Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: The membrane FAS-R (blue) forms a trimer upon ligand binding (TNFSF13, red). The adaptor molecule FADD (green) then binds to the death domain of FAS-R through its own death domain. The amino terminus of FADD facilitates binding to caspase-8 (maroon oval). Caspase-8 self-activates through proteolytic cleavage into active caspase subunits, which can cleave other effector caspases (3, 6, 9), eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis. In most cell types (Type II), caspase-8 catalyzes the cleavage of the pro-apoptotic BH3-only protein BID (orange) into its truncated form, tBID. tBID engages anti-apoptotic members of the family (e.g. BCL-2), allowing pro-apoptotic members (e.g. BAX) to translocate to the outer mitochondrial membrane, thus permeabilizing the membrane and facilitating release of pro-apoptotic proteins such as cytochrome c (tan). Cytochrome c release reinforces the cellular apoptotic drive through the intrinsic apoptotic pathway. Death receptors 4/5 (TNFSF10) appear to have identical intracellular pathway signaling to FAS receptor .

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Membrane, Ligand Binding Assay, Binding Assay

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: Genes analyzed with ABI Taqman Gene Expression assay part numbers.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Gene Expression, Binding Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: cDNA samples obtained from the combined SMRI and NSW TRC collections from individuals with schizophrenia and unaffected controls were subjected to qRT-PCR. Expressions of TNFSF13 (A), FAS receptor (C), CFLAR pan (E), CFLAR long (F) and BID (G) relative to three housekeeping genes (β-actin, TATA box binding protein and ubiquitin C) were calculated using the deltadelta Ct method. Horizontal lines indicate the population median, except for panel (G) where they indicate the mean as those data were normally distributed. Frequency distributions for TNFSF13, FAS receptor and BID are displayed in panels (B), (D), and (H). ** p<0.01.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Quantitative RT-PCR, Binding Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: cDNA samples obtained from the SMRI collection from individuals with bipolar disorder and unaffected controls were subjected to qRT-PCR. Expressions of TNFSF13 (A), FAS receptor (B), BID (C), CFLAR pan (D), and CFLAR long (E) relative to three housekeeping genes (β-actin, TATA box binding protein and ubiquitin C) were calculated using the deltadelta Ct method. Horizontal lines indicate the population median, except for panel (E) where they indicate the mean as those data were normally distributed. * p<0.05.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Quantitative RT-PCR, Binding Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: cDNA samples from individuals with schizophrenia, bipolar disorder and unaffected controls were subjected to qRT-PCR. Expressions of TNFSF13 (A), FAS receptor (B) and BID (C) relative to three housekeeping genes (β-actin, TATA box binding protein and ubiquitin C) were calculated using the deltadelta Ct method. Horizontal lines indicate the population median, except for panel (C) where they indicate the mean as those data were normally distributed.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Quantitative RT-PCR, Binding Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: Variance (R 2 ) in parvalbumin, somatostatin, PPP1R9B and DLG4 mRNA transcript levels accounted for by TNFSF13 expression and tissue pH in the NSW TRC collection.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

doi: 10.1371/journal.pone.0035511

Figure Lengend Snippet: Intracellular pH of U-87 MG cells was artificially decreased using nigericin and phosphate buffer to pH 6.4, 6.9 and 7.3 for 0.5, 3, 12 or 24 hours and transcript levels of the TNFSF13 (A) or FAS receptor (B) genes measured by qRT-PCR. Expression levels were calculated relative to three housekeeping genes (β-actin, TATA box binding protein and ubiquitin C) using the deltadelta Ct method. a Transcript levels at the 0.5 hours time point were significantly greater than at all other time points, b Decreasing pH significantly suppressed transcript levels of TNFSF13, c There was no significant effect of pH on FAS receptor transcripts. U-87 MG glioblastoma cell cultures were treated with TNFSF13 (1 pg or 100 pg TNFSF13/microliter of culture media) for up to 48 hours and the intracellular pH determined (C). *p<0.05, ***p<0.001.

Article Snippet: Human recombinant TNFSF13 (Catalogue Number 884-AP, RD Systems) was added (in 50% fresh media) at final concentrations of 0, 1 and 100 ng TNFSF13/mL media 48, 24 and 12 hours prior to pH determination.

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Eomes and Brachyury control pluripotency exit and germ layer segregation by changes of chromatin state

doi: 10.1101/774232

Figure Lengend Snippet: a , Schematic of Eomes Gfp/Δ (EoKO) and Bra Tom/Δ (BraKO) loss-of-function alleles in mouse embryonic stem cells (ESCs). One allele contains a fluorescent reporter ( Gfp or Tomato , Tom ) knock-in, the second allele contains frame-shift deletions in exon 1 of each gene. b , Differentiation protocol to mesoderm and definitive endoderm (ME) cell types: mouse ESCs are maintained in LIF+2i medium, followed by aggregation to embryoid bodies (EBs) in serum-free (SF-basal) medium for 2 days, and treatment with ActivinA (ActA) from day 2 to day 5. Arrows indicate time points of analysis. c , Relative mRNA expression of ME markers during differentiation of WT, BraKO, EoKO, and double knock-out (dKO) cells measured by qRT-PCR in a 5-day time-course showing the complete absence of ME differentiation in dKO cells. Error bars indicate SEM. p-values for differences of mean expression between WT and dKO samples were calculated by Student’s t-test. *:p≤0.05; **:p≤0.01; ***: p≤0.001; ****: p≤0.0001. d , Immunofluorescence (IF) staining of definitive endoderm (SOX17 and FOXA2) and mesoderm (FOXC2) markers at day 4 of differentiation. Scale bars 100 μm. e , Heatmap showing clustered groups (I to V) of downregulated genes (adjusted p-value≤0.05, log 2 (FC)≤-2.5) in dKO compared to WT cells at day 5 of differentiation analyzed by RNA-seq. Scale indicates centered scaled counts normalized by library size and gene-wise dispersion. Important genes in each group are indicated to the right. f , Gene ontology (GO) enrichment analysis of downregulated genes in dKO compared to WT cells indicating the significantly reduced signatures of ME developmental programs in dKO cells. p-values for each term are indicated.

Article Snippet: EBs were subsequently transferred into 60 mm non-adhesive dishes and allowