cd66a (cc1) antibody  (Thermo Fisher)

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: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Livers from groups of WT and CC1-KO C57BL/6 donor mice, stored in UW solution (4°C/18 hours), were transplanted to WT C57BL/6 recipient mice. OLT and serum samples were analyzed 6 hours after reperfusion. The sham group underwent the same procedures except for OLT. (A) Representative H&E staining (original magnification ×100). (B) Suzuki’s histological grading of liver IRI. (C) sAST and sALT levels (IU/L). (D) Representative TUNEL staining and immunohistochemical staining of OLT-infiltrating CD11b+ and Ly6G+ cells (original magnification ×200). (E) Quantification of TUNEL-positive cells/HPF. (F) Serum HMGB1 (ng/mL) and MCP1 (pg/mL) levels measured by ELISA. (G) Quantification of hepatic CD11b+ and Ly6G+ cells/HPF. (H) Real-time reverse transcription PCR–assisted (qRT-PCR–assisted) detection of mRNA coding for MCP1, CXCL1, CXCL2, and CXCL10 in OLT. Data were normalized to HPRT gene expression. Data are mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B, C, and E–G) or Student’s t test (H), n = 5–6/group.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Staining, TUNEL Assay, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO liver grafts stored in UW solution (4°C/18 hours) were perfused with physiological saline (2 mL) via a cuff placed at the portal vein to collect liver flush from inferior vena cava. (B) Liver flush samples (20 μL) from cold-stressed WT or CC1-deficient livers were screened by Western blots for HMGB1/Histone H3 levels (n = 4/group, *P < 0.05, Student’s t test). (C) WT or CC1-KO liver grafts were collected after cold storage (4°C/18 hours). Representative (n = 3/group) immunohistochemical staining of CC1/4HNE (a ROS metabolite), CC1/HMGB1, and TUNEL is shown. Arrowheads indicate extranuclear HMGB1 localization. (D) BMDM cultures (WT) were stimulated (6 hours) with liver flush obtained from WT or CC1 KO cold-stored grafts. qRT-PCR–assisted detection of mRNA coding for MCP1, CXCL2, CXCL10 with β2M normalization (n = 4–6, *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Saline, Western Blot, Immunohistochemical staining, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours). Liver samples were collected right after cold storage (before OLT) or 3 hours after reperfusion (post-OLT). (B and C) Western blot–assisted detection and relative intensity ratio of cleaved caspase-3, RIP3, CC1, ASK1, p-p38 in naive liver, cold-stored liver or postreperfusion OLT (WT). Vinculin (VCL) expression served as an internal control and was used for normalization (n = 3/group). (D) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in naive WT or CC1-KO liver. VCL expression served as an internal control and was used for normalization (n = 4/group). (E) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in cold-stored WT or CC1-KO livers. VCL expression served as an internal control and used for normalization (n = 3/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B and C) or Student’s t test (D and E).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Primary mouse hepatocytes (WT) with or without cold stimulation (4°C/4 hours) were incubated for the indicated time periods. Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 2/group). (B–D) Cold-stimulated WT or CC1-KO hepatocytes were pretreated with or without siRNA against ASK1. (B) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 3/group). (C) Representative (n = 3/group) immunohistochemical staining of 4HNE (red, upper panels), HMGB1 (red, middle panels), and dead cell detection (red, lower panels). (D) Quantification of dead cells/HPF (n = 4–5/group). *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Incubation, Western Blot, Expressing, Control, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A–C) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours) with or without ASK1 inhibitor (10 μg/15 mL). (A) Western blot–assisted detection and relative intensity ratio of CC1 and p-p38. VCL expression served as an internal control and used for normalization (n = 3–4/group). (B) Representative (n = 3/group) immunohistochemical staining of CC1/4HNE and CC1/HMGB1. (C) Liver flush (20 μL) from cold-stressed WT or CC1-KO livers with or without ASK1 inhibitor were analyzed by Western blots for HMGB1 levels (n = 3–4/group). (D–H) Cold-stored (4°C/18 hours) WT or CC1-KO livers were transplanted into recipient mice, and OLT and serum samples were analyzed at 6 hours after reperfusion. Some CC1-KO grafts were preincubated with ASK1 inhibitor (10 μg/15 mL) during cold storage (4°C/18 hours). Separate OLT recipient groups were monitored for 20-day survival. (D) Representative H&E (original magnification ×100) and TUNEL staining. (E) sAST and sALT levels (IU/L; n = 7–8/group). (F) Suzuki’s histological grading of liver IRI (n = 7–8/group). (G) Quantification of TUNEL-positive cells/HPF (n = 7–8/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test. (H) Recipient mice were monitored for 20 days and cumulative survival was analyzed (Kaplan-Meier method). Dotted line: WT → WT; solid line: CC1-KO → WT; bold line: CC1-KO+ASK1 inhibitor → WT (n = 6–9/group; *P < 0.05 vs. CC1-KO → WT, log-rank test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1, ASK1, and p-p38 levels (see Supplemental Figure 1A). (A) Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups based on the relative CEACAM1/β-actin levels (cutoff = 0.85, median). (B) Western blot–assisted expression of ASK1 and p-p38. Data shown in dot plots and bars indicate mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Four representative Western blots are shown (case 1/2: low CEACAM1, case 3/4: high CEACAM1). (D) Representative (n = 3) CEACAM1/4HNE staining (original magnification ×200). (E) Representative (n = 3) CEACAM1/HMGB1 staining (original magnification ×400).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, MANN-WHITNEY, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups, based on the relative CEACAM1/β-actin levels (see Figure 6A). (A) Serum AST levels at POD1–7. (B) Serum ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding clinical cases. Representative (n = 3) TUNEL staining (original magnification ×400). (D) Incidence of EAD (Fisher’s exact test). (E) The cumulative probability of overall graft survival. (F) The cumulative probability of rejection-free graft survival. Solid line indicates low CEACAM1; dotted line indicates high CEACAM1 human OLT patient group (Kaplan-Meier method, log-rank test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Expressing, MANN-WHITNEY, TUNEL Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were classified into low (n = 30) and high (n = 30) CEACAM1 (CC1) expression groups (see Figure 6A for details). Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding cases, followed by qRT-PCR–assisted detection of mRNA coding for TLR4, CD80, CD86, CXCL10, CD68, Cathepsin G, CD28, CD4, and IL17. Data normalized to GAPDH gene expression are shown in dot plots and bars indicative of mean ± SEM. #P < 0.05 (Mann-Whitney U test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1 levels. (A) ROC analysis of CEACAM1/β-actin for predicting EAD. Based on a ROC curve and Youden index on the basis of best accuracy in relation to EAD incidence, the CEACAM1/β-actin cutoff value of 0.71 was determined. AUROC, area under the receiver operating characteristic curve. (B) Based on the optimal cutoff value (0.71), 60 human OLTs were classified into CEACAM1/β-actin less than 0.71 (n = 20) and CEACAM1/β-actin greater than 0.71 cases (n = 40), and the incidence of EAD was evaluated. #P < 0.05 (Fisher’s exact test). (C) Serum AST and ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (D) Stepwise multivariate logistic regression analysis was performed to identify independent risk factors of EAD.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, MANN-WHITNEY