Journal: Cancer Cell

Article Title: TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

doi: 10.1016/j.ccell.2023.09.001

Figure Lengend Snippet: Analysis of the role of TIM-3 in the tumorigenesis of human and murine DIPG cell lines (A) Western blot analysis of TIM-3 expression in DIPG human and murine cell lines, using GRB-2 as a housekeeping protein. (B) Viability assay (MTS) of TP54 and DIPG007 cells 7 days after TIM-3-specific shRNA knockdown. (C) Viability assay (MTS) of TP54 and DIPG007 cells 7 days after treatment with a human anti-TIM-3 antibody (provided by BMS, BMS-986258). (D) Western blot analysis of TIM-3 expression in control guide NP53 and TIM-3KO (G2C1, G2C9, and G1C7) NP53 different cell lines. (E) Clonogenicity assay with control guide NP53 cells versus three different TIM-3KO NP53 clones at 2 weeks. (F) Evaluation of the proliferative capacity by a BrdU assay performed with TIM-3KO clones normalized to control guide cells (mean ± SEM) (G) Apoptosis analysis of TIM-3KO cells compared to control guide cells. Viable (Annexin V − 7AAD − ), early apoptosis (Annexin V + 7AAD − ), and late apoptosis (Annexin V + 7AAD + ). (H) Viability assay (MTS) of NP53 control guide, G2C9 and G1C7 TIM-3KO cells 7 days after treatment with galectin-9 and CEACAM1 (TIM-3 main ligands). (I) Kaplan-Meier survival curves of immunocompetent mice bearing control guide NP53 cells (n = 6), G2C9 TIM-3KO cells (n = 5, log rank; p = 0.0012), or G1C7 TIM-3KO cells (n = 5, log rank; p = 0.0012). (J) Gene pathways enriched in the set of genes with differential expression between TIM-3 + and TIM-3KO cells. Results obtained from RNA-seq data. (K) Heatmap of the differentially expressed genes between TIM-3 + and TIM-3KO cells related to the MAPK and PI3K-AKT pathways. Results were obtained from previous RNA-seq data. (L) Western blot analysis of PTEN, MEK1, pMEK, ERK1/2, pERK1/2, and c-JUN expression in TIM-3+ and TIM-3KO murine DIPG cell lines. Standardized quantification against its own vinculin (housekeeping) and a Student’s t test is performed for TIM-3+ versus TIM-3KO statistical analysis. (M) Kaplan-Meier survival curves of immunocompetent mice bearing HSJD-007 tumors and treated with an anti-human TIM-3 antibody (BMS-986258) (n = 9) or IgG1 (n = 9 log rank; p = 0.0001). Student’s t test (panel B, C), one-way ANOVA (panel E) and two-way ANOVA (panel F, H) were performed. Bar graphs indicate the mean ± SEM. (ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). See also Figures S2 and .

Article Snippet: Similarly, the same number of cells were seeded for Gal-9 (3535-GA-50, R&D System) and CEACAM1 (50571-M08H, SinoBiological) recombinant proteins experiment.

Techniques: Western Blot, Expressing, Viability Assay, shRNA, Knockdown, Control, Clone Assay, BrdU Staining, RNA Sequencing Assay

Journal: Cancer Cell

Article Title: TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

doi: 10.1016/j.ccell.2023.09.001

Figure Lengend Snippet:

Article Snippet: Similarly, the same number of cells were seeded for Gal-9 (3535-GA-50, R&D System) and CEACAM1 (50571-M08H, SinoBiological) recombinant proteins experiment.

Techniques: Virus, shRNA, Recombinant, BrdU Cell Proliferation Assay, Enzyme-linked Immunospot, Expressing, Control, Mouse Assay, Software

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: Genetic abrogation of CEACAM1 leads to decreased body surface temperature and increased diarrhea and IL-6 production in response to LPS ( a ) Body surface temperature of WT and Ceacam1 −/− mice in response to LPS (i.p. injection, 10 mg/kg) ( n = 10 each group). * p < 0.05 in comparison with WT treated with LPS. # p < 0.05 in comparison with WT treated with normal saline. ( b ) Diarrheogenic activity of Ceacam1 −/− mice in response to LPS (i.p. injection, 10 mg/kg) ( n = 17, each group). ( c - f ) Quantification of 4 serum cytokines of Wild type (WT) and Ceacam1 −/− mice (KO) in response to LPS (i.p. injection, 10 mg/kg) (n = 10, each group). * p < 0.05 in comparison with WT

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques: Injection, Comparison, Saline, Activity Assay

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: IL-6 expression levels of organs in Ceacam1 −/− mice in response to LPS challenge. ( a ) IL-6 mRNA expression levels of mouse organs after i.p. injection of LPS (10 mg/kg) for 2 h ( n = 4). (B) IL-6 mRNA expression level in PBS perfused liver and splenocytes after treated with 500 ng/mL LPS for 2 h in vitro ( n = 3). ( c ) IL-6 mRNA expression level in bone marrow cells after treated with 500 ng/mL LPS for 2 h in vitro ( n = 3). ( d ) Percentage of bone marrow CD11b + Ly6G − cells in WT and Ceacam1 −/− mice ( n = 4). ( e ) Intracellular staining of IL-6 in bone marrow cells after treated with Brefeldin A (BFA) plus 500 ng/mL LPS for 5 h in vitro ( n = 3). ( f ) Intracellular staining of IL-6 of bone marrow cells after i.p. injection of BFA plus LPS (10 mg/kg) for 5 h in vivo ( n = 3). (g) Intracellular staining of IL-6 of splenocytes after i.p. injection of BFA plus LPS (10 mg/kg) for 5 h in vivo ( n = 3)

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques: Expressing, Injection, In Vitro, Staining, In Vivo

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: Production of IL-6 in monocytes and their progenitors in wild type and Ceacam1 −/− mice. ( a ) Flow cytometry gating of monocytes (Mo), common monocyte progenitors (cMoP), monocyte-macrophage DC progenitors (MDP) and common DC precursors (CDP) in bone marrow cells (n = 4). ( b ) Percentage of Lin − cells in bone marrow cells (n = 4). (Note: Lin = lineage markers: including CD3, CD19, B220, Ly6G, NK1.1, TER119) ( c ) Percentage of CD115 + cells in Lin − bone marrow cells (n = 4). ( d ) Percentage of MDP, cMoP, Mo, and CDP in Lin − CD115 + bone marrow cells (n = 4). ( e ) Percentage of intracellular IL-6 staining in sorted MDP, cMoP, Mo, and CDP cells after treatment with Brefeldin A (BFA) plus 500 ng/mL LPS for 5 h in vitro (n = 4). (F) Flow analysis of intracellular IL-6 staining of sorted MDP, cMoP, Mo, and CDP cells after treatment with BFA plus 500 ng/mL LPS for 5 h in vitro (n = 4)

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques: Flow Cytometry, Staining, In Vitro

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: Cytokine expression of bone marrow monocytes. Expression of RP105 but not TLR4. ( a ) IL-6 , IFN-β , IFN-α and CCL5 mRNA expression levels of bone marrow monocytes in WT, Ceacam1 −/− and Il6ra −/− mice after treatment of 500 ng/mL LPS in vitro (n = 3). ( b ) IL-6 mRNA expression levels of bone marrow monocytes in WT, Ceacam1 −/− and Stat3 flox/flox ( Stat3 −/− ) mice after treatment of 500 ng/mL LPS in vitro (n = 3). ( c ) Surface TLR4, CD14, RP105 and MD1 staining of RAW264.7 cells and bone marrow monocytes of WT and Ceacam1 −/− mice (n = 3). ( d ) Immunoblot analysis for TLR4 detection in RAW264.7 cells and bone marrow monocytes of WT mice (1, 2, 3 mean three separated experiments; MO: monocytes; Mϕ: RAW264.7 macrophages). ( e ) TLR4 mRNA expression level in bone marrow monocytes of WT and Ceacam1 −/− mice(n = 4). ( f ) IL-6 mRNA expression level in bone marrow monocytes pretreated with TLR4 blocking antibody (10 μg/mL) or TLR4 inhibitor C34 (100 μM) for 20 min following 500 ng/mL LPS treatment for 30 min in vitro (n = 3)

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques: Expressing, In Vitro, Staining, Western Blot, Blocking Assay

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: Co-immunoprecipitation and immunoblot analyses of RP105 and its signaling partners on bone marrow monocytes in response to LPS in Ceacam1 −/− and wild type mice ( a ) Immunoblot analysis of RP105, MD1 and Syk after immunoprecipitation (IP) with RP105 antibody in WT and Ceacam1 −/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min. ( b ) Immunoblot analysis of RP105, pVAV1 and β-actin after IP with RP105 antibody in WT and Ceacam1 −/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). ( c ) Immunoblot analysis of CEACAM1 and β-actin after IP with CEACAM1 antibody in WT bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). ( d ) IL-6 mRNA expression of bone marrow monocytes pretreated with pVAV1 inhibitor azathioprine and 6-thio-GTP for 20 min following 500 ng/mL LPS treatment for 30 min in vitro (n = 3). ( e ) IL-6 mRNA expression of bone marrow monocytes treated with RP105 activating antibody (clone RP/14, 20 μg/mL) or 500 ng/mL LPS for 30 min (n = 3). ( f ) IL-6 mRNA expression of bone marrow monocytes treated with blocking CD14 antibody (clone M14-23, 10 μg/mL) or blocking MD1 antibody (clone MD113, 10 μg/mL) for 20 min following 500 ng/mL LPS for 30 min (n = 3). ( g ) Immunoblot analysis of CD14 and Src after IP with RP105 antibody in WT and Ceacam1 −/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). ( h ) IL-6 mRNA expression of bone marrow monocytes pretreated with 50 nM Src l1 (Src inhibitor) for 20 min following treatment of 500 ng/mL LPS for 30 min (n = 3). ( i ) Anti-CEACAM1 IPs from bone marrow monocytes after treatment with 500 ng/mL LPS for 15 min were followed by immunoblot analysis with 4G10 or anti-pSHP1 antibodies. (Upper); IP with anti-CEACAM1, immunoblot analysis with 4G10. Loading control: immunoblot with anti-CEACAM1. (Lower): IP with anti-CEACAM1 and immunoblot with anti-pSHP-1. Loading control: immunoblot with anti-SHP1. (TL: total cell lysate)

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques: Immunoprecipitation, Western Blot, Expressing, In Vitro, Blocking Assay, Control

Journal: BMC Immunology

Article Title: CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

doi: 10.1186/s12865-019-0287-y

Figure Lengend Snippet: Model for regulation of RP105 signaling in LPS treated murine monocytes. RP105 (blue) associates with MD1 (purple) and CD14 (black). In response to LPS, the complex recruits SRC (black), VAV1 (grey), actin (lt. blue) and CEACAM1 (mauve). SRC phosphorylates tyrosines (red) on RP105, VAV1, and CEACAM1. SHP1 (orange) is recruited to the pITIM on CEACAM1, and in turn, is phosphorylated on its tyrosine by SRC. Downstream signaling of VAV1, dependent on recruitment of actin, is reduced by sequestration of actin by CECAM1 (shown by arrows)

Article Snippet: After 24 h, 48 h and 72 h of transfection, cells were harvested and stained with mouse CEACAM1 APC-conjugated antibody (R&D systems, Inc., Minneapolis, MN 55413, USA) to verify the silencing effect of CEACAM1.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: One Year Follow-Up Risk Assessment in SKH-1 Mice and Wounds Treated with an Argon Plasma Jet

doi: 10.3390/ijms18040868

Figure Lengend Snippet: Unchanged tumor marker levels in liver, lung, brain, and skin of plasma-treated mice. Representative images of immunohistochemistry of different tumor markers (TM): AFP ( I ) in liver, β2M in brain ( II ), CEA in lung ( III ), as well as NSE staining in skin tissue ( IV ) after one year in positive control (+ve ctrl, left) and plasma-treated (right) animals ( A ). Scale bar 50 µm (right columns) or 100 µm (left columns). Using ELISA, we analyzed AFP ( I ), and calcitonin ( CT ) ( II ), a TM of medullary thyroid carcinoma, in blood serum ( B ; * p < 0.05; ( n > 9). The mRNA expression levels of five TM and β-actin in liver, brain, lung, and ear skin tissues from plasma- and untreated (ctrl) mice were compared with organs from a HCC-bearing mouse (+ve ctrl). At least three independent experiments were performed and summarized in the indicated experimental groups (m, male; f, female). AFP , α-fetoprotein; NOPE , neighbor of Punc 11 ; β2M , β2-microglobulin; NSE , neuron specific enolase; CEA , carcinoembryonic antigen ( C ).

Article Snippet: Prior to imaging by light microscopy, sections were incubated with α-fetoprotein (MAB1368, R&D Systems, Germany), β-2-microglobulin (sc-15366, Santa Cruz, Heidelberg, Germany), neuron-specific enolase (#R30242, NSJ Bioreagents, Hamburg, Germany), and carcinoembryonic antigen (AF6480, R&D Systems, Wiesbaden, Germany) overnight at 4 °C.

