Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 3. Characterization and cytokine expression profiles of the Th17 cell subset among PBMCs and TILs from NPC patients. A, T cell surface markers were detected in Th17 cells from NPC patients. T cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin for 4 h, then stained with fluorescence- conjugated antibodies against the markers shown, and analyzed by flow cytometry. B, graph of the percentage of cytokine-secreting Th17 cells among PBMCs and TILs from NPC patients (n 5) and PBMCs from healthy donors (n 5). Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.001).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Expressing, Staining, Fluorescence, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 4. Generation and migration of Th17 cells mediated by NPC tumor cell lines. A, NPC tumor cell lines induce the differentiation of naive T cells into Th17cellsinvitro.PurifiedCD4naiveTcellsfromhealthydonorswerestimulatedwithOKT3andthenco-culturedwiththeirradiatedNPCcelllineC666(EBV) or CNE2 (EBV) in IL-2-containing medium for 7 days. The NP69 and LCL lines and the cytokines IL-1 and TGF were used as controls. All cultured cells were stained for Foxp3 and IL-17 for FACS analysis after stimulation of phorbol 12-myristate 13-acetate/ionomycin. The data represent one of three independent experiments. B, mean percentage of cytokine-secreting Th17 cells induced from CD4 naive T cells from three experiments. C, migration of Th17 cells was increased in response to cultured supernatants from the NPC cell lines C666 and CNE2, relative to that from the normal NP cell line NP69 or with medium alone. The data represent one of three independent experiments. D, mean percentage of cytokine-secreting Th17 cells in the inner well and outer well after migration induced by supernatants from NP69, C666, or CNE2 cells from three experiments. Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.05).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Migration, Cell Culture, Staining

Journal: bioRxiv

Article Title: A 50-gene high-risk profile predictive of COVID-19 and Idiopathic Pulmonary Fibrosis mortality originates from a genomic imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease

doi: 10.1101/2023.10.22.563156

Figure Lengend Snippet: A . Study design of the 50-gene signature and cytokine analysis in COVID-19 patients (Cohort 1). B. Heatmap of COVID-19 patients based on the 50-gene signature discriminates three risk groups (low, intermediate, and high) based on SAMS. Every column represents a patient, and every row represents a gene. Log-based two-color scale is adjacent to the heatmap. Red denotes increased expression and green denotes decreased expression. Gene expression data is represented as Log2 normalized expression values. C-D. Time to death and time to discharge by day 60 in hospitalized patients with COVID-19, respectively. E-H. Plasma cytokine concentrations (IL6, IP-10, SPP1 and TGFβ) in low, intermediate, and high-risk profile patients with COVID-19 at days 2, 6 and 13 post admission. The data is presented as an average of triplicate values ± SEM for each group. Two-way ANOVA test (GraphPad software) Tukey’s multiple comparisons were used; * p<0.05. I. Study design of 7-gene signature analysis by RT-qPCR in PBMCs from COVID-19 patients (Cohort 2). J. Heatmap of COVID-19 patients based on the 50-gene signature discriminates three risk groups (low, intermediate, and high) based on SAMS Up scores. Heatmap nomenclature is the same as in . K-L. Time to death and time to discharge by day 60 in hospitalized patients with COVID-19 respectively in cohort two. The data is presented as an average of triplicated TUs values ± SEM for each group. * p<0.05

Article Snippet: We measured cytokine concentrations of 121 plasma samples from COVID-19 patients from Cohort 1 using a customized, Bioplex 200 compatible, human cytokine panel including, IL6, IP10, SPP1 and TGFβ-1 (#FCSTM18-06, R&D Systems).

Techniques: Expressing, Gene Expression, Clinical Proteomics, Software, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Xanthine Oxidoreductase Promotes the Inflammatory State of Mononuclear Phagocytes through Effects on Chemokine Expression, Peroxisome Proliferator-activated Receptor-γ Sumoylation, and HIF-1α

doi: 10.1074/jbc.m110.150847

Figure Lengend Snippet: FIGURE 9. Inhibition of XOR activity increases levels of HIF-1 protein in rat inflammatory MNP and in PMA-differentiated U937 cells. A, I-MNP were purified from rat lungs 24 h following insufflation of Th-1 cytokines and were plated in 12-well plates at 1.0 106 cells/well and grown under normoxic conditions. Nonadherent cells were removed by washing after 1 h, and cells were treated with the indicated doses of MIG132. Cells were harvested after 6 h; whole cell lysates were prepared and Western immunoblots run with antibody to HIF-1. In addition, washed, adherent cells were exposed to MIG132 (50 M) for the indicated times, and Western immunoblots were run on whole cell lysates and probed with antibody to HIF-1. Blots were subsequently stripped and re-probed with antibody to GAPDH to control for protein loading. B, I-MNP were purified and plated as in A. Plates were placed in either nor- moxic (21% O2, 5% CO2) or hypoxic culture (1% O2, 5% CO2, 94% N). After 1 h, cells were treated with the XOR inhibitors allopurinol (Allo, 150 M), oxypuri- nol (Oxy, 150 M), or Y-700 (50 nM). One h later cells were treated with MIG132 (50 M) and grown for 6 h. Whole cell lysates were then prepared and West- ern immunoblots run with independent duplicate samples as indicated. Blots were first probed with antibody to HIF-1 and subsequently with antibody to GAPDH. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of duplicate samples. C, I-MNP were purified, plated, and grown in normoxia in the presence of MIG132 as in A. Cells were exposed to the XOR inhibitor Y-700 (50 nM) for 1 h and subsequently treated with IL-1 (10 ng/ml), IFN- (20 ng/ml), or LPS (1.0 g/ml). Whole cell lysates were pre- pared and Western immunoblots run after 24 h of exposure to cytokines. Blots were run on triplicate samples, and representative blots are shown. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. D, U937 cells were plated in 12-well plates at 1 106 cells/well and treated with PMA (30 nM) for 48 h. Cells were then washed; the medium was replaced, and cells were grown under normoxic or hypoxic conditions for 1 h in the presence of 50 M MIG132. Subsequently, cells were treated with Y-700 (50 nM) for 1 h and cytokines added as above. Whole cell lysates were prepared after 24 h of exposure to cytokine/MIG132, and Western immunoblots were run sequentially with antibody to HIF-1 and GAPDH. Representative blots are shown of three independent blots for each experiment. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. E, I-MNP were purified and plated in normoxia as in B. Levels of VEGF were quantitated in the cell-free supernatant over a period of 48 h in culture. In addition, cells were treated independently with MIG132 in the absence or presence of allopurinol, oxypurinol, or Y-700 as in B, and levels of VEGF were measured in the cell-free supernatant 6 or 24 h after treatment. Data show the mean S.D. of three independent experiments. F, I-MNP were plated as above in the presence or absence of Y-700 (1.0 mM) and in the absence of MIG132. After 24 h, cell-free supernatants were collected and analyzed with the rat-specific proteome profiler. Experiments were performed in quadruplicate, and each cytokine was analyzed from two spots on each filter (boxed for VEGF), and thus all data reflect eight independent determinations for VEGF alone. Spots were quantitated as described by the supplier using an R&D transmission mode scanner and image analysis software from R&D. Data show the mean S.D. of light transmission signals (arbitrary units) from eight spots for VEGF only and both control and Y-700 groups and were normalized first to the mean positive control spots (A1, A2, A19, A20, D1, and D2) and subsequently to the signal obtained from the control samples, which was thereby set at 1.00. ***, p 0.02.

Article Snippet: VEGF was also measured using the rat specific proteome profiler array (catalog no. ARY008, R&D Systems, Inc.).

Techniques: Inhibition, Activity Assay, Purification, Western Blot, Control, Transmission Assay, Software, Positive Control

Journal: JAIDS Journal of Acquired Immune Deficiency Syndromes

Article Title: Frailty and Circulating Markers of Inflammation in HIV+ and HIV− Men in the Multicenter AIDS Cohort Study

doi: 10.1097/qai.0000000000001261

Figure Lengend Snippet: FIGURE 2. Relative percentile estimates for the HIV2 frail group, the HIV+ nonfrail group, and the HIV+ frail group (left, middle, and right d, respectively), and 95% confidence limits of the estimates (error bars), for exposure groups relative to the nonfrail HIV2 group, obtained by generalized gamma regression models for each biomarker, adjusted for age, race, study sites, and education, and BMI, current smoking, depressive symptoms, HCV infection, hypertension, diabetes, dyslipidemia, kidney disease, liver disease, and cancer. Significant differences (P # 0.002) are indicated by #. Marginally significant differences (0.002 , P , 0.05) are those for which the 95% confidence intervals do not include 1.0. Relative percentile estimates are given for differences that were significant or marginally significant. The biomarkers that had $1 significant (P , 0.05) comparison in any analysis are shown.

Article Snippet: Serum levels of soluble (s) CD14, sCD27, sgp130, sIL6 receptor (sIL-6R), sIL-2 receptor-a (sCD25), and sTNF receptor-2 (sTNFR2), plus a cytokine known as B-cellactivating factor or B-lymphocyte stimulator (BAFF/BLyS), and the chemokine CXCL13 (B-lymphocyte chemoattractant/ B-cell-attracting chemokine 1; BLC/BCA1) were measured in one custom-designed multiplexed assay panel (Human Biomarker Custom Premix Kit A, R & D Systems, Minneapolis, MN) per manufacturer’s directions at the University of California, Los Angeles, using a Bio-Plex 200 Luminex instrument and Bio-Plex analysis software (Bio-Rad, Hercules, CA).

Techniques: Biomarker Discovery, Infection, Comparison

Journal: JAIDS Journal of Acquired Immune Deficiency Syndromes

Article Title: Frailty and Circulating Markers of Inflammation in HIV+ and HIV− Men in the Multicenter AIDS Cohort Study

doi: 10.1097/qai.0000000000001261

Figure Lengend Snippet: FIGURE 3. Relative percentile estimates (d) and 95% confidence limits (error bars) of the estimates for the frail HIV+ men compared with nonfrail HIV+ men. Data were generated using generalized gamma regression models for each biomarker adjusted for age, race, study sites, and education (left bars with d, model 1) and further adjusted for BMI, current smoking, presence of depressive symptoms, HCV infection, hypertension, diabetes, dyslipidemia, kidney disease, liver disease, and cancer (right bars with d, model 2). Relative percentile differences between the frail and nonfrail HIV+ men that were significantly different at P # 0.002 are indicated by #; those that were significantly different at 0.002 # P #0.05 are those for which the 95% confidence intervals do not include 1.0.

Article Snippet: Serum levels of soluble (s) CD14, sCD27, sgp130, sIL6 receptor (sIL-6R), sIL-2 receptor-a (sCD25), and sTNF receptor-2 (sTNFR2), plus a cytokine known as B-cellactivating factor or B-lymphocyte stimulator (BAFF/BLyS), and the chemokine CXCL13 (B-lymphocyte chemoattractant/ B-cell-attracting chemokine 1; BLC/BCA1) were measured in one custom-designed multiplexed assay panel (Human Biomarker Custom Premix Kit A, R & D Systems, Minneapolis, MN) per manufacturer’s directions at the University of California, Los Angeles, using a Bio-Plex 200 Luminex instrument and Bio-Plex analysis software (Bio-Rad, Hercules, CA).

Techniques: Generated, Biomarker Discovery, Infection