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12 protocols using interleukin 6 (il 6)

1

Multiplex Cytokine Profiling

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We performed multiplex assays to measure IL-1β, IL-2, IL-6, TNFα, and IFN-γ purchased from Meso Scale Discovery. All experimental operations were in accordance with standard protocols. R2s of the standard curves for each plate were greater than 0.999.
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2

Biomarker Analysis in Blood Samples

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Blood samples were collected into EDTA and serum tubes and were centrifuged for 15 min at 1000g at 4°C and 21°C, respectively. Aliquots were immediately frozen in liquid nitrogen and subsequently stored at −80°C until analysis. Plasma glucose, FFA, TAG and total cholesterol were analyzed with standard enzymatic methods (ABX Pentra 400 autoanalyzer, Horiba ABX, Montpellier, France). Plasma glycerol was analyzed with an enzymatic assay (Enzytec Glycerol, Roche Biopharm, Basel, Switzerland) automated on a Cobas Fara spectrophotometric autoanalyzer. Plasma insulin, leptin, and adiponectin concentrations were analyzed with commercially available radioimmunoassay (RIA) kits (Human insulin specific RIA, human leptin RIA, human adiponectin RIA, Millipore Corporation, Billerica, MA). Plasma concentrations of IL‐6 (Meso Scale Discovery, Gaithersburg, MD), apelin‐12 (Phoenix pharmaceuticals Inc., Burlingame, CA), RBP4 (R&D systems, Minneapolis, MN) and Vaspin (Adipogen, San Diego, IL) were determined by ELISA. The apelin‐12 ELISA has 100% cross‐reactivity with human apelin‐12, apelin‐13, and apelin‐36. Serum ACE activity was measured with a standard enzymatic assay (Bühlmann, Basel, Switzerland) while serum nesfatin‐1 (Phoenix pharmaceuticals Inc., Burlingame, CA) concentrations were measured by ELISA.
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3

Measuring Neuroinflammatory Markers in ART-Naïve Individuals

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We selected monocyte and macrophage activation markers, as well as specific inflammatory markers that have previously been associated with NCI in ART-naïve individuals35 (link)36 (link)37 (link). Levels of selected monocyte activation and inflammatory markers were measured in CSF and blood plasma by enzyme-linked immunosorbent assay (ELISA): sCD163 (Trillium Diagnostics, Brewer, ME, USA); or by electrochemiluminescence multiplex assay: MCP-1, IL-8, IL-6 and TNF-α, (Meso Scale Diagnostics, Rockville, MD, USA), according to the manufacturer’s procedures.
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4

Profiling Immune Cytokine Responses

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1.5 × 106 Jurkat, THP1 (ATCC), THP1-Dual or THP1-Dual STING KO cells were treated with 3x GI50 V158411 or 50 μg/mL cGAMP for 24 h. Cells were harvested by centrifugation and lysed in 100 μL ice cold lysis buffer on ice for 30 min. Cell debris was pelleted and 25 μL of the supernatant added to a 10 spot U-PLEX biomarker plate pre-conjugated with anti-TNF-α, MCP-3, IL-6, IL-22, IL-18, IL-1β, IL-1α, IFN-γ, IFN-β and IFN2a capture antibodies (MesoScale Discovery, Rockville, MD). The plate was developed according to the manufacturer’s instructions and read using a SECTOR Imager 2400.
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5

Biomarker Evaluation in HIV Cohort

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At each study visit, blood was collected into EDTA tubes and tested for CD4 count (PIMA, Alere) and HIV-1 RNA viral load (Cobas Taqman platform in Uganda and the Roche CAP/CTM HIV-1 v2 assay in South Africa). Additional blood was centrifuged for plasma separation, and frozen at −80oC. Due to resource constraints that prevented us from testing the entire cohort, only non-pregnant individuals underwent additional testing of cryopreserved plasma for: 1) IL-6 (MesoScale Discovery, Rockville, MD); 2) soluble CD14 (sCD14, R&D Systems, Minneapolis, MN); and 3) D-dimer (Diagnostica Stago, Parsippany, NY. Biomarker assays were performed at the Laboratory for Clinical Biochemistry Research at the University of Vermont.
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6