Techniques: Marker, Clinical Proteomics, Immunohistochemistry, Staining, Positive Control, Enzyme-linked Immunosorbent Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: One Year Follow-Up Risk Assessment in SKH-1 Mice and Wounds Treated with an Argon Plasma Jet

doi: 10.3390/ijms18040868

Figure Lengend Snippet: Murine primer sequences or catalog numbers (Biomol, Germany) for use in quantitative real-time PCR.

Article Snippet: Prior to imaging by light microscopy, sections were incubated with α-fetoprotein (MAB1368, R&D Systems, Germany), β-2-microglobulin (sc-15366, Santa Cruz, Heidelberg, Germany), neuron-specific enolase (#R30242, NSJ Bioreagents, Hamburg, Germany), and carcinoembryonic antigen (AF6480, R&D Systems, Wiesbaden, Germany) overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: A single-cell atlas of the mouse and human prostate reveals heterogeneity and conservation of epithelial progenitors

doi: 10.7554/eLife.59465

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse Cd66a (CEACAM1)-PE (recombinant monoclonal) , Miltenyi , cat 130-106-209, lot 5190208411, clone REA410 , FACS (1:700).

Techniques: Recombinant, Software, Gene Expression

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 1. Recipient CC1 signaling mitigates hepatic IRI and suppresses NF-kB p65 in mouse OLT. Mouse WT livers subjected to 18 hours of cold storage were transplanted into WT or CC1KO syngeneic recipients (n ¼ 6–8/group). OLT/serum samples were analyzed 6 hours after reperfusion. The sham group (n ¼ 5) underwent the same procedures except for OLT. (A) Representative H&E and TUNEL staining. DAPI, 40,6-diamidino-2-phenylindole. Scale bars ¼ 100 mm. (B) Suzuki’s histologic grading of liver IRI and quantification of TUNELþ cells/HPF. (C) sAST/sALT and (D) lactate dehydrogenase (LDH) levels (U/L). Quantitative reverse transcription polymerase chain reaction–assisted OLT detection of (E) IFN-gamma, IL6, IL17, and gran- zyme B/perforin 1, and (F) TLR4, IL1b, and TNF-a (n ¼ 6–7/group). Data normalized to hypoxanthine guanine phosphor- ibosyltransferase gene expression. (G) Western blot–assisted detection of phosphorylated (p)-IkBa, IkBa, p-NF-kB p65, NF-kB p65, and vinculin (VCL). The relative intensity ratio of p-IkBa/IkBa and p-NF-kB p65/NF-kB p65 (n ¼ 4/group) is shown. Data are shown as mean ± standard error of the mean *P < .05, **P < .01, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: TUNEL Assay, Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 2. CC1 signaling enhances TIM-3 expression and suppresses CD4þ T-cell inflammatory signature. (A) Representative (n ¼ 4/group) flow cytometry of CC1 and TIM-3 expression in peripheral blood lymphocytes from naive WT and CC1KO mice. TIM-3þ frequency in (B) WT CC1þ and CC1 CD4þ T cells and in (C) WT and CC1KO CD4þ and CD8þ T cells. (D) Repre- sentative (n ¼ 4/group) flow cytometry of CC1 and TIM-3 expression in activated peripheral blood lymphocytes from WT and CC1KO mice subjected to hepatic IRI. TIM-3þ frequency in (E) WT CC1þ and CC1 CD4þ T cells and in (F) WT and CC1KO CD4þ and CD8þ T cells. (G) CC1þ frequency in naive vs post-IRI in WT CD4þ T cells. (H) TIM-3þ frequency in naive vs post-IRI in WT and CC1KO CD4þ T cells. (I) Quantitative reverse-transcription polymerase chain reaction–assisted detection of IFN-g, T-box protein (T-bet) expressed in T cells, IL6, IL17, IL22, and TNF-a in CD4þ T cells from WT and CC1KO mice before and after stimulation (n ¼ 6/group). White square: WT; black square: CC1KO mouse. Data shown as mean ± standard error of the mean. *P < .05, ****P < .0001, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Expressing, Cytometry, Reverse Transcription, Polymerase Chain Reaction

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 4. Enhanced T cell–specific TIM-3 alleviates IRI-OLT and suppresses Kupffer cell NF-kB p65 in CC1-deficient re- cipients. WT livers after 18 hours of cold storage were transplanted into WT, CC1KO, T cell–specific TIM-3Tg/CC1KO, and TIM-3Tg/CC1KO þ anti–TIM-3 antibody (n ¼ 6–8/group). OLT/serum samples were analyzed at 6 hours. (A) Representative H&E and TUNEL staining. DAPI, 40,6-diamidino-2-phenylindole. Scale bars ¼ 100 mm. (B) Suzuki’s histologic grading of liver IRI and quantification of TUNELþ cells/HPF. (C) sAST/sALT (U/L). Quantitative reverse-transcription polymerase chain reaction–assisted detection of (D) IFN-gamma, IL6, and IL17, and (E) TLR4, IL-1b, and TNF-a in OLT (n ¼ 6/group). Data in D and E were normalized to hypoxanthine guanine phosphoribosyltransferase gene expression. (F) Western blot-assisted detection of CC1, phosphorylated (p)-NF-kB p65, NF-kB p65, and vinculin (VCL). The relative intensity ratio of p-NF-kB p65/NF-kB p65 (n ¼ 3–4/group) is shown. (G) Representative NF-kB staining in OLT. Arrows indicate nuclear NF-kB locali- zation in nonparenchymal cells. Scale bars ¼ 100 mm. (H) Representative C-type lectin domain family 4 member F (CLEC4F; Kupffer cell) and p-NF-kB p65 staining. Arrowheads indicate Kupffer cells augmenting p-NF-kB p65. Scale bars ¼ 100 mm (left panels) and 20 mm (enlarged images). Data are shown as mean ±standard error of the mean. *P < .05, **P < .01, ***P < .01, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: TUNEL Assay, Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 5. Donor liver CC1 deficiency compromises T cell–specific TIM-3 regulation in CC1-deficient recipients. (A) CC1KO livers after 18 hours of cold storage were transplanted into CC1KO or TIM-3Tg/CC1KO mice. OLT/serum samples were analyzed at 6 hours (n ¼ 6/group). The sham group (n ¼ 5) underwent the same procedures, except for OLT. (B) Representative H&E staining. Scale bars ¼ 100 mm. (C) Suzuki’s histologic grading of liver IRI and sAST/sALT (U/L). (D) Representative C-type lectin domain family 4 member F (CLEC4F; Kupffer cells) and phosphorylated (p)-NF-kB p65 staining in OLT. DAPI, 40,6- diamidino-2-phenylindole. Arrowheads indicate p-NF-kB p65þ cells. Scale bars ¼ 100 mm. Quantative reverse-transcription polymerase chain reaction–assisted detection of (E) IFN-gamma, IL6, and IL17 (n ¼ 6/group), and (F) TLR4, IL-1b, and TNF-a (n ¼ 6/group) in OLT. Data were normalized to hypoxanthine guanine phosphoribosyltransferase (HPRT) gene expression. (G) Western blot–assisted detection of CC1, p-NF-kB p65, NF-kB p65, and b-actin. The relative intensity ratio of p- NF-kB p65/NF-kB p65 (n ¼ 3/group) is shown. Data are shown as mean ± standard error of the mean. ***P < .001, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 6. Perioperative increase of CC1 promotes anti-inflammatory phenotype in human OLT. (A) Pretransplant (after cold storage) and posttransplant (2 hours after reperfusion) hepatic biopsy specimens were collected from OLT patients. Post-/pre- CC1 ratios were analyzed at the gene (n ¼ 27) and protein (n ¼ 50) levels. Relationship between post-/pre-CC1 gene ratio and (B) CD154, CD28, IFN-gamma, and IL-17, (C) TLR2, TLR4, TLR9, and CD68, and (D) cathepsin G and HO-1 gene expression with b-actin normalization (n ¼ 27), *P < .05, **P < .01; nonparametric Spearman’s method.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Gene Expression

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 7. Perioperative increase of CC1 attenuates hepatocellular injury and improves rejection-free human OLT survival. Post-/pre- CC1 ratios were assessed by Western blots with b-actin normalization. (A) OLT patients were divided into low (n ¼ 25) and high (n ¼ 25) post-/pre-CC1 ratio groups, based on the median value of the CC1 ratio (cutoff ¼ 1.05). (B) Representative Western blots and case patient-related clinical parameters (case patients 1 and 2: low post-/pre-CC1 ratio, case patients 3 and 4: high post-/pre-CC1 ratio). (C) sAST/sALT at POD 17. (D) Representative CD4/CC1 staining in OLT. Arrows: CC1-negative CD4þ T cells; arrowheads: CC1-positive CD4þ T cells. Scale bars ¼ 100 mm. DAPI, 40,6-diamidino-2-phenylindole. (E) Incidence of EAD. (F) The cumulative rejection rate (Kaplan-Meier method). The solid line indicates high and dotted line low post-/pre-CC1 ratio in human OLT. Data shown as mean ± standard error of the mean. *P < .05, Mann-Whitney U test in C, Fisher’s exact test in E, and log-rank test in F.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Western Blot, Staining, MANN-WHITNEY

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

Article Title: The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

doi: 10.1097/CJI.0000000000000100

Figure Lengend Snippet: DIATHIS1 reactivity on natural killer (NK) and melanoma cells. A, Intact/living melanoma cells were stained with 10 μg/mL of single-chain variable fragment (scFv) DIATHIS1 or with the same amount of an irrelevant human scFv antibody and processed for the identification of the binding pattern profiles by flow cytometry studies. The gray histograms represent cells reacted with secondary antibodies, whereas black lines show the scFv antibodies binding. B, The specificity of the scFv DIATHIS1 for CEACAM1 was evaluated with a competitive binding assay. MelC cells were exposed to 10 μg/mL of scFv DIATHIS1 and in separated samples soluble rCEACAM1 was also added at increasing concentrations ranging from 5 to 50 μg/mL. Negative control (ctr) is represented by MelC cells reacting with secondary antibodies only. The upper panel shows the specific scFvDIATHIS1 binding revealed with the FITC fluorophore, whereas in the lower panel the counterstaining of nuclei with DAPI is shown. C, The binding pattern of scFv DIATHIS1 was also evaluated on IL-2-dependent NK-92 and NKL cell lines as well as on freshly isolated and in in vitro–activated NK cells derived from different healthy donors. Cells were stained with 10 μg/mL of scFv DIATHIS1 and processed for conventional flow cytometry studies. The gray histograms represent cells reacted with secondary antibodies (ctr), whereas black lines show the level of the scFvDIATHIS1 antibody binding. d indicates donor.

Article Snippet: CEACAM1 expression was also assessed by a rabbit polyclonal antibody to human CEACAM1 N -terminal domain (Antibodies-online, Aachen, DE) or the anti-CEACAM1 mAb clone 4D1/C2 (Merk Millipore, Darmstadt, Germany).

Techniques: Staining, Binding Assay, Flow Cytometry, Competitive Binding Assay, Negative Control, Isolation, In Vitro, Derivative Assay

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

Article Title: The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

doi: 10.1097/CJI.0000000000000100

Figure Lengend Snippet: Determination of CEACAM1 isoforms. RNAs isolated from MelC and Mel501 cell lines and from STA primary melanoma cells were analyzed in RT-PCR assays for the relative quantities determination of CEACAM1 isoforms by using the primer sequences and the method described and validated in. A, The gels represent a typical PCR result obtained with cDNAs from MelC, Mel501, and STA as template. The common sense primer was FP49. The antisense primers, L and S, were BP60 and BP59, respectively. PCRs were performed by using 3 primers (L+S) for the relative quantification. B, The Western blot analysis of CEACAM1 isoforms on total cellular lysates from MelC, Mel501, and STA melanoma cells reacted with the anti-CEACAM1 mAb 4D1/C2. The right panel shows the signals obtained on Mel501 and STA samples after a long time exposure. Total proteins were loaded at equal amounts and equal loading was monitored by antiactin blotting. C, The histograms reporting the relative quantities of CEACAM1 isoforms mRNA expression in percentage on the total CEACAM1mRNA expression determined by quantitative scanning of the bands in the gels. All data are representative of 3 independent experiments. All data are the mean±SD; * P <0.05; ** P <0.01; *** P <0.0001 (2-tailed Student t test).

Article Snippet: CEACAM1 expression was also assessed by a rabbit polyclonal antibody to human CEACAM1 N -terminal domain (Antibodies-online, Aachen, DE) or the anti-CEACAM1 mAb clone 4D1/C2 (Merk Millipore, Darmstadt, Germany).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Western Blot, Expressing

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Knock-Out, Infection, Bacteria, Derivative Assay, Expressing, Flow Cytometry, Control