Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Quantitative proteomic analysis identifies significantly down‐regulated lipid transporter proteins in SAH. (A) Partial list of proteins differentially regulated (>1.5‐fold; P < 0.05) between SAH, AC, HC, and its GO analysis, which documents enrichment of lipid transporters in SAH. (B) KEGG enrichment analysis of the up‐regulated (n = 32) and down‐regulated (n = 176) proteins in patients with SAH represents significant ( P < 0.05) enrichment. (C) Plasma level of PON1 was down‐regulated in patients with SAH (median, 23.2; range, 2.3‐139.4) compared to those with AC (median, 44.7; range, 25.8‐131) and HC (median, 139.5; range, 46.6‐196.0) ( P < 0.05). (D) Plasma level of apolipoproteins (APOB, APOE, APOA1, APOA2, APOC1, APOC3) in SAH compared to AC and HC ( P < 0.05 is significant). (E) IHC showing expression of PON1, LDL, CD36, SRA1, ABCA1, and ABCG1 in SAH (n = 5) and AC (n = 5). For all IHC analyses, relative quantization of positively stained cells are expressed as mean number of positive cells/10 high‐power field (40×) and * P < 0.05. Abbreviations: COA, coenzyme A; HBG1, hemoglobin subunit gamma 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; LBP, lipopolysaccharide binding protein; LPS, lipopolysaccharide; TLR4, toll‐like receptor 4;

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Clinical Proteomics, Expressing, Staining, Binding Assay

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Plasma levels of lipid transporter proteins correlate to severity and outcome in SAH. (A) Plasma level of PON1 was down‐regulated in nonsurvivors of SAH (median, 13.1; range, 2.3‐97) versus survivors (median, 33.4; range, 5.7‐139.4; P < 0.01). (B) Plasma levels of apolipoproteins (APOB, APOE, APOA1, APOA2, APOC1, and APOC3) in nonsurvivors compared to survivors. (C) PON1 interaction network and the levels of the ratios of PON1:lipid transporter proteins (PON1:APOB, PON1:APOE, PON1:APOA1, PON1:APOC1, PON1:APOC3) in nonsurvivors and survivors. (D) Correlation of PON1 and lipid transporter proteins with severity and outcome in SAH; pink represents P < 0.05. (E) Diagnostic efficacy of PON1 compared to other lipid transporter proteins in predicting severity and mortality in patients with SAH; P < 0.05. (F) Kaplan‐Meier survival analysis of patients with SAH based on PON1 levels of <18 μg/mL versus >18 μg/mL (log rank <0.01). Abbreviations: ALB, albumin; ALT, alanine aminotransferase; CC, correlation coefficient; CI, confidence interval; LB, lower bound; MTTP, microsomal triglyceride transfer protein; NS, not significant; Plat_B, platelet_baseline; Sig., significance; UB, upper bound.

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Clinical Proteomics, Diagnostic Assay

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Decreased PON1 levels associated with an increase in lipid oxidation and accumulation in macrophages in SAH. (A) Plasma oxidized LDL levels in SAH (median, 227; range, 150‐430) compared to AC (median, 175; range, 200‐100) and HC (median, 172; range, 185‐96), and nonsurvivors of SAH (median, 256; range, 183‐430) compared to survivors (median, 198; range, 155‐310); P < 0.05 is significant. (B) Accumulation of lipid bodies in CD11b+CD163+ macrophages in SAH (median, 4.6%; range, 1.01%‐22.7%) compared to AC (median, 3.07%, range, 1.02%‐4.51%) and HC (median, 1.85%; range, 1.01%‐2.22%), and accumulation of lipid bodies in nonsurvivors of SAH (median, 10.93%; range, 2.44%‐22.70%) compared to survivors (median, 2.24%; range, 0.85%‐7.62%); P < 0.05 is significant. (C) Accumulation of lipid bodies in CD11b+HLA‐DR+ macrophages in SAH (median, 24.55%; range, 4.65%‐68.60%) compared to AC (median, 6.38%; range, 3.79%‐8.41%) and HC (median, 0.87%; range, 0.10%‐2.22%), and accumulation of lipid bodies in nonsurvivors of SAH (median, 45.95%; range, 16.60%‐68.60%) compared to survivors (median, 18.70%; range, 4.65%‐39.00%); P < 0.05 is significant. (D) Relative expression of linked genes to lipid uptake, transport, metabolism, biosynthesis, macrophage activation, and inflammation in PBMCs of nonsurvivors versus survivors. (E) Immunofluorescent analysis of Nile red staining of healthy MDMs treated with plasma samples of nonsurvivors and survivors of SAH. Mean florescent intensity of Nile red staining represents the amount of lipid bodies stored. (F) IHC showing expression of CD68, TNF‐α, and NOD1 in SAH (n = 5) and AC (n = 5). For all IHC analyses, relative quantization of positively stained cells are expressed as mean number of positive cells/10 high‐power field (40×); * P < 0.05. Abbreviations: EPO, erythropoietin; FITC, fluorescein isothiocyanate; H&E, hematoxylin and eosin; LB, lipid body.

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Clinical Proteomics, Expressing, Activation Assay, Staining

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: PON1 substitution modulates macrophage lipid homeostasis and prevents accumulation of lipid bodies in patients with SAH. (A) Workflow to estimate the effect of PON1 substitution in THP1 macrophages treated with plasma samples of SAH, AC, and HC. (B) Frequency of CD11b+CD163+ macrophages in SAH plasma (median, 17.41%; range, 14.30%‐19.40%) compared to SAH+PON1 (median, 13.55%; range, 6.98%‐14.70%), AC plasma (median, 13.55%; range, 8.39%‐19.90%) compared to AC+PON1 (median, 13.70%; range, 7.63%‐19.00%), and HC plasma (median, 8.20%; range, 6.79%‐8.82%) compared to HC+PON1 (median, 6.86%; range, 6.20%‐9.10%). Lipid body content:SAH plasma (median, 90%; range, 85.50%‐92.90%) compared to SAH+PON1 (median, 83.90%; range, 72.80%‐87.90%), AC plasma (median, 82.70%; range, 80.20%‐85.20%) compared to AC+PON1 (median, 76.05%; range, 72.40%‐78.60%), and HC plasma (median, 86.80%; range, 83.90%‐87.80%) compared to HC+PON1 (median, 77.65%; range, 73.30%‐81.80%); P < 0.05 is significant. (C) Frequency of CD11b+HLA‐DR+ macrophages was not significant in SAH plasma compared to SAH+PON1 or in AC plasma compared to AC+PON1 and HC plasma compared to HC+PON1. The lipid body content was SAH plasma (median, 67.35%; range, 63.6%‐70.3%) compared to SAH+PON1 (median, 57.7%; range, 55.2%‐63.4%), AC plasma (median, 65.55%; range, 63.1%‐67.3%) compared to AC+PON1 (median, 65.55%; range, 63.1%‐67.3%), and HC plasma (median, 64.35%; range, 61.6%‐68.8%) compared to HC+PON1 (median, 61%; range, 59.7%‐64.9%). (D) Validation of accumulation of lipid bodies by Nile red staining in macrophages treated with SAH, AC, or HC plasma in the presence and absence of PON1 ( P < 0.05 is significant. Scale 20×.), and percentage intensity of oxidative burst calculated by DHR in THP1 macrophages treated with SAH and AC plasma in the presence and absence of PON1 (* P < 0.05). (E) Relative expression of linked genes to lipid uptake, transport, metabolism, biosynthesis, and inflammation in THP1 macrophages treated with SAH, AC, or HC plasma (upper panel). The effect of PON1 on the expression of these genes is shown in the lower panel (bright red, green, and blue are the expressions on PON1 treatment). (F) Relative protein expression of CD36, SRA1, ABCA1, and ABCG1 in THP1 macrophages treated with SAH, AC, or HC plasma in the presence or absence of PON1; * P < 0.05. Abbreviations: DHR, dihydrorhodamine; EPO, erythropoietin; FACS, fluorescence‐activated cell sorting; FITC, fluorescein isothiocyanate; FSC, forward scatter; LXL, luxate‐like; MFI, mean fluorescence intensity; NS, not significant; PMA, phorbol 12‐myristate 13‐acetate; SSC, side scatter.

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Clinical Proteomics, Biomarker Discovery, Staining, Expressing, Fluorescence, FACS

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Macrophage proteomic analysis validates the decrease in lipid metabolism and biosynthesis after PON1 treatment in THP1‐derived macrophages. (A) Workflow documents eight‐plex proteomic analysis of THP1 macrophages treated with plasma samples of SAH, AC, and HC in the presence or absence of PON1. This was followed by clustering analysis of 1,817 proteins. To estimate the effect of PON1 expression of THP1 macrophages treated with SAH plasma+ PON1/SAH plasma treatment, THP1 macrophages treated with AC plasma+ PON1/AC plasma treatment, THP1 macrophages treated with HC plasma+ PON1/HC plasma treatment, PON1‐induced and PON1‐suppressed proteins were identified. Red venny diagram documents the integration of PON1‐induced genes in SAH, AC, and HC plasma, and green venny documents the integration of PON1‐suppressed genes. (B) Network of GO (biological, molecular, and cellular function) functional enrichment of the PON1‐induced genes in SAH, AC, and HC; significant enrichment is highlighted with shades of yellow with a darker shade having more significance ( P < 0.05). (C) Network of GO (biological, molecular, and cellular function) functional enrichment of the PON1‐suppressed genes in SAH, AC, and HC; significant enrichment is highlighted with shades of yellow with a darker shade showing greater significance ( P < 0.05). (D) Functional analysis based on the proteins induced or suppressed by PON1 treatment. Red bar documents fold regulation, the effect of PON1 in SAH plasma‐treated THP1 macrophages. Blue bar documents fold regulation, the effect of PON1 in AC plasma‐treated THP1 macrophages. Green bar documents fold regulation, the effect of PON1 in AC plasma‐treated THP1 macrophages for lipid metabolism and inflammation and immune regulation linked pathways. Fold regulation is calculated based on the percentage enrichment and mean expression of proteins in the pathways. (E) Functional analysis based on proteins induced or suppressed by PON1 treatment. Red bar documents fold regulation, the effect of PON1 in SAH plasma‐treated THP1 macrophages. Blue bar documents fold regulation, the effect of PON1 in AC plasma‐treated THP1 macrophages. Green bar documents fold regulation, the effect of PON1 in AC plasma‐treated THP1 macrophages for apoptosis, cellular growth and development, and energy metabolism linked pathways. Fold regulation is calculated based on the percentage enrichment and mean expression of proteins in the pathways. Abbreviations: Abl, albumin; AMPA, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor; CoA, coenzyme A; CXCR2, chemokine (C‐X‐C motif) receptor 2; EGFR1, epidermal growth factor receptor1; GDP, Glyceraldehyde‐3‐phosphate dehydrogenase; GIP, Gastric inhibitory polypeptide; GluR2, Glutamate receptor 2; GTPase, guanosine triphosphatase; iTRAQ, isobaric tag for relative and absolute quantitation; KSRP, Far upstream element‐binding protein 2; LC‐MS/MS, liquid chromatography–tandem mass spectrometry; MHC, major histocompatibility complex; mRNA, messenger RNA; NICD, Neurogenic locus notch homolog protein 1; NuMA, Nuclear mitotic apparatus protein 1; PECAM1, Platelet endothelial cell adhesion molecule; PIP2, Phosphatidylinositol (4,5)‐bisphosphate; PKR, protein kinase R; PLC, Phospholipase C; PRPP, Phosphoribosyl pyrophosphate; STAT3, signal transducer and activator of transcription 3; TP, thromboxane receptor; VEGFR3, Vascular endothelial growth factor receptor 3.

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Derivative Assay, Clinical Proteomics, Expressing, Cell Function Assay, Functional Assay, Multiplex sample analysis, Quantitation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Immunopeptidomics

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Causality for PON1‐mediated decrease in accumulation of lipid bodies, oxidative stress, and inflammation. Paradigm for the role of PON1 treatment in SAH plasma‐treated macrophages. In patients with SAH under low PON1, lipid transport and metabolism is perturbed. This is followed by an increase in plasma LDL oxidation, which significantly induces circulating macrophages for lipid uptake by increasing expression of CD36 and SRB1. Lipid bodies accumulate in the macrophages due to a decrease in lipid metabolism and lipid export receptor ABCA1. Increase in the content of lipid bodies increases inflammation and oxidative stress and cellular apoptosis (TRAIL, BAD‐mediated apoptosis) and shifts macrophages to alternate energy metabolism. Resubstitution of PON1 recalibrates the macrophage–lipid homeostasis by mediating a decrease in lipid uptake receptor and an increase in lipid export receptor, thereby neutralizing macrophage inflammation and oxidative stress, apoptosis, and a shift in energy metabolism. Abbreviation: ACAT, acyl‐coenzyme A:cholesterol acyltransferase.

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Clinical Proteomics, Expressing

Journal: Hepatology Communications

Article Title: Dysregulated Lipid Transport Proteins Correlate With Pathogenesis and Outcome in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1438

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of the Most Significant Parameters for the Determination of Mortality in Severe Alcoholic Hepatitis

Article Snippet: A total of 10 6 THP1 cells in triplicate were treated with study plasma samples at a 10% concentration in the presence or absence of PON1 (cat. no. ENZ‐299; Prospec, Israel) at 2.5 units/mL (50 ng/mL) for the next 24 hours.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death

doi: 10.1371/journal.pone.0211090

Figure Lengend Snippet: (A-C) ENZ induces telomere damage in CRPC cells. Based on the dose-response data shown in , CRPC cells were treated for 24 hr with 5 μM ENZ (22Rv1) or 10 μM ENZ (C4-2B and LNCaP/AR), then labeled with antibodies to DNA damage marker γ-H2AX and the telomere marker TIN2. Colocalization of γ-H2AX and TIN2 indicate DNA damage at telomeres. Cells with a TIF response to ENZ (>5 dual-labeled foci) were counted in enlarged (1000X) photomicrographs of representative fields. Data are expressed as mean ± SD of 3 independent experiments. (D-E) Combining ENZ with ATMi KU60019 leads to cell death. 22Rv1 (D) , C4-2B (E) , and LNCaP/AR (F) cells were treated with 5 μM ENZ in the presence or absence of 10 μM KU60019 for 24 hr, then washed to remove drugs and allowed to grow for 14 days (colony formation assay). The survival fraction is plotted relative to vehicle-treated controls; mean ± SD of 3 independent experiments.

Article Snippet: In order to test the effect of AR antagonist ENZ and ATM inhibitor KU59403 (Medkoo Bioscience, NC) on 22Rv1 tumors, athymic nude mice (Charles River) were inoculated subcutaneously with 4 X 10 6 22Rv1 cells as described by Wu et al.[ ], and when tumor size reached about 200 mm 3 , tumor-bearing mice were randomly assigned to the following 4 treatment groups: vehicle (control, 6 mice), ENZ (enzalutamide, Selleckchem, TX) alone (7 mice); ATMi KU59403 alone (7 mice); ENZ + KU59403 (6 mice).

Techniques: Labeling, Marker, Colony Assay

Journal: PLoS ONE

Article Title: Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death

doi: 10.1371/journal.pone.0211090

Figure Lengend Snippet: 22Rv1 tumor-bearing athymic nude mice were randomly assigned to vehicle control (Cont), ENZ, ATMi KU59403 (KU), or combined ENZ+KU treatment for 4 weeks. A) Tumor size over time is presented as mean tumor volume (mm 3 ) of each treatment group (n = 6 or 7 mice/group). Error bars represent standard deviation. Statistical analysis was performed for comparison between ENZ alone or KU alone and KU+ENZ treatments: *, p<0.05; **, p<0.001; ***, p<0.0001. B) The doubling time of each tumor was calculated from a plot of log tumor volume vs. time. Doubling times of each treatment group are presented as Box-Whisker plots; horizontal lines represent mean, first and third quartiles, and whiskers represent the minimum and maximum doubling time of each group. The doubling time (days, mean ± SEM) of each group was: Control, 4.94 ± 1.0; KU, 5.50 ± 1.36; ENZ, 5.46 ± 1.36; ENZ+KU, 12.71 ± 4.38. P values are shown in the chart. C) Body weight of mice during the treatment period, relative to day 0 of treatment of each group. D) ENZ induces ATM activation in 22Rv1 xenograft tumors. Immunostaining of pATM is shown in a representative 22Rv1 xenograft tumor section from each treatment group. E) Evaluation of cell death in serial sections of 22Rv1 tumors. TUNEL assay to detect cell death was performed as described in the manufacturer’s protocol (InVitrogen). Images show ~1, 000 cells (blue) in a representative tumor tissue section from each treatment group. Cell death was analyzed by counting the percentage of cells that were dead (red) in each image.

Article Snippet: In order to test the effect of AR antagonist ENZ and ATM inhibitor KU59403 (Medkoo Bioscience, NC) on 22Rv1 tumors, athymic nude mice (Charles River) were inoculated subcutaneously with 4 X 10 6 22Rv1 cells as described by Wu et al.[ ], and when tumor size reached about 200 mm 3 , tumor-bearing mice were randomly assigned to the following 4 treatment groups: vehicle (control, 6 mice), ENZ (enzalutamide, Selleckchem, TX) alone (7 mice); ATMi KU59403 alone (7 mice); ENZ + KU59403 (6 mice).

Techniques: Control, Standard Deviation, Comparison, Whisker Assay, Activation Assay, Immunostaining, TUNEL Assay