Biomarker Testing in HIV Cohort

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At each study visit, blood was collected into EDTA tubes and tested for CD4+ cell count (Pima, Abbott Laboratories, Lake Bluff, Illinois, USA) and HIV-1 RNA viral load [Cobas Taqman platform in Uganda (Applied Biosciences, Beverley Hills, California, USA) and the CAP/CTM HIV-1 v2 assay (Roche Laboratories, Basel, Switzerland) in South Africa]. Additional blood was centrifuged for plasma separation, and frozen at -80oC. Due to resource constraints that prevented us from testing the entire cohort, only nonpregnant individuals underwent additional testing of cryopreserved plasma for interleukin (IL-6; MesoScale Discovery, Rockville, Maryland, USA); soluble CD14 (sCD14; R&D Systems, Minneapolis, Minnesota, USA); and D-dimer (Diagnostica Stago, Parsippany, New York, USA). Biomarker assays were performed at the Laboratory for Clinical Biochemistry Research at the University of Vermont.
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7

DXA Scanning for Body Composition

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All patients underwent whole-body DXA scanning (Hologic, QDR 4500A) at baseline (week 0) and at week 48 to assess total body and subtotal (total minus head) BMD, as well as fat and lean body mass. Fasting plasma samples were collected and frozen at −70°C. Markers of inflammation and bone turnover were measured at weeks 0, 16, and 48. All assays were performed blinded to treatment group and BMD measurements. Inflammatory markers measured were: soluble tumor necrosis factor (TNF)-α, interleukin 1b (IL1-B), and IL-6 (Meso Scale Discovery, Rockville, MD). BTMs measured included C-terminal telopeptide of type 1 collagen (CTX, marker of bone resorption), and N-terminal type 1 procollagen (P1NP, marker of bone formation). CTX was measured using a luminometric assay on Elecsys 2010 (Hoffmann-La Roche); reference range: men 0.158–0.584 ng/mL, pre-menopausal women 0.162–0.573 ng/ml, post-menopausal women 0.330–1.008 ng/ml; Inter-assay coefficient of variance <8.9%. P1NP was measured with radioimmunoassay (UniQ P1NP RIA kit; Orion Diagnostica) reference range 25.91–132.5 ng/mL; Inter-assay coefficient of variance <12.4%.
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8

Biomarker Evaluation in HFpEF Subtypes

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hs‐CRP, IL‐6,TNF‐α and PTX3 were measured at baseline exams by the NHLBI Heart Failure Research Network Biomarker Core Laboratory at the University of Vermont using commercially available kits (hs‐CRP, Siemens, Indianapolis; IL‐6, Meso Scale Discovery, Gaithersbury, MD; TNF‐α, EMD Millipore, Billerica, MA; PTX3, R&D systems, Minneapolis, MN). Samples were shipped on dry ice and stored at −80°C until analysis at the Biomarker Core Laboratory.
In addition to determining whether there are differences in pro‐inflammatory biomarkers in AD‐HFpEF versus S‐HFpEF, we also tested whether biomarker levels are associated with echocardiographic‐Doppler abnormalities of left ventricular diastolic function in all comers and are predictive of clinical outcomes in AD‐HFpEF. In the DOSE and ROSE trials short‐term clinical outcomes included urine volume, change in cystatin‐C, change in creatinine, change in NT‐proBNP, and change in weight over the 72 hours after randomization. In addition, post‐discharge clinical status with respect to survival and re‐hospitalizations was determined by telephone call 60 days after randomization for each trial.
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9

Intradermal LPS Administration Safety and Cytokine Response

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Safety and tolerability were monitored by tracking adverse events, measuring vital signs, and standard laboratory tests (i.e. haematology) at 3, 6, 10, 24 and 48 hours after LPS administration. Circulating cytokines (IL‐1β, IL‐6, IL‐8, IL‐10, IFN‐γ and TNF; Meso Scale Discovery, Rockville, Maryland, USA) were measured in blood samples to detect a possible systemic effect of intradermal LPS administration. The blood samples for cytokine analysis were analysed in 2 batches with each having a different dilution, therefore the lower limit of quantitation (LLOQ) of the samples was the following: IL‐1β 0.298 or 0.745 pg/mL; IL‐6 1.52 or 3.81 pg/mL; IL‐8 1.25 or 3.12 pg/mL; IL‐10 0.702 or 1.76 pg/mL; IFN‐γ 10 or 25 pg/mL; and TNF 0.760 or 1.90 pg/mL.
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10

BALF Cytokine Profiling via MSD

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The BALF supernatant samples were analysed using MSD pro-inflammatory panel V-Plex including the following analytes: IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO and TNF-α (Meso Scale Diagnostics, Rockville, MD). The analysis was performed according to the manufacturer’s instruction.
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