Journal: Translational Oncology

Article Title: STIL facilitates the development and malignant progression of triple-negative breast cancer through activation of Fanconi anemia pathway via interacting with KLF16

doi: 10.1016/j.tranon.2024.102010

Figure Lengend Snippet: STIL knockdown inhibits the Fanconi anemia signaling pathway. (A) KEGG pathway enrichment analysis of down-regulated genes sets upon STIL knockdown in MDA-MB-231 cells. (B) qPCR validation of STIL-dependent regulation for selected FA pathway genes. (C) Western blotting of FA pathway proteins FANCD2, FANCA, FANCB, and FANCI after STIL knockdown in indicated breast cancer cell lines. The data represented the mean ± SD of three independent experiments, and the level of significance was indicated by **** P <0.0001, *** P < 0.001,** P < 0.01, * P < 0.05.

Article Snippet: Paraffin-embedded tumor tissues were processed for immunohistochemical staining with primary antibodies against STIL (1:100, Proteintech Group, USA), FANCD2 (1:100, Santa Cruz Biotechnology) and Ki-67 (1:100, Abcam) overnight at 4 °C and stained with 3, 3′-diaminobenzidine for color development.

Techniques: Knockdown, Biomarker Discovery, Western Blot

Journal: Translational Oncology

Article Title: STIL facilitates the development and malignant progression of triple-negative breast cancer through activation of Fanconi anemia pathway via interacting with KLF16

doi: 10.1016/j.tranon.2024.102010

Figure Lengend Snippet: STIL regulated breast cancer cells progress by targeting FANCD2. (A) Western blotting was used to detect the proportion of STIL protein expression in the cytoplasm and nucleus of breast cancer cell lines MDA-MB-231 and HCC1806. β-Actin and Lamin B1 are used as internal references for cytoplasm and nucleus, respectively. ( B ) Dual-luciferase reporter assay detect the effect of STIL overexpression on the activation activity of FANCD2 promoter. (C) The expression of FANCD2 in TNBC tissues and adjacent normal tissues (BRCA, Breast cancer; T, Tumor, red; N, Normal, blue). (D) Correlation of gene expression between STIL and FANCD2 in human breast cancer. (E-F) MDA-MB-231 cells and HCC1806 cells were co-transfected with STIL shRNA and FANCD2 overexpression plasmids, and then colony formation and wound scratch assay were performed. (G-J) MDA-MB-231 cells with stable knockdown of STIL and overexpression of FANCD2 simultaneously were injected subcutaneously into mice, and (G) the tumor volume was calculated within 27 days after tumors were injected, (H) Tumor images at the end of the experiment were presented, (I) the tumor volume was calculated after being removed from the mice. (J) the representative photographs of IHC staining for STIL, FANCD2 and Ki67 in tumor tissues were shown. (K) Mice were injected with tumor cells through the tail vein. Images of a representative lung metastasis mouse model (left). Representative H&E staining of metastatic nodules in the lungs (right). Scale bars in ( F ), 200 μm. Scale bars in ( J ), 50 μm. Scale bars in (K), 200 μm and 100 μm. The data represented the mean± SD of three independent experiments, and the level of significance was indicated by **** P <0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Paraffin-embedded tumor tissues were processed for immunohistochemical staining with primary antibodies against STIL (1:100, Proteintech Group, USA), FANCD2 (1:100, Santa Cruz Biotechnology) and Ki-67 (1:100, Abcam) overnight at 4 °C and stained with 3, 3′-diaminobenzidine for color development.

Techniques: Western Blot, Expressing, Luciferase, Reporter Assay, Over Expression, Activation Assay, Activity Assay, Gene Expression, Transfection, shRNA, Wound Healing Assay, Knockdown, Injection, Immunohistochemistry, Staining

Journal: Translational Oncology

Article Title: STIL facilitates the development and malignant progression of triple-negative breast cancer through activation of Fanconi anemia pathway via interacting with KLF16

doi: 10.1016/j.tranon.2024.102010

Figure Lengend Snippet: The interaction between STIL and KLF16 regulates the transcription and expression of FNACD2. ( A ) Screen for differentially expressed gene for both STIL immunoprecipitation Mass Spectrometry and Transcription Factors of FANCD2 in JASPAR Database. ( B ) MDA-MB-231 cell lysates were immunoprecipitated with anti-STIL (left), anti-KLF16 (right), or control IgG antibody, and then precipitates were probed with anti-KLF16 or anti-STIL antibody. ( C ) STIL (red) and KLF16 (green) colocalize in MDA-MB-231 cells shown by confocal images. Nuclei were stained with DAPI (blue). ( D and E ) The expression levels of FANCD2 mRNA and protein after KLF16 knockdown in MDA-MB-231 and HCCC1806 cell lines. ( F ) Dual-luciferase reporter assay detect the effect of KLF16 overexpression on the activation activity of FANCD2 promoter. ( G ) Proteins from pull-down assay were separated by polyacrylamide gel electrophoresis and reveled by anti-STIL and anti-KLF16 antibodies. (H) Pull‐down assay to evaluate the interaction of STIL and KLF16 with the promoter of FANCD2 after overexpressing and knocking down STIL. ( I and J ) FANCD2 mRNA and promoter activity were measured by qPCR and dual-luciferase reporter assay. Scale bars in ( C ), 50 μm.The data represented the mean ± SD of three independent experiments, and the level of significance was indicated by **** P <0.0001, *** P < 0.001,** P < 0.01, * P < 0.05. “NS” means no significant difference with the control group.

Article Snippet: Paraffin-embedded tumor tissues were processed for immunohistochemical staining with primary antibodies against STIL (1:100, Proteintech Group, USA), FANCD2 (1:100, Santa Cruz Biotechnology) and Ki-67 (1:100, Abcam) overnight at 4 °C and stained with 3, 3′-diaminobenzidine for color development.

Techniques: Expressing, Immunoprecipitation, Mass Spectrometry, Control, Staining, Knockdown, Luciferase, Reporter Assay, Over Expression, Activation Assay, Activity Assay, Pull Down Assay, Polyacrylamide Gel Electrophoresis

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Palmitic acid-conjugated peptides induce enhanced T cell activation in vitro and in vivo (A) moDCs were pulsed with different concentrations of gp100 280–288/45–59 or C16:0 gp100 280–288/45–59 for 3 h and co-cultured o/n with gp100-specific CD8 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data are presented as mean ± SEM of six individual experiments, measured in triplicate. Statistical analyses were performed using a two-way ANOVA with a Sidak post hoc multiple comparison test. ∗∗∗p < 0.001. (B) OVA 257–264/323–339 - and C16:0 OVA 257–264/323–339 -loaded BMDCs were co-cultured with OT-I T cells for 3 days. Mean ± SEM measured in triplicate. Data are representative of three individual experiments. Statistical analyses were performed using a two-way ANOVA with a Sidak post hoc multiple comparison test. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) moDCs were pulsed with different concentrations of gp100 280–288/45–59 or C16:0 gp100 280–288/45–59 for 3 h and co-cultured o/n with gp100-specific CD4 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data are presented as mean ± SEM measured in triplicate and representative of three individual experiments (D) gp100 280–288/45–59 and C16:0 gp100 280–288/45–59 (30 μM) were injected intradermally into human skin with (black line) or without (blue line) GM-CSF/IL-4. At the site of injection punch biopsies were taken and cultured for 2 days. The migrated skin APCs were collected and co-cultured o/n with gp100-specific CD8 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data of two individual donors per condition are shown.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Activation Assay, In Vitro, In Vivo, Cell Culture, Comparison, Injection

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: The addition of a single palmitic acid affects the processing pathway of antigens moDCs were treated with different inhibitors or DMSO control (ctrl) 30 min prior to and during the 3 h antigen pulse. Thereafter, moDCs were washed and co-cultured o/n with gp100-specific CD8 + T cells. IFNγ secretion was determined by ELISA as a measure of T cell activation. (A) Illustration of interference in antigen processing by the different inhibitors. (B–F) Relative IFNγ secretion induced by co-culture of gp100-specific CD8 + T cells with gp100 280–288/45–59 - or C16:0 gp100 280–288/45–59 -pulsed moDCs that were pre-treated with (B) bortezomib, (C) cathepsin S inhibitor, (D) chloroquine, (E) primaquine, and (F) brefeldin A. Data are represented as relative to the short peptide control after subtraction of background (IFNγ levels of unpulsed moDCs). Symbols represent the mean of triplicates of seven donors. Statistical analyses were performed with a paired Student’s t test. ∗p < 0.05, ∗∗∗p < 0.001. (G) Overview of experimental setup for antigen presentation with DCs from Batf3 KO mice. (H) Proliferation of OT-I cells after co-culture with C16:0 or unmodified peptide in WT and Batf3 KO BMDCs. Statistical analysis was performed with a Student’s t test. ∗∗p < 0.01.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Co-Culture Assay, Immunopeptidomics

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Palmitoylation of peptides is a membrane-targeting moiety that allows for continuous loading and processing (A) HA signal at different time points in fixed and permeabilized moDCs that were pulsed with 10 μM gp100 280–288/45–59 (black circles) or C16:0 gp100 280–288/45–59 (black squares) for 45 min at 4°C, after which moDCs were transferred to a 37°C incubator without washing away the product. Data are shown as mean ± SEM of three individual experiments. (B) Membrane-bound HA signal (AF647, upper panel) and intracellular HA signal (AF488, lower panel) of moDCs that were pulsed with C16:0 peptide or left untreated for 45 min at 4°C with and without permeabilization. (C) Visualization of the membrane localization of the C16:0 gp100 280–288/45–59 -HA peptide in BMDCs after a 45-min incubation at 4°C determined by STED confocal microscopy. (D) Mannose receptor (MR) and C16:0 peptide HA detection in moDCs treated with glucose depletion medium prior to the antigen pulse (45 min at 4°C). Data are shown as mean ± SEM of four individual experiments. Statistical analyses were performed with a paired Student’s t test. ∗∗p < 0.01. (E) Decay of membrane and total (membrane + intracellular) HA signal at different time points in moDCs that were pulsed with 10 μM C16:0 gp100 280–288/45–59 . Graphs are presented as mean ± SEM of three individual experiments.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Membrane, Incubation, Confocal Microscopy

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Exploiting the membrane-integrating capability of C16:0 peptides as a tool for antigen enrichment in tumor cells and tumor-derived apoptotic vesicles (A) HA signal in DCs (humans and mice) and different tumor cell lines (JY, THP-1, Mel-JuSo, and Mel-BRO cells) that were pulsed with 10 μM gp100 280–288/45–59 or 16:0 gp100 280–288/45–59 for 45 min at 4°C. Data shown are representative of multiple individual experiments. (B) Viability of human THP-1 cells (leukemia) and (C) murine GL261 (glioblastoma) tumor cells that were pulsed with 10 μM C16:0 gp100 280–288/45–59 or C16:0 OVA 257–264/323–339 and co-cultured with antigen-specific T cells (gp100 CD8 + T cells/CD8 + T cells from OVA-immunized mice). Data show representative experiment measured in triplicate. (D) Overview of experimental setup for antigen enrichment of apoptotic extracellular vesicles. (E) IFNγ secretion by gp100-specific CD8 + T cells after co-culture with moDCs that were incubated with apoptotic cell-derived extracellular vesicles (ApoEVs) which were first loaded with 10 μM C16:0 gp100 280–288/45–59 . Data are presented as mean ± SEM of three individual experiments, measured in triplicate.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Membrane, Derivative Assay, Cell Culture, Co-Culture Assay, Incubation

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Schematic displays the computational approach (BECC) and rigor (diversity and number of datasets) used to identify the 166-gene ViP and a subset of 20-gene severe (s)ViP signatures, and the subsequent experimentally validated inferences and impact of the same in a recent study . The numbers in gray circles denote the total number of datasets analyzed in each category. b Schematic displays the various pathogenic triggers that induce ViP signatures (many of which are triggers also for KD) and the prominent induction of IL15/IL15RA as an invariant nature of the cytokine storm. c Bubble plots of ROC-AUC values (radii of circles are based on the ROC-AUC) demonstrating the strength of classification and the direction of gene regulation (Up, red; Down, blue) for the classification based on the 20-gene severe ViP signature (top) and 166-gene ViP signature (bottom) in numerous publicly available historic datasets. ViP signatures classified KD vs. healthy children (left), acute vs. convalescent KD (middle) and treatment response in the setting of combination therapy with IV steroids (MP methylprednisone) and IV IgG alone (IVIG), but not IVIG alone. Numbers on top of bubble plots indicate number of subjects in each comparison group. d , e Bar (top) and violin (bottom) plots display the classification of blood samples that were collected during acute (AV), sub-acute (SA; ~10–14 days post-discharge) and convalescent (CV; 1 year post-onset) visits from two independent KD cohorts ( d ; Historic Cohort 1; e ; Prospective Cohort 2) using ViP (left) or sViP (right) signatures. f Bar (top) and violin (bottom) plots display the sub-classification of blood samples in Cohort 1 based on coronary artery aneurysm (CAA) status using ViP (left) or sViP (right) signatures. Welch’s two sample unpaired two-sided t -test is performed on the composite gene signature score to compute the p values. In multi-group setting each group is compared to the first control group and only significant p values are displayed on the right. Additional pvalues are displayed on the left.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: Comparison, Control

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Schematic displays the workflow for patient blood collection and analysis by RNA Seq (this figure) and cytokine array by mesoscale (Figs. and ). b , c Bar (top) and violin (bottom) plots display the classification of blood samples that were collected during collected during acute (AV) and sub-acute (SA; ~10–14 days post-discharge) visits of KD subjects and from patients diagnosed with MIS-C. The p value for comparison between acute KD (AV) and MIS-C (M) is displayed in red font. d , e Heatmaps display the patterns of expression of the 166 genes in ViP ( d ) and 20 gene sViP (e) signatures in the KD and MIS-C samples. The only cytokine–receptor pair within the signature, i.e., IL15/IL15RA , are highlighted on the left in red font in ( d ). f Schematic displays the 13-transcript whole blood signature (no overlaps with ViP signature genes) previously demonstrated to distinguish KD from other childhood febrile illnesses . g and h Bar (top) and violin (bottom) plots display the classification of blood samples that were collected during acute (AV) and convalescent (CV) visits from two independent KD cohorts ( g ; Historic Cohort 1 ; e ; Prospective Cohort 2) using 13-transcript KD signature. FC, febrile control. See also Supplementary Fig. for co-dependence analysis of ViP and KD-13 signatures. Welch’s two sample unpaired two-sided t -test is performed on the composite gene signature score to compute the p values. In multi-group setting each group is compared to the first control group and only significant p values are displayed. The p value for comparison between acute KD (AV) and MIS-C (M) is displayed in red font.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: RNA Sequencing, Comparison, Expressing, Control

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Heatmap displays the results of unsupervised clustering of sub-acute and acute KD (KD-SA, KD-AV; n = 10 each) and MIS-C ( n = 10) subjects using the cytokine profiles determined by mesoscale (MSD). Red = cytokines differentially expressed between MIS-C and KD. See also Supplementary Fig. for violin plots for individual cytokines. b Source data are provided as a Supplementary Data . Violin plots display the shared (top panels; IL15, MIP1a, IL2, IL6 and VEGF) and distinct (bottom panels; IFNγ, IL1β, IL8, IL10, and TNFα) features of the cytokine storm in MIS-C vs. KD subjects. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. c Schematic shows the process used to integrate serum cytokine array results with whole blood RNA Seq data; cytokines that were differentially expressed in MIS-C were used to inform GSEA of the corresponding pathways. d – f Gene set enrichment analysis (GSEA pre-ranked analysis) of three pathways derived from MSigDB: SANA TNF SIGNALING UP ( d ), TIAN TNF SIGNALING VIA NFkB ( e ), and SANA RESPONSE TO IFNG UP ( f ) demonstrate the significance of TNF ( d , e ) and IFNG ( f ) pathway activation in MIS-C. g , h Down-regulated genes after IL1B ( g ) and IL10 ( h ) stimulation were derived from differential expression analysis of GSE44722 ( n = 269 genes), and GSE61298 ( n = 208 genes) respectively. GSEA pre-ranked analysis to test the significance of IL1B and IL10 pathway is performed like panels d – f using the down-regulated genes. GSEA pre-ranked analysis computes nominal pvalue and FDR using an empirical phenotype-based permutation test procedure. No adjustments were made for multiple comparisons because of single hypothesis testing. Source data are provided as a Source Data file.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: RNA Sequencing, Derivative Assay, Activation Assay, Quantitative Proteomics

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Heatmap displays the results of hierarchical agglomerative clustering of acute KD (KD-AV; n = 10) and MIS-C ( n = 10) subjects using the cytokine profiles determined by mesoscale (MSD) and the laboratory features. Source data are provided as a Source Data file. b Violin plots display PLT (platelet) and AEC (absolute eosinophil counts) in KD and MIS-C (unpaired two-sided Student’s t -test used to test significance). c – e Correlation test (two-sided test of the slope of the regression line compared to zero) between AEC and PLT ( c ; left) and IL15 and PLT ( c ; right), and MIP1α and PLT ( d ) and MIP1α and IL15 ( e ) are shown, and significance was calculated and displayed using GraphPad Prism 9. Significance: ns: non-significant, **** p < 0.0001. See Supplementary Fig. for all possible correlation tests between clinical and cytokine data in KD, MIS-C and COVID-19. f Correlation tests between PLT (left) or AEC (right) on the Y -axis and gene signature scores on the X -axis [either ViP (top), sViP (middle) or a IL15/IL15RA composite (bottom)] were calculated and displayed as scatter plots using python seaborn lmplots with the p -values. The confidence interval around the regression line is indicated with shades. g Schematic summarizing the findings in MIS-C based on laboratory and RNA seq analysis.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: RNA Sequencing

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a – c Severe (s)ViP signature can classify severe MIS-C based on in two independent studies ( GSE166489 and GSE167028 ). Schematic in a summarizes the definition of severe MIS-C. b , c Classification of blood samples in two cohorts of MIS-C subjects, based on the need for ICU management due to the presence (MYO+) or recovery in the absence (R or MYO−) of myocardial dysfunction using sViP signature. Welch’s two sample unpaired two-sided t -test is performed to compute the p values. d Bubble plots of ROC-AUC values (radii of circles are based on the ROC-AUC) and the direction of gene regulation (Up, red; Down, blue) in publicly available datasets using 4 gene signatures: the 166-gene ViP signature, the 20-gene sViP signature, the KD-13 signature, and finally the IL15/IL15RA composite score. Numbers on top of bubble plots indicate number ( n ) of control vs. disease samples in each dataset. Abbreviations: PBMCs peripheral blood mononuclear cells, Mac macrophages, WB whole blood, MTb M. tubercutosis , Flu Influenza, HIV human immunodeficiency virus, RSV respiratory syncytial virus, JM juvenile myositis, sjia systemic juvenile idiopathic arthritis, SLE systemic lupus erythematosus, IBD Inflammatory bowel disease, COPD chronic obstructive pulmonary disesase, JDM juvenile dermatomyositis, MS multiple sclerosis, BAL bronchoalveolar lavage, NOMID neonatal onset multisystem inflammatory disease, MAS macrophage activation syndrome, NLRC4 NLR Family CARD Domain Containing 4. e Schematic showing the experimental design for studying differentially expressed genes (DEGs) in between KD and MIS-C subjects. f , g PCA ( f ) and a clustered heatmap analysis ( g ) of KD (green, f ) and MIS-C (orange, f ) samples are shown based on top 2242 genes according to mean absolute deviation identified using StepMiner algorithm . Source data are provided. h Reactome pathway analysis of the DEGs between seven KD and seven MIS-C subjects in f (marked on the PCA). i Venn diagram between 166-gene ViP signature against the DEGs. Number of genes are indicated for each group in the Venn diagram. 11 overlapping genes between ViP signature and up-regulated in MIS-C are listed at the top.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: Control, Virus, Activation Assay

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Violin plots display the left ventricular ejection functions (LVEF) in KD and MIS-C patients. Statistical significance was determined by unpaired two-sided Student’s t -test. b – d Correlation tests (two-sided test of the slope of the regression line compared to zero) between LVEF ( Y -axis) and gene signature scores on the X -axis [either ViP ( b ), sViP ( c ), or a IL15/IL15RA composite ( d )] are displayed as a scatter plot and significance was calculated and displayed as in Fig. f. The confidence interval around the regression line is indicated with shades. e Bar and violin plots show how a IL15/IL15RA compositive score varies between KD samples. The score classifies KD-AV with giant CAAs from control (KD-CV) samples with a ROC AUC 0.95. Welch’s two sample unpaired two-sided t -test is performed on the composite gene signature score to compute the p values. In multi-group setting each group is compared to the first control group and only significant p values are displayed.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: Control

Journal: Nature Communications

Article Title: An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease

doi: 10.1038/s41467-022-30357-w

Figure Lengend Snippet: a Summary of datasets used (publicly available prior ones and new original cohorts) to support the conclusions in this work. Numbers in circles denote the number of subjects in each cohort. b Venn diagram displays the major findings from the current work. ViP/sViP signatures, and more specifically, the IL15/IL15RA specific gene induction are shared between patients in all three diagnostic groups. While these signatures are known to be associated with diffuse alveolar damage in the lungs of patients with COVID-19 , it is associated with CAA in KD and with reduction in cardiac muscle contractility in MIS-C. Overlapping features between each entity are displayed.

Article Snippet: Formalin-fixed, paraffin-embedded heart tissue sections from COVID19 and KD patients were stained anti-human IL15 receptor A polyclonal antibody (11:200 dilution; proteintech ® , Rosemont, IL, USA; catalog# 16744-1-AP) and anti-human IL15 monoclonal antibody (1:10 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog# sc-8437) after heat-induced antigen retrieval with Tris buffer containing EDTA (pH 9.0).

Techniques: Diagnostic Assay

Journal: Journal of Inflammation Research

Article Title: The Relationship Between Soluble Interleukin-17 Receptor Levels and CD3-Positive T Cells and Lymphocytes in Patients with Sepsis and Their Predictive Clinical Significance

doi: 10.2147/JIR.S479310

Figure Lengend Snippet: Characteristics of Patients with Sepsis in the Survival and Death Groups on Admission Day 1

Article Snippet: sIL-7R levels were quantitatively measured using a sandwich enzyme immunoassay (human sIL-7R ELISA kit, CUSABIO, Wuhan, China).

Techniques:

Journal: Journal of Inflammation Research

Article Title: The Relationship Between Soluble Interleukin-17 Receptor Levels and CD3-Positive T Cells and Lymphocytes in Patients with Sepsis and Their Predictive Clinical Significance

doi: 10.2147/JIR.S479310

Figure Lengend Snippet: Characteristics of Patients with Sepsis in the Survival and Death Groups on Admission Day 5

Article Snippet: sIL-7R levels were quantitatively measured using a sandwich enzyme immunoassay (human sIL-7R ELISA kit, CUSABIO, Wuhan, China).

Techniques:

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: A, Schematic illustration of a series of eukaryotic expressing plasmids (DNA vaccines) harboring the genes of HMGN1, gp100, or HMGN1-gp100 fusion. The eukaryotic expressing vector used in the DNA vaccines is pcDNA3.1/myc-His B (Invitrogen), which provides a myc epitope and a polyhistidine-tag at the C-terminus. The signal peptide of insulin was introduced in front of target genes to ensure secretion. The Kozak sequence was optimized. In the fusion gene HMGN1-gp100, a flexible linker (Gly4Ser)3 was inserted between HMGN1 and gp100. B, HEK293 cells (106/flask) were transfected with 10 µg of pcDNA3.1, pcDNA3.1-HMGN1, pcDNA3.1-gp100, or pcDNA3.1-HMGN1-gp100 plasmid using Lipofectamine™ 2000. Six hours after the transfection, the culture medium was replaced with DMEM containing 1% FBS and after an additional 24 hour-incubation, supernatants were analyzed by Western Blot. Purified human HMGN1 (1 µg) was used as a positive control. Left panel: lanes loaded with the supernatant of HEK293 cells transfected with pcDNA3.1-HMGN1 or purified HMGN1 were probed with α-polyHis (left two lanes) or α-HMGN1 (right two lanes). Right panel: lanes loaded with the supernatants of HEK293 cells transfected with indicated plasmids were probed with α-HMGN1. C, Transfection and subsequent analysis were similar as in B. Supernatants of HEK293 cells transfected with indicated plasmids were analyzed Western blot using anti-HMGN1 (left panel) or anti-gp100 (right panel) antibodies.

Article Snippet: Plasmid construction, purification, transfection, and cell line establishment The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: Expressing, Plasmid Preparation, Sequencing, Transfection, Incubation, Western Blot, Purification, Positive Control

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: C57BL/6 mice (7-week old, female, 5 mice/group) were DNA vaccinated with indicated plasmids via gene gun once a week for 3 consecutive weeks. Five days after the last vaccination, single cell suspensions of spleens and draining (inguinal) lymph nodes of vaccinated mouse were stimulated in vitro with 1 µM gp100(25–33) peptide for 6 h at 37°C in humidified air containing 5% CO2, with the addition of GolgiPlug™ for the last 4 h of culture. The cells were immunostained with fluorophore-conjugated antibodies against mouse CD3 and CD8, permeabilized, and followed by staining with fluorophore-conjugated anti-mouse IFNγ and anti-IL-13 antibodies as detailed in the Materials and Methods section. The data were analyzed for the presence of IFNγ+ or IL-13+ CD8 T cells. Shown is the average (mean ± SD) of each group (n=5), representative of one of two separate experiments.

Article Snippet: Plasmid construction, purification, transfection, and cell line establishment The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: In Vitro, Staining