device (San Jose, CA, USA), according to the manufacturer's instructions. Coefficients of variation were 6.9–7.8% for IL-6, 4.8–7.4% for sCD14, 4% for IP-10 and 9–13% for MIG, while the limits of detection were 0.039, 125, 0.50 and 1.10 pg/mL, respectively. Plasma samples were allowed to thaw for 50 min at room temperature before centrifugation for 5 min at 101 g, and then placed in Eppendorf tubes in the amounts required for each assay kit. Plasma was diluted as recommended by the kit manufacturers. Standards provided with the kits were measured in duplicate and the mean value was used as a reference. Samples with values higher than the highest standard value were further diluted and retested. D0 and M24 samples were tested in the same run. The same biomarkers in the healthy blood donors were measured with the same kits.
To investigate relationships between biomarker levels and patient characteristics at cART initiation, biomarker levels were compared among the HIV-infected patients according to their sex, age, body mass index (BMI), smoking status, hepatitis B virus surface antigen (HbsAg) serology, prior AIDS-defining events, CD4 cell count, CD4:CD8 ratio and viral load, using Wilcoxon tests. When more than one factor was associated with higher levels of a given biomarker (P < 0.15) in univariable models, the relevant factors were entered in a backward stepwise regression model, and variables with P values > 0.10 were removed from the model. The two-sample Wilcoxon rank-sum test was used to compare biomarker levels between the HIV-infected patients and controls.
Among the HIV-infected patients, changes in the level of each biomarker during the first 2 years of cART were expressed as the difference between M24 and D0, and median differences were tested with a one-sample sign rank test.
Biomarker levels were considered 'elevated' if they exceeded the mean value plus two standard deviations in the healthy subjects. Changes in the proportion of patients with elevated biomarker values between D0 and M24 were assessed using McNemar's test. Logistic regression was used to identify factors associated with elevated biomarker values after 2 years of cART. Factors associated with elevated biomarker values in univariable analysis (P < 0.15) were included in multivariable analysis. A sensitivity analysis excluded patients who experienced viral 'blips' (VL transiently above 50 copies/mL) between month 6 and month 24. stata 12 software (StataCorp LP, College Station, TX, USA) was used for all statistical analyses, and P values < 0.05 were considered to denote statistical significance.
Methods
Study Population
With their informed consent, all HIV-infected patients receiving care in the Infectious Diseases Department of Pitié-Salpêtrière Hospital (Paris, France) have their clinical, biological and therapeutic data prospectively recorded in standardized electronic medical records (NADIS database). Biological data obtained in Pitié-Salpêtrière Hospital laboratories, including HIV RNA levels and CD4 and CD8 cell counts, are directly imported into the database from the laboratory computer system, thus minimizing collection errors. The quality of the database is ensured by automated checks during data capture, and by regular controls and annual assessments. Routine blood tests are performed at each hospital visit, and residual plasma is stored frozen, and identified by a serial number.
All HIV-1-infected patients who started a first line of cART between January 2006 and December 2009 were screened for eligibility for this study, using the NADIS database. Patients were eligible if they received a first-line combination recommended by contemporary French guidelines (2006 and 2008) [tenofovir/emtricitabine (TDF/FTC) or abacavir/lamivudine (ABC/3TC), combined with either efavirenz or a ritonavir-boosted protease inhibitor (atazanavir (ATV/r), fosamprenavir (FPV/r) or lopinavir (LPV/r))] and had a rapid and sustained virological response, defined by plasma HIV-1 viral load (VL) < 400 HIV-1 RNA copies/mL at 6 months and < 50 copies/mL at 24 months, with no values > 1000 copies/mL between month 6 and month 24, to control for the possible effect of residual viral replication on immune activation and inflammation. Hepatitis C virus (HCV) coinfection (positive HCV antibody) was a noninclusion criterion, as HCV infection can also influence biomarker levels. Eligible patients were enrolled if frozen plasma samples obtained at the time of cART initiation [day 0 (D0)] and at month 24 (M24) were available. Biomarker levels among HIV-infected patients were compared with their levels among 20 HIV-seronegative healthy blood donors. In France, donors can donate blood between 18 and 70 years of age. The study was approved by the Pitié-Salpêtrière institutional review board, and eligible patients were asked to give their written consent to the use of their medical information and plasma samples, as required by French law.
Sample Collection and Plasma Soluble Biomarker Assays
We selected biomarkers of inflammation and immune activation that can be reliably measured in frozen plasma. We evaluated IL-6 as a biomarker of inflammation, IP-10 and MIG as biomarkers of immune activation including T lymphocytes and monocytes, and sCD14 as a biomarker of monocyte activation.
Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IL-6 and sCD14, according to their manufacturer's instructions (HS600B and DC140, respectively; R&D Quantikine, Minneapolis, MN, USA). IP-10 and MIG levels were determined in thawed diluted plasma with Cytometric Bead Array (CBA) kits (BD) on a BD FACS Canto I device (San Jose, CA, USA), according to the manufacturer's instructions. Coefficients of variation were 6.9–7.8% for IL-6, 4.8–7.4% for sCD14, 4% for IP-10 and 9–13% for MIG, while the limits of detection were 0.039, 125, 0.50 and 1.10 pg/mL, respectively. Plasma samples were allowed to thaw for 50 min at room temperature before centrifugation for 5 min at 101 g, and then placed in Eppendorf tubes in the amounts required for each assay kit. Plasma was diluted as recommended by the kit manufacturers. Standards provided with the kits were measured in duplicate and the mean value was used as a reference. Samples with values higher than the highest standard value were further diluted and retested. D0 and M24 samples were tested in the same run. The same biomarkers in the healthy blood donors were measured with the same kits.
Statistical Analysis
To investigate relationships between biomarker levels and patient characteristics at cART initiation, biomarker levels were compared among the HIV-infected patients according to their sex, age, body mass index (BMI), smoking status, hepatitis B virus surface antigen (HbsAg) serology, prior AIDS-defining events, CD4 cell count, CD4:CD8 ratio and viral load, using Wilcoxon tests. When more than one factor was associated with higher levels of a given biomarker (P < 0.15) in univariable models, the relevant factors were entered in a backward stepwise regression model, and variables with P values > 0.10 were removed from the model. The two-sample Wilcoxon rank-sum test was used to compare biomarker levels between the HIV-infected patients and controls.
Among the HIV-infected patients, changes in the level of each biomarker during the first 2 years of cART were expressed as the difference between M24 and D0, and median differences were tested with a one-sample sign rank test.
Biomarker levels were considered 'elevated' if they exceeded the mean value plus two standard deviations in the healthy subjects. Changes in the proportion of patients with elevated biomarker values between D0 and M24 were assessed using McNemar's test. Logistic regression was used to identify factors associated with elevated biomarker values after 2 years of cART. Factors associated with elevated biomarker values in univariable analysis (P < 0.15) were included in multivariable analysis. A sensitivity analysis excluded patients who experienced viral 'blips' (VL transiently above 50 copies/mL) between month 6 and month 24. stata 12 software (StataCorp LP, College Station, TX, USA) was used for all statistical analyses, and P values < 0.05 were considered to denote statistical significance.
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