Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Elevated soluble CD14 (sCD14) concentration in the serum of cirrhotic patients. Concentration of sCD14 was measured in healthy controls (n=31), patients with chronic viral hepatitis (n=26) and patients with cirrhosis (n=50) as described in patients and methods. Data is presented graphically as Whisker box-plots

Article Snippet: Absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human CD14 (R&D Systems).

Techniques: Concentration Assay, Whisker Assay

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: High correlation between the levels of human beta defensin-1 (hBD-1) and soluble CD14 (sCD14) in the hepatic veins of cirrhotic patients . Concentrations of hBD-1 and sCD14 were measured as described in patients and methods. Each dot corresponds to individual patients with cirrhosis (n=45). Analysis was performed in samples collected from peripheral veins (n=25, ) and from hepatic veins (n=20, )

Article Snippet: Absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human CD14 (R&D Systems).

Techniques:

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) strongly correlate in serum of patients with cirrhosis. Concentrations of sCD14 were measured as described in Patients and Methods. Concentrations of LBP were measured by a commercially available ELISA, according to manufacturer’s instructions. Each dot corresponds to individual patients with cirrhosis (n=36). Analysis was performed in samples collected from peripheral veins

Article Snippet: Absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human CD14 (R&D Systems).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Macrophages treated with interferons induce different responses in lymphocytes via extracellular vesicles

doi: 10.1016/j.isci.2024.109960

Figure Lengend Snippet:

Article Snippet: Beads CD14 + , Miltenyi Biotec, Germany , Cat. # 130-050-201.

Techniques: Recombinant, Protein Extraction, Protease Inhibitor, Pore Size, Electrophoresis, Staining, Software, Cytometry

Journal: Cell

Article Title: A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression

doi: 10.1016/j.cell.2017.06.049

Figure Lengend Snippet: Key Resources Table

Article Snippet: Primary human monocytes Primary human CD14+ monocytes were isolated from peripheral blood (Leukopacs, MGH blood bank) by positive selection using CD14 MicroBeads (Miltenyi) according to the manufacturer's instructions.

Techniques: Virus, Clinical Proteomics, Recombinant, Knock-Out, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Reporter Assay, RNA Sequencing, Control, Plasmid Preparation, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium promotes hepatic steatosis and fibrosis in mice fed a high-fat plus high-sucrose diet (HFHS). (A) Schematic representation of HFHS experimental design. (B) Representative images of liver tissue stained with H&E and Oil Red O after short-term HFHS treatment ( n = 5). (C) Relative mRNA levels of Col1a1 , β-Pdgfr , Tgf-β , and Timp-1 in the liver after short-term HFHS treatment ( n = 5). (D) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after short-term HFHS treatment ( n = 5). (E) Body weight of mice after long-term HFHS treatment ( n = 15). (F) Liver weight of mice after long-term HFHS treatment ( n = 15). (G) The length of the small intestine after long-term HFHS treatment ( n = 15). (H) Serum endotoxin levels after long-term HFHS treatment ( n = 15). (I) The endotoxin levels in the liver after long-term HFHS treatment ( n = 15). (J) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after short-term HFHS treatment ( n = 5). (K) Relative mRNA levels of Col1a1, β-Pdgfr , Tgf-β , and Timp1 in the liver after the HFHS treatment for 16 weeks ( n = 5). (L) Representative images of liver tissue stained with H&E, Oil Red O, and Masson's trichrome and immunohistochemical staining for α -SMA and TGF- β after long-term HFHS treatment ( n = 5). (M) Levels of α -SMA and TGF- β were quantified using Image J software and expressed as AODs. Data are shown as the mean ± SD. For the two groups, statistical significance was tested by unpaired Student's t -test; for more than two groups, statistical significance was tested by one-way ANOVA followed by the least significant difference (LSD) test; ∗ P < 0.05 , ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Staining, Immunohistochemical staining, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium activates inflammatory signaling pathways in both the ileum and liver. (A) Endotoxin in serum and liver ( n = 10). (B) Immunofluorescent staining of F4/80 (red) in the liver of 8-week-old mice ( n = 5). (C) Protein levels of F4/80 in the liver of 8-week-old mice ( n = 3). (D) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver of 8-week-old mice ( n = 5). (E) Relative mRNA levels of Il-1β and Tnf-α in the liver of 8-week-old mice ( n = 5). (F) Relative mRNA levels of Lbp in the liver of 8-week-old mice ( n = 5). (G) Representative H&E staining, immunohistochemical staining for AP, and immunofluorescent staining for CD14 (green) in the ileum of 8-week-old mice ( n = 5). (H) Protein levels of CD14 and relative mRNA levels of Lbp and CD14 in the ileum of 8-week-old mice ( n = 5). (I) Enrichment graph of NOD-like receptor signaling dataset performed with ileum samples from 8-week-old Pparα ΔIE mice ( n = 3). (J) Heatmap representation of genes involved in innate antibacterial defense in 8-week-old Pparα ΔIE ileum relative to Pparα fl/fl ( n = 3). (K) Relative mRNA levels of Defa21 , Defa22 , Defa3 , Defa5 , and Reg3β in the ileum from 8-week-old mice ( n = 5). (L) Schematic representation of high-fructose corn syrup (HFCS) experimental design. (M) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in liver tissue from 8-week-old mice exposed to HFCS for 14 days ( n = 5). (N) Representative H&E image and immunohistochemical staining for AP in the ileum of 8-week-old mice exposed to HFCS for 14 days ( n = 5). (O) Serum endotoxin levels in 8-week-old mice exposed to HFCS for 14 days ( n = 8). AP: alkaline phosphatase staining. Data are shown as the mean ± SD. An unpaired two-tailed Student's t -test; ∗ P < 0.05 , ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Protein-Protein interactions, Staining, Immunohistochemical staining, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium increases the translocation of gut-derived antigens into the liver. (A) Intestinal permeability assessment (FITC-dextran, 4 kD) in 8-week-old mice ( n = 10). (B) Relative mRNA levels of Zo-1 and Cldn8 in the ileum from 8-week-old mice ( n = 5). (C) The relative proportion of bacterial species in the cecum content by 16S rRNA gene sequencing ( n = 6). (D) Bugbase phenotypic prediction of gut microbiota in 8-week-old mice ( n = 6). (E) The mRNA and protein levels of PV1 in the ileum of 8-week-old mice ( n = 5). (F) Transmission electron microscopy images of the diaphragm (red star) in the capillaries from ileum sections in 24-week-old mice ( n = 3). (G) Representative images of fluorescence microscopy and transmission electron in 8-week-old mice treated with FITC-LPS (green) or Au-LPS ( n = 3–5). (H) Portal HDL-C levels in 8-week-old mice ( n = 10). (I) The protein levels of APOA1 and ABCA1 in the ileum of 8-week-old mice ( n = 5). (J) Relative mRNA levels of Apoa1 , Pon1 , and Pon3 in the ileum of 8-week-old mice ( n = 5). (K) Serum APOA1 levels in 8-, 16- and 32-week-old mice ( n = 8–10). (L) Serum APOA1 levels in 8-week-old mice exposed to HFCS for 14 days ( n = 8). (M) Serum APOA1 levels in 16-week-old Pparα Δhep mice ( n = 10). (N) Schematic representation of a cocktail of broad-spectrum antibiotics (Abx) experimental design. (O) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in the liver from 8-week-old mice treated with Abx ( n = 5). (P) Triglyceride in serum and liver treated with Abx ( n = 10). (Q) Relative mRNA levels of genes related to triglyceride accumulation in the liver from 8-week-old mice treated with Abx ( n = 5). (R) Hepatic levels of cytokines from 8-week-old mice treated with Abx ( n = 5). (S) Protein levels of F4/80 in the liver of 8-week-old mice treated with Abx ( n = 3). (T) Relative mRNA levels of F4/80, Clec4f , and Cd14 in the liver of 8-week-old mice treated with Abx ( n = 5). LD: lipid droplet; M: mitochondria; FITC-LPS: fluorescein isothiocyanate (FITC)-LPS; Au-LPS: LPS-gold-complexes. Data are shown as the mean ± SD. An unpaired two-tailed Student's t -test; ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Translocation Assay, Derivative Assay, Permeability, Sequencing, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Staining, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Butyric acid attenuates PPAR α -induced necroptosis in HIEC-6 cells and ameliorates hepatic steatosis in Pparα ΔIE mice. (A) Relative CREB3L3 mRNA level in HIEC-6 cells transfected with the PPAR α expression vector ( n = 3). (B) Relative mRNA level of apoptotic and necroptosis signaling components in HIEC-6 cells transfected with the CREB3L3 expression vector ( n = 3). (C) Relative mRNA level of apoptotic and necroptosis signaling components in HIEC-6 cells treated with TNF- α ( n = 3). (D) Effects of GW6471 on RIPK3 and MLKL promoter-driven luciferase activities in human intestinal epithelial HIEC-6 cells ( n = 3). (E) Relative CREB3L3 mRNA level in HIEC-6 cells treated with butyric acid ( n = 3). (F) Relative mRNA level of necroptosis signaling components in HIEC-6 cells treated with TNF- α and butyric acid ( n = 3). (G) Schematic representation of the experimental design for butyric acid treatment. H, Relative CREB3L3 mRNA level in Pparα fl/fl and Pparα ΔIE mice treated with butyric acid ( n = 5). (I) Relative mRNA level of necroptosis signaling components in Pparα ΔIE mice treated with butyric acid ( n = 5). (J) Representative immunohistochemical staining for cleaved-caspase 3 and MLKL, and immunofluorescent staining for cleaved-caspase 8 (green), p-RIPK3 (red), and p-MLKL (red) in the ileum after butyric acid treatment ( n = 5). (K) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in liver tissue after butyric acid treatment ( n = 5). (L) Relative mRNA levels of genes related with lipolysis, lipogenesis, and lipid droplet proteins in the liver after butyric acid treatment ( n = 5). (M) Triglyceride in serum and liver after butyric acid treatment ( n = 10). (N) Hepatic levels of cytokines in mice after butyric acid treatment ( n = 5). (O) Protein levels of F4/80 in the liver after butyric acid treatment ( n = 3). (P) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after butyric acid treatment ( n = 5). Buty: butyric acid. Data are shown as the mean ± SD. For the two groups, statistical significance was tested by unpaired Student's t -test; for more than two groups, statistical significance was tested by one-way ANOVA followed by the least significant difference (LSD) test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Immunohistochemical staining, Staining

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: Lin- c-Kit+ progenitors in bone marrow express TLRs and their co-receptors.(A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin- c-Kit+ bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLRs and related molecules are expressed by hematopoietic stem cells and progenitor cells.(A) Lineage marker negative cells were enriched from bone marrow suspensions before staining with antibodies to hematopoietic subsets and TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the appropriate isotype matched Abs. The results shown are representative of three independent experiments. (B) Total RNA was extracted from each progenitor subset and semi-quantitative RT-PCR was conducted to detect mRNA encoding TLR4, MD-2, TLR2, and CD14. The results are shown as values normalized to peak expression for each of the transcripts and representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Marker, Staining, Quantitative RT-PCR, Expressing

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation causes a progression of Lin+ cells from LKS+ cells and soluble CD14 augments the acquisition of Mac-1 and F4/80.(A) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin+ or Lin- cells are indicated. (B) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam3CSK4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam3CSK4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures (*P<0.001). The results are representative of three independent experiments. (D) Left, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with 1 μg/ml LPS (Left, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles). After 24, 48, or 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. The graph depicts cell yields and the results are representative of two independent experiments. Right, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Right, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of Mac-1 and F4/80. The graph depicts cell yields and the data are representative of two independent experiments. (E) Sorted Flk-2- or Flk-2+ LKS+ cells (10,000 cells/well) were cultured with medium alone, LPS (10 μg/ml) or Pam3CSK (1 μg/ml). Flk-2- LKS+ cells were cultured with SCF. Flk-2 LKS+ cells were cultured with SCF and FL. After 72 h in culture, cells were analyzed by flow cytometry for expression of lineage markers (left panels). Percentages given in quadrants indicate the frequencies of Lin+ or Lin- cells and the bar graphs on the right depict cell yields. The data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments. (*P<0.03: Flk-2- LKS+, *P<0.002: Flk-2+ LKS+)

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Flow Cytometry, Expressing, Recombinant

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis.(A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88-/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam3CSK4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19+ cells or Mac-1+ cells (Right) and the data represent mean values with standard deviations from triplicate cultures (*P<0.02). The results are representative of those obtained in five independent experiments. (B) Subsets of the recovered cells described in panel (A) were sorted and used to prepare Giemsa-May-Grünwal stained slides. (C) Cultured cells from C57BL/6 mice were also analyzed by flow cytometry for expression of Gr-1 and CD11c. (D) Sorted CLPs from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with a range of concentrations of LPS or combinations of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graph depicts cell yields for CD19+ cells or Mac-1+ cells (Right). Data represent mean values with standard deviations from triplicate cultures (*P<0.003). The results are representative of three independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Staining, Cell Culture, Recombinant