Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 6. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 7. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient- derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56 + CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ +NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay, MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 8. 7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 9. Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Cytotoxicity Assay, Control, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Elevated IL8 transcript levels in the course of NAFLD progression. ( A ) mRNA expression levels of the IL8 gene in the livers of patients with fatty liver ( n = 47) and NASH ( n = 51) from a publicly available RNA sequencing database (GSE167523). Statistical significance was assessed using Student’s t -tests (** p < 0.01). ( B ) GSEA plots showing the enrichment of genes associated with neutrophil chemotaxis and neutrophil activation in gene rank based on differential expression between NASH vs. NAFL livers. ( C ) mRNA expression levels of the IL8 gene in the liver among different stages of NAFLD obtained from a publicly available RNA sequencing database (GSE135251). F0–F1, F2, F3, and F4 represent different NASH groups classified by the severity of fibrosis. TPM refers to transcripts per million base pairs. For comparisons involving more than three groups, a one-way analysis of variance (ANOVA) was conducted, followed by post-hoc Tukey’s tests to determine specific group differences. (* p < 0.05, ** p < 0.01). IL8, interleukin 8; GSEA, gene set enrichment analysis; NAS, NAFLD activity score; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Expressing, RNA Sequencing, Chemotaxis Assay, Activation Assay, Quantitative Proteomics, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression induces increased hepatic neutrophil infiltration in the livers of HFD-fed mice. C57BL/6 mice fed an HFD for 3 months were infected with an adenovirus overexpressing the human IL8 gene (Ad- IL8 ) or an adenovirus expressing green fluorescence protein (Ad- GFP ) via the tail vein and sacrificed for analysis 2 weeks post-infection. ( A ) Schematic illustration of the experimental design. ( B ) Serum IL8 levels measured using ELISA analysis. ( C ) Hepatic IL8 transcript levels were calculated using the ΔCt method (Ct IL8 – Ct Gapdh ). ( D ) Paraffin-embedded liver sections were subjected to immunohistochemical analysis of MPO and LY6G ( n = 5/group). Red arrows indicate MPO- or LY6G-positive cells. Scale bars indicate 200 μm. The number of MPO- or LY6G-positive cells per 100× field was counted and illustrated in the graph on the right-hand side. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (** p < 0.01). ELISA, enzyme-linked immunosorbent assay; HFD, high-fat diet; IL8, interleukin 8; MPO, myeloperoxidase.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Infection, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression enhances liver injury and oxidative stress in the livers of HFD-fed mice. ( A ) Serum ALT and AST levels ( n = 5/group). ( B ) Body weight and liver weight ( n = 5/group). ( C , D ) Paraffin-embedded liver sections were subjected to the TUNEL assay (panel C ) and immunohistochemical analyses of MDA and 4-HNE (panel D ) ( n = 5/group). Representative images of each staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for each staining is illustrated in graphs ( right ). ( E ) Mouse liver homogenates were subjected to RT-qPCR analysis of the genes involved in NOX2 complex formation and neutrophil oxidative burst. ( n = 5/group). ( F ) Immunoblot analysis of mouse liver homogenates. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (** p < 0.01). 4-HNE, 4-hydroxynonenal; ALT, alanine transaminase; AST, aspartate transaminase; HFD, high-fat diet; IL8, interleukin 8; MDA, malondialdehyde; NOX2, NADPH oxidase 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, TUNEL Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression enhances hepatic inflammation in the livers of HFD-fed mice. ( A ) Paraffin-embedded liver sections were subjected to immunohistochemical analysis of F4/80 ( n = 5/group). Representative images of F4/80 staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for F4/80 staining is illustrated in graphs ( right ). ( B , C ) Mouse liver homogenates were subjected to RT-qPCR analysis of proinflammatory genes (panel B ) and chemokine genes (panel C ) ( n = 5/group). Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (* p < 0.05, ** p < 0.01). HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Immunohistochemical staining, Staining, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Fibrogenic effects of IL8 overexpression in the livers of HFD-fed mice. ( A ) Paraffin-embedded liver sections were subjected to Sirius Red staining and immunohistochemical analysis of α-SMA ( n = 5/group). Representative images of each staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for each staining is illustrated in graphs ( right ). ( B ) Mouse liver homogenates were subjected to RT-qPCR analysis of fibrogenic genes ( n = 5/group). Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (* p < 0.05, ** p < 0.01). α-SMA, alpha-smooth muscle actin; HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Staining, Immunohistochemical staining, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Effects of recombinant human IL8 protein on stress kinase activation and fibrogenic gene induction in vitro. ( A ) Schematic illustration of neutrophil chemotaxis assay. Mouse bone marrow (BM)-derived neutrophils were placed on the Transwell insert (pore size: 3 µm), and mouse Cxcl1 or human IL8 recombinant protein (100 ng/mL) was added in the bottom chamber. The number of neutrophils that migrated to the lower chamber over 6 h was counted and illustrated graphically (right). ( B ) AML12 mouse hepatocytes were treated with human IL8 recombinant protein (100 ng/mL) for 24 h and subjected to cell viability measurements using the CCK-8 reagent. ( C ) AML12 mouse hepatocytes were treated with human IL8 recombinant protein (100 ng/mL) for 15 min or 30 min and lysed in RIPA buffer for immunoblot analyses of stress kinases. ( D ) LX-2 human hepatic stellate cells were treated with human IL8 recombinant protein (100 ng/mL) for 24 h, and the total RNA was isolated for RT-qPCR analysis of fibrosis-related genes. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons or Student’s t -tests (** p < 0.01). HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Recombinant, Activation Assay, In Vitro, Chemotaxis Assay, Derivative Assay, Pore Size, CCK-8 Assay, Western Blot, Isolation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Primer sequences for quantitative polymerase chain reactions.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques:

Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 4. TLR5 immunostaining, increased chemokine expression and mucosal neutrophil influx in H7 flagellin stimulated human colon xenografts. A. Sections of unstimulated human colon xenograft were stained with anti-TLR5 (red) (panel A), an isotype control antibody (panel D), or anti- TLR5 antibody preabsorbed with TLR5 peptide (panel G). Panels B, E and H are the same sections stained with Alexa 488-phalloidin for F-actin (green). Panels C, F and I are merged images of panels A and B, D and E and G and H respectively. Original magnification 400 ×. B. EHEC H7 flagellin or PBS was instilled in the lumen of human colon xenografts. Xenografts were harvested 6 h later, epithelial cells were isolated and CXCL8 mRNA expression levels were determined by real-time PCR. Data are mean + SEM from three xenografts. *P < 0.01 compared with PBS control. C. Human intestinal xenografts were injected with EHEC H7 flagellin (panel A) or PBS (panel C), harvested 6 h later and immunostained for CCL20. No immunostaining was seen with an isotype control (not shown). Panels B and D are nuclear counterstaining of panels A and C, respectively, with Hoechst 33258 dye. Original magnification 200 ×. D. Human intestinal xenografts injected with EHEC H7 flagellin (top panel) or PBS (bottom panel), were harvested after 6 h and stained with hematoxylin and eosin. Top panel shows a small cell infiltrate consisting mostly of neutrophils (arrows); original magnification 400 ×.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

Techniques: Immunostaining, Expressing, Staining, Control, Isolation, Real-time Polymerase Chain Reaction, Injection

Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 5. Induction of CXCL8 by H7 flagellin, Stx2 and isogenic mutants of EHEC. A. Caco-2 cells were stimulated with titrated doses of H7 flagellin alone or in combination with titrated concentrations of Stx2 for 6 h after which CXCL8 protein in culture media was assayed by ELISA. Data are mean + SEM of three repeated experiments. B. Caco-2 cells were infected with wild-type EHEC 86–24 (stx+/fliC+), or isogenic mutants of EHEC 86–24 that lack fliC (stx+/fliC–), Stx (stx–/fliC+) or both (stx–/fliC–) at an moi of 100 for 3 h after which cells were washed and cultures were incubated in media containing gen- tamicin for an additional 6 h. CXCL8 protein in culture supernatants was measured by ELISA. Data are mean + SEM of three repeated experiments. *P < 0.05 compared with wild-type EHEC. Motility in soft agar is shown for each mutant.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Incubation, Mutagenesis

Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 6. TLR5 distribution and flagellin stimula- tion of polarized Caco-2 cells. A. Polarized Caco-2 cells were immunostained for TLR5 (red) and F-actin (green) and analy- sed by confocal microscopy. Top panels show merged apical, middle and basal sections. Bot- tom panel is a merged X-Z reconstruction. Orig- inal magnification 600 ×. B. Titrated concentrations of EHEC H7 flagellin were added to either the apical or basolateral chambers of polarized Caco-2 cells. Superna- tants from the basolateral chamber were removed after 6 h and CXCL8 was assayed by ELISA. Transepithelial resistance of the mono- layers remained unchanged during the time course of the experiments. Values are mean + SEM of three repeated experiments.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

Techniques: Confocal Microscopy, Enzyme-linked Immunosorbent Assay