Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a Expression of TF from RNA-seq data of ileal biopsies from Crohn’s Disease (CD); illeal (iCD, n = 162), colonic (cCD, n = 56), Ulcerative Colitis (UC, n = 62), and query IBD healthy control cohort (CTRL, n = 42) patients in the RISK study (GEO ID GSE57945). b Expression of coagulation and inflammation-associated genes from RNA sequencing of paediatric CD ( n = 11), UC ( n = 7), and inflamed query non-IBD biopsies ( n = 12). c TF, CD3 and CD4 co-staining and d the average number of TF + CD3 + CD4 + T cells per field in colonic biopsies from paediatric IBD patients (IBD, n = 4, non-IBD, n = 3). e – h Lamina propria cells were isolated from colonic biopsies from paediatric IBD patients ( n = 5) and inflamed query non-IBD biopsies ( n = 2). e – g The percentage of TF expressing CD3 + CD4 + TF + T cells and h their cell surface TF expression was measured by flow cytometry and compared. Student’s t -test (two-tailed) ( d , h ) or Mann–Whitney U Test ( a , g ) was used to determine statistical significance from a 322 biological donors (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42), d 7 biological donors ( n = 4 IBD, n = 3 inflamed non-IBD), g , h 7 biological donors ( n = 5 IBD, n = 2 inflamed non-IBD) and expressed as mean ± s.e.m. Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Expressing, RNA Sequencing, Control, Coagulation, Staining, Isolation, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a Schematic diagram of the T cell transfer model of colitis. CD4 + T effector cells were isolated from donor C57BL/6 wild-type (WT) mice and i.p. injected into host Rag1 −/− mice ( Rag1 −/− T cells). Rag1 −/− mice i.p. injected with PBS were used as a control ( Rag1 −/− vehicle). b Colitis developed over a 4-week period, and disease progression was measured by % weight loss compared to original weight. c This was confirmed by subsequent colon histology analysis. d TF staining and e Corrected total fluorescence (CTF) of mice colons following T cell transfer-induced colitis. f TF and CD3 co-staining of mouse colons following T cell transfer-induced colitis (reproduced n = 4 Rag1 −/− T cells and n = 3 Rag1 −/− vehicle). 2-way ANOVA ( b ) or two-tailed Student’s t -test ( c , e ) was used to determine statistical significance from 3–4 biological replicates, n = 4 male Rag1 −/− T cells and n = 3 male Rag1 −/− vehicle, expressed as mean ± s.e.m. Scale bars: 100 µm ( d , f ). Source data are provided in the Source Data file. Created in BioRender. Preston, R. (2025) https://BioRender.com/o91u731 .

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Injection, Control, Biomarker Discovery, Staining, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor human blood and plated at a density of 0.8 × 10 6 /ml with IL-2 for unactivated conditions (θ). Plated cells were activated by anti-CD3/anti-CD28 activation beads and stimulated with IL-2 for Th0 conditions, and differentiation cytokines (αIL-4 + IL-12) were added to skew cells to a Th1 lineage. a – d Following 5 days in culture, θ, Th0 and Th1 cells were washed with EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in normal pooled platelet-poor plasma. b Lagtime, c peak thrombin levels and d endogenous thrombin potential (ETP) were measured and compared between θ, Th0 and Th1 cells. e The rate of clot formation was measured in θ, Th0 and Th1 cells. f – i θ, Th0 and Th1 cell-mediated thrombin generation was analysed by calibrated automated thrombinography using FVII-deficient platelet-poor plasma. g Lagtime, h peak thrombin levels and i ETP were measured and compared between θ, Th0 and Th1 cells. j F3 gene expression, k , l the percentage of cells expressing TF, m cell surface TF expression and n T cell-dependent FXa generation was measured in θ, Th0 and Th1 cells. Student’s paired t -test (two-tailed) ( c – e , g – i , l – n ), Wilcoxon test (two-tailed) ( b ), or Mann–Whitney U Test (two-tailed) ( j ) was used to determine statistical significance. Data is expressed as mean ± s.d. ( b – e , g – i , l – n ) for 7 ( a – d ), 10 ( e ), 6 ( g – i ), 8 ( j ) and 4 ( l – n ) biological donors/group. Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Activation Assay, Clinical Proteomics, Gene Expression, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor human blood and skewed to θ (unactivated cells), Th0, and Th1 cell subtypes as described previously. a ASMase translocation to the cell surface, d PS exposure on the outer membrane leaflet, and g PDI recruitment to the cell surface were assessed. TF decryption pathways in θ, Th0 and Th1 were analysed by b , c ASMase cell surface expression, e , f fluorescently labelled lactadherin binding to exposed cell surface PS, and h , i cell surface PDI expression. Student’s paired t -test (two-tailed) ( c , f , i ) was used to determine statistical significance. Data is expressed as mean ± s.d. ( c , f , i ) for 5 ( c ), 8 ( f ), and 4 ( i ) biological donors/group. Source data are provided in the Source Data file. Created in BioRender. Preston, R. (2025) https://BioRender.com/x11c079 .

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Translocation Assay, Membrane, Expressing, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor adult human peripheral blood, inflamed non-IBD paediatric peripheral blood and paediatric IBD peripheral blood were plated at a density of 1 × 10 6 /ml and incubated for 24–48 h at 37 °C in AIM-V media supplemented with immune replacement serum. a – d The percentage of cells expressing TF and e their cell surface TF expression was measured by flow cytometry and compared between groups. f – j Plated cells were washed in EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in FXII-deficient plasma. g Lagtime, h peak thrombin levels and i ETP were measured. Furthermore, the ability of isolated T cells to facilitate FXa generation in which the T cells were the sole source of TF, was measured ( j ). Mann–Whitney U Test (two-tailed) ( d , e , g ) or Student’s t -test (two-tailed) ( j , h , i ) was used to determine statistical significance. Data is expressed as mean ± s.e.m. ( d , e , g – j ) for d , e , j 17 biological donors ( n = 12 IBD, n = 2 inflamed non-IBD, N = 3 healthy adult), g – i 21 biological donors ( n = 13 IBD, n = 2 inflamed non-IBD, N = 6 healthy adult) and j 18 biological donors ( n = 12 IBD, n = 2 inflamed non-IBD, N = 4 healthy adult). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Incubation, Expressing, Flow Cytometry, Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a PROC (Protein C; PC) expression in colonic biopsies from patients in the RISK study (GEO ID GSE57945) (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42). b , c Following cell culture, T cells were washed and incubated with fluorescently labelled activated PC. Binding was measured by flow cytometry. Following activated PC pre-treatment, activated PC was removed, θ (unactivated cells) and Th0 cells were washed with EDTA-containing PBS, and their capacity to initiate clotting was analysed by d – g thrombin generation and h clot formation assays. Activated PC pre-treated Th1 cell-dependent thrombin generation was analysed by i – l thrombin generation assays and m clot formation assays. Following activated PC pre-treatment, Th0 procoagulant activity was analysed by n FXa generation assay, o F3 gene expression and r , s PDI cell surface expression by flow cytometry. Similarly, Th1 cell thrombogenicity was analysed by p FXa generation assay, q F3 gene expression and t , u PDI cell surface expression by flow cytometry. The contribution of PDI-mediated TF decryption was assessed by treating Th0 ( v , w ) and Th1 ( x , y ) cells with 10 mM rutin for 1 h. Cells were washed with EDTA-containing PBS, and their capacity to initiate clotting was analysed by v – y thrombin generation. Student’s t -test (two-tailed) ( c ), Mann–Whitney U Test (two-tailed) ( a , n – q ), Student’s paired t -test (two-tailed) ( e – g , j – l , m , s , u , w , y ), or Wilcoxon test ( h ) was used to determine statistical significance. Data is expressed as mean ± s.e.m. ( a , c , e – h , j – l , m – q , s , u , w , y ) for a 322 biological donors (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42), c 5–10 biological donors ( n = 5 θ, n = 10 Th0 and n = 9 Th1), e – g , j – l 6 biological donors, h , m 10 biological donors, n , p , s , u 4 biological donors, o , q 8 biological donors and w , y 3–5 biological donors ( n = 3 θ, n = 5 Th0/Rutin and n = 5 Th1/Rutin). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Expressing, Control, Cell Culture, Incubation, Binding Assay, Flow Cytometry, Coagulation, Activity Assay, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor adult human peripheral blood, inflamed non-IBD paediatric peripheral blood and paediatric IBD peripheral blood, plated at a density of 1 × 10 6 /ml and incubated at 37 °C in AIM-V media supplemented with immune replacement serum, +/- 20 nM of activated PC for 24–48 h. a – d Cells were washed in EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in FXII-deficient plasma. b Lagtime, c ETP and d peak thrombin levels were measured and compared, as was their ability to facilitate FXa generation ( e ). Mann–Whitney U Test (two-tailed) ( b – d ) or Student’s t -test (two-tailed) ( e ) was used to determine statistical significance for all conditions except IBD vs IBD + activated PC. For these conditions, either the Wilcoxon test (two-tailed) ( b – d ) or the Student’s paired t -test (two-tailed) ( e ) was used as the data is matched. Data is expressed as mean ± s.e.m. for b – d 21 biological donors ( n = 13 IBD/IBD activated PC, n = 2 inflamed non-IBD, N = 6 healthy adult) and e 19 biological donors ( n = 13 IBD, n = 2 inflamed non-IBD, N = 4 healthy adult). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Incubation, Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: Journal of Virology

Article Title: NFAT4 Is Required for JC Virus Infection of Glial Cells

doi: 10.1128/jvi.01456-06

Figure Lengend Snippet: FIG. 2. NFAT4 is functionally active in glial cells. (a) NFAT expression in SVG-A and U-87MG whole-cell lysates. NFATs 1 to 4 were detected using antibodies (NFATc1 7A6, NFATc2 4G6-G5, NFATc3 F-1, and NFATc4 H-74; Santa Cruz Biotech Inc.) diluted 1:200. Ramos cell lysates were used as a positive control. (b) Luciferase reporter gene assays using an NFAT-responsive reporter construct were used to compare NFAT activity in SVG-A cells and U-87MG cells in the presence or absence of 292 mg/liter glutamate. A control construct that lacked the NFAT binding site was used to measure basal transcriptional activity. (c) The NFAT reporter construct was cotransfected with either a control construct ( SV40 T-Ag) or a construct expressing the SV40 large T-Ag ( SV40 T-Ag).

Article Snippet: Ramos cell lysates were purchased from Santa Cruz Biotech Inc. Whole-cell extracts were separated on Tris-HCl-ready gels (Bio-Rad).

Techniques: Expressing, Positive Control, Luciferase, Construct, Activity Assay, Control, Binding Assay

Journal: Vaccines

Article Title: An Antibody Persistent and Protective Two rSsCLP-Based Subunit Cocktail Vaccine against Sarcoptes scabiei in a Rabbit Model

doi: 10.3390/vaccines8010129

Figure Lengend Snippet: Variation of specific IgG ( a and b ) and total IgE ( c ) levels in the sera of immunized rabbits. a: the OD 450 nm value of the specific IgG antibodies was detected by an rSsCLP5-based indirect ELISA; b: the OD 450 nm value of specific IgG antibodies was detected by an rSsCLP12-based indirect ELISA; c: the total IgE antibody concentration (pg/mL) was detected by an ELISA kit (Elabscience, Wuhan, China). The rSsCLP 5 and rSsCLP 12 refer to rSsCLP5 and rSsCLP12, respectively.

Article Snippet: The serum samples collected from the vaccination trial were analyzed for the presence of specific IgG antibodies with a rSsCLP5-based indirect ELISA [ ] and rSsCLP12-based indirect ELISA [ ], total IgE antibodies with an ELISA kit (Elabscience, Wuhan, China), and the level of IL-4, IL-10, IFN-γ, and TNF-α with an ELISA kit (Elabscience, Wuhan, China).

Techniques: Indirect ELISA, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: An Antibody Persistent and Protective Two rSsCLP-Based Subunit Cocktail Vaccine against Sarcoptes scabiei in a Rabbit Model

doi: 10.3390/vaccines8010129

Figure Lengend Snippet: Variation of cytokines IL-10 ( a ), TNF-α ( b ), IL-4 ( c ), and IFN-γ ( d ) in the sera of immunized rabbits. a, the concentration of IL-10 (pg/mL) was detected by an ELISA kit (Elabscience, Wuhan, China); b, the concentration of TNF-α (pg/mL) was detected with an ELISA kit (Elabscience, Wuhan, China); c, the concentration of IL-4 (pg/mL) was detected using an ELISA kit (Elabscience, Wuhan, China); d, the concentration of IFN-γ (pg/mL) was detected using an ELISA kit (Elabscience, Wuhan, China). The rSsCLP 5 and rSsCLP 12 refer to rSsCLP5 and rSsCLP12, respectively.

Article Snippet: The serum samples collected from the vaccination trial were analyzed for the presence of specific IgG antibodies with a rSsCLP5-based indirect ELISA [ ] and rSsCLP12-based indirect ELISA [ ], total IgE antibodies with an ELISA kit (Elabscience, Wuhan, China), and the level of IL-4, IL-10, IFN-γ, and TNF-α with an ELISA kit (Elabscience, Wuhan, China).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Device

Article Title: CRISPR-Cas12a immunosensing on glass fiber for point-of-care quantification of multiple inflammation biomarkers in osteoarthritis

doi: 10.1016/j.device.2024.100319

Figure Lengend Snippet: Figure 6. Detection of inflammation biomarkers in clinical samples of OA patients by the GUSCI platform and ELISA (A) Heatmap of the levels of protein expression. F indicates GUSCI platform results, while E indicates ELISA results. The color scale bar represents the con- centration of biomarkers (the concentration unit for IL-4, IL-6, TNF-a, IFN-g is pg mL1, and the unit for MMP-3 is ng mL1). (B–F) Correlation between the GUSCI platform and ELISA to measure IL-4, IL-6, TNF-a, IFN-g, and MMP-3 concentration.

Article Snippet: The human IL-6 ELISA kit (catalog number VAL102), human IL-4 ELISA kit (catalog number VAL123), and human IFN-g ELISA kit (catalog number VAL104C) were obtained from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay