epithelial cell line caco2  (ATCC)

Journal: Clinical & Translational Immunology

Article Title: Aberrant intra‐epithelial lymphocytes cause enterocyte cell death in refractory celiac disease by CD103 ‐β7‐receptor‐mediated granzyme‐B degranulation which can be restored by etrolizumab

doi: 10.1002/cti2.70099

Figure Lengend Snippet: Granzyme‐B is the key cell death mediator in enterocyte‐induced cell death and is only secreted by aberrant intra‐epithelial T‐lymphocyte (IEL) in the presence of enterocytes. (a) Cytoplasmic localisation of granzyme‐B granules in refractory celiac disease type II (RCDII) cells P2 using immunofluorescence (red, granzyme‐B; blue, DAPI nucleus staining, 100× magnification). (b) Expression of degranulation marker CD107a on RCDII cell lines P1 and P2 in the absence and presence of epithelial Caco2 cells after 4 h of incubation. (c) CD107a expression on aberrant IEL of a duodenal biopsy from a representative RCDII patient with villous atrophy after 4 h of incubation; left panel, isotype‐matched control. (d) Granzyme‐B secretion by RCDII cell lines in the absence and presence of enterocyte cell line Caco2 after 6 h of incubation. (e) RCDII cell lines P1 and P2 induce killing of epithelial Caco2 cells. The control cell line SUDHL4 showed no cytotoxicity against the Caco2 cells. (f) Enterocyte cell death by RCDII cells incubated with increasing concentrations of degranulation blocker hydroxychloroquine sulphate (HCQ). (g) Killing of intestinal Caco2 cells by RCDII cells in the presence of increasing concentrations of the granzyme‐B inhibitor Z‐AAD‐CH2Cl. For cell death assays, cytotoxicity was measured after 16 h of co‐incubation at an effector:target ratio 2:1. Cell experiments were done in triplicate. *** P ≤ 0.001, unpaired t ‐test, results are shown as mean + sem.

Article Snippet: The intestinal epithelial cell line Caco2 was obtained from the American Type Culture Collection (ATCC) and cultured with DMEM medium (BioWhittaker) containing 10% FBS (GE Healthcare Life Sciences) and 100 IU penicillin/100 μg/mL streptomycin (1% P/S) at 37°C.

Techniques: Immunofluorescence, Staining, Expressing, Marker, Incubation, Control

Journal: Clinical & Translational Immunology

Article Title: Aberrant intra‐epithelial lymphocytes cause enterocyte cell death in refractory celiac disease by CD103 ‐β7‐receptor‐mediated granzyme‐B degranulation which can be restored by etrolizumab

doi: 10.1002/cti2.70099

Figure Lengend Snippet: Aberrant intra‐epithelial T‐lymphocyte (IEL) demonstrate upregulated expression of CD103 and require cell–cell binding to induce enterocyte killing. (a) Refractory celiac disease type II (RCDII) cell‐induced cytotoxicity of Caco2 cells in the presence of a transwell system. Cell death was measured after 16 h of co‐incubation at an effector:target ratio 2:1. (b) Degranulation by RCDII cells in the presence of a transwell system. CD107a expression was measured after 4 h of co‐incubation with Caco2 cells at an effector:target ratio 2:1. (c) NKG2D expression on aberrant IEL of RCDII patients and patients with celiac disease (CD) on gluten‐free diet (GFD), using flow cytometry analysis. Activated CD8+ T cells served as positive control. (d) Representative histograms of NKG2D expression on RCDII cell lines P1 and P2 using flow cytometry; grey shaded peak, isotype‐matched control. (e) Killing of epithelial cells Caco2 by RCDII cell line P1 in the presence of 20 μg/mL NKG2D‐blocking mAb or isotype control mAb. (f) CD103 expression on aberrant IEL of RCDII patients and patients with CD on GFD, using flow cytometry analysis. (g) Follow‐up of CD103 expression on aberrant IEL from a representative RCDII patient, showing persistent villous atrophy after first‐line treatment (cladribine; non‐responding) and complete mucosal recovery after second‐line treatment (autologous stem cell transplantation; responding). t = 0 at diagnosis and start treatment, t = 1 is 3 months after first‐line treatment, t = 2 and t = 3 is 3, respectively, 6 months after stem cell transplantation. (h) Histograms of CD103 expression on RCDII cell lines P1 and P2 using flow cytometry analysis; grey shaded peak, isotype‐matched control. Cell experiments were done in triplicate. * P ≤ 0.05, *** P ≤ 0.001, unpaired t ‐test, results shown as mean + sem.

Article Snippet: The intestinal epithelial cell line Caco2 was obtained from the American Type Culture Collection (ATCC) and cultured with DMEM medium (BioWhittaker) containing 10% FBS (GE Healthcare Life Sciences) and 100 IU penicillin/100 μg/mL streptomycin (1% P/S) at 37°C.

Techniques: Expressing, Binding Assay, Incubation, Flow Cytometry, Positive Control, Control, Blocking Assay, Transplantation Assay, Biomarker Discovery

Journal: Clinical & Translational Immunology

Article Title: Aberrant intra‐epithelial lymphocytes cause enterocyte cell death in refractory celiac disease by CD103 ‐β7‐receptor‐mediated granzyme‐B degranulation which can be restored by etrolizumab

doi: 10.1002/cti2.70099

Figure Lengend Snippet: Aberrant intra‐epithelial T‐lymphocyte (IEL)‐enterocyte binding via CD103 induces granzyme‐B‐mediated enterocyte cell death. (a) Killing of Caco2 epithelial cells by refractory celiac disease type II (RCDII) cell lines in the presence of 10 μg/mL CD103‐blocking mAb or isotype control. Cell death was measured after 16 h of co‐incubation at an effector:target ratio 2:1. (b) Degranulation by RCDII cell lines, measured by CD107a expression, in the presence of 10 μg/mL CD103‐blocking mAb or the matching isotype control. Degranulation was measured after 4 h of co‐incubation with Caco2 cells at an effector:target ratio 2:1. (c) Secretion of granzyme‐B by RCDII cell lines co‐incubated with Caco2 cells in the presence of 10 μg/mL CD103‐blocking mAb compared to the isotype control. Secretion was measured after 6 h of co‐incubation at an effector:target ratio 2:1. (d) Upper left picture: small intestinal organoids; upper right picture: attachment of RCDII cells to an organoid (blue arrow); lower left picture: RCDII cells induce killing of organoids (red arrows); lower right picture: in the presence of 10 μg/mL CD103‐blocking mAb RCDII–induced organoid cell death is evidently reduced, illustrated by the presence of viable organoids (green arrows). (e) Induction of organoid cell death by RCDII cells P2 in the presence of 10 μg/mL CD103‐blocking antibody or an isotype control, measured by cell count via microscopy. Killing was measured after 24 h of co‐incubation at an effector:target ratio 50:1. Figure , upper pictures 25× magnification, lower pictures 10× magnification, using an Olympus microscope. Cell experiments for Figure performed in duplicate, other cell experiments performed in triplicate. *** P ≤ 0.001, unpaired t ‐test, results shown as mean + sem.

Article Snippet: The intestinal epithelial cell line Caco2 was obtained from the American Type Culture Collection (ATCC) and cultured with DMEM medium (BioWhittaker) containing 10% FBS (GE Healthcare Life Sciences) and 100 IU penicillin/100 μg/mL streptomycin (1% P/S) at 37°C.

Techniques: Binding Assay, Blocking Assay, Control, Incubation, Expressing, Cell Characterization, Microscopy

Journal: Clinical & Translational Immunology

Article Title: Aberrant intra‐epithelial lymphocytes cause enterocyte cell death in refractory celiac disease by CD103 ‐β7‐receptor‐mediated granzyme‐B degranulation which can be restored by etrolizumab

doi: 10.1002/cti2.70099

Figure Lengend Snippet: Etrolizumab inhibits granzyme‐B secretion and restores enterocyte viability. (a) Histograms of β7 expression on refractory celiac disease type II (RCDII) cell lines P1 and P2; grey shaded peak, isotype‐matched control. (b) Caco2 cell death by RCDII cells in the presence of 50 μg/mL etrolizumab or isotype control. Cell death was determined after 16 h of co‐incubation at an effector:target ratio 2:1. (c) Degranulation by RCDII cell lines in the presence of 50 μg/mL etrolizumab or the matching isotype control. Degranulation was measured after 4 h of co‐incubation with Caco2 cells at an effector:target ratio 2:1. (d) Secretion of granzyme‐B by RCDII cell lines co‐incubated with enterocyte Caco2 cells in the presence of 50 μg/mL etrolizumab compared to the isotype control. Secretion was measured after 6 h of co‐incubation at an effector:target ratio 2:1. (e) Upper picture: RCDII cells attach to the organoid surface and cause membrane disruption (red arrow), inducing organoid cell death; lower picture: the attachment of RCDII cells to organoids and subsequent organoid cell death is decreased in the presence of 50 μg/mL etrolizumab antibody. (f) Induction of organoid cell death by RCDII cells P2 in the presence of 50 μg/mL etrolizumab or an isotype control, measured by cell count via microscopy. Killing was measured after 24 h of co‐incubation at an effector:target ratio 20:1. Figure , pictures 10× magnification, using an Olympus microscope. Cell experiments were done in triplicate. *** P ≤ 0.001, unpaired t ‐test, results shown as mean + sem.

Article Snippet: The intestinal epithelial cell line Caco2 was obtained from the American Type Culture Collection (ATCC) and cultured with DMEM medium (BioWhittaker) containing 10% FBS (GE Healthcare Life Sciences) and 100 IU penicillin/100 μg/mL streptomycin (1% P/S) at 37°C.

Techniques: Expressing, Control, Incubation, Membrane, Disruption, Cell Characterization, Microscopy

Journal: Scientific Reports

Article Title: Increase of intracellular Zn 2+ concentration directly inhibits phospholipase Cε and suppresses inflammation and tumour formation in mice

doi: 10.1038/s41598-025-25886-5

Figure Lengend Snippet: Zinc as an Active Principle of ZPT. (A) Chemical structures of dipyrithione and sodium pyrithione are shown. The activities of varying concentrations of ZPT, ZnSO 4 , dipyrithione and sodium pyrithione to inhibit the hydrolysis of Compound 1 by purified PLCε were measured as described in Methods. ( B ) The activities of varying concentrations of ZnSO 4 to inhibit the hydrolysis of Compound 1 by purified PLCε, PLCβ4, PLCδ1 or PLCγ1 were measured as described in Methods. (C) The activities of 5 µM ZnSO 4 to inhibit the hydrolysis of Compound 1 by purified PLCε were measured in the presence of varying concentrations of sodium pyrithione. ( D ) Caco2 cells were treated with 5 µM ZnSO 4 in the presence or absence of 5 µM sodium pyrithione or with 5 µM ZPT for 30 min. Intracellular Zn 2+ concentrations of the treated cells were measured by using Zinquin ethyl ester as described in Methods. The values obtained are expressed as fold changes over the average value of untreated cells. All the experiments described above were performed three times in duplicates.

Article Snippet: A human colon cancer epithelial cell line Caco2 was purchased from ATCC (HTB-37) and maintained in 5% CO 2 at 37 °C in modified Eagle’s minimum essential medium (MEM) (Nacalai tesque) supplemented with 20% fetal bovine serum (FBS) (Sigma), non-essential amino acids (Gibco) and 100 μg/ml penicillin-streptomycin (Nacalai tesque).

Techniques: Purification

Journal: Scientific Reports

Article Title: Increase of intracellular Zn 2+ concentration directly inhibits phospholipase Cε and suppresses inflammation and tumour formation in mice

doi: 10.1038/s41598-025-25886-5

Figure Lengend Snippet: Effect of ZPT on PKD–NF-κB Signaling and Proinflammatory Gene Expression. (A) Caco2 and SW480 cells were serum-starved for 3 h in the presence of the indicated concentrations of ZPT or a vehicle (DMSO) and subsequently stimulated by 20 µM LPA for 30 min. PKD phosphorylated at Ser916 and total PKD in the cell lysates were quantified by immunoblotting with anti-phospho-PKD (Ser916) and anti-PKD Abs, respectively, as described in Methods. Numbers below the immunoblots indicate fold increase of the phospho-PKD signals divided by the total PKD signals over that at 0 min after LPA stimulation of the control cells. Each of the experiments was performed three times yielding equivalent results. A representative result is shown. The original blots are presented in Supplementary Figures S4 and S5. (B) Caco2 cells treated with 5 µM ZPT or the vehicle as described in (A) were subjected to subcellular fractionation and the resulting nuclear and cytoplasmic fractions were subjected to immunoblotting with the anti-NF-κB p65 Ab. TATA-binding protein (TBP) and α-tubulin were used as markers for the nuclear and the cytoplasmic fractions, respectively. A fold change of the nuclear NF-κB caused by 5 µM ZPT treatment is shown in numbers. Each of the experiments was performed three times yielding equivalent results. A representative result is shown. The original blots are presented in Supplementary Figure S6. (C) Total cellular RNAs isolated from Caco2 cells treated as described in (B) were subjected to qRT-PCR for quantification of the mRNA levels of the proinflammatory molecules: CXCL1, CXCL8, CCL2, CCL20, TNF-α and COX-2, by using the b- actin mRNA as an internal control. The obtained values are expressed as relative fold changes of nuclear NF-κB normalized to TATA-binding protein (TATA), with the value from the − LPA/−ZPT condition set to 1. The experiments were performed three times in duplicates.

Article Snippet: A human colon cancer epithelial cell line Caco2 was purchased from ATCC (HTB-37) and maintained in 5% CO 2 at 37 °C in modified Eagle’s minimum essential medium (MEM) (Nacalai tesque) supplemented with 20% fetal bovine serum (FBS) (Sigma), non-essential amino acids (Gibco) and 100 μg/ml penicillin-streptomycin (Nacalai tesque).

Techniques: Gene Expression, Western Blot, Control, Fractionation, Binding Assay, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Increase of intracellular Zn 2+ concentration directly inhibits phospholipase Cε and suppresses inflammation and tumour formation in mice

doi: 10.1038/s41598-025-25886-5

Figure Lengend Snippet: Effect of ZPT Administration on Proliferation of Xenografted Caco2 cells. Caco2 cells (5 × 10 6 cells) were implanted subcutaneously into the right flanks of female nude mice. One week later, the mice were administered intraperitoneally with ZPT, dipyrithione or the vehicle 3 days per week for 8 weeks, during which the body weight (A) and the tumour volumes (B) were measured every 7 days. The number of mice ( n ) for each group is shown in parenthesis. The number of mice ( n ) for each group is shown in parenthesis. Thereafter, the tumours were excised and weighed (C) .

Article Snippet: A human colon cancer epithelial cell line Caco2 was purchased from ATCC (HTB-37) and maintained in 5% CO 2 at 37 °C in modified Eagle’s minimum essential medium (MEM) (Nacalai tesque) supplemented with 20% fetal bovine serum (FBS) (Sigma), non-essential amino acids (Gibco) and 100 μg/ml penicillin-streptomycin (Nacalai tesque).

Techniques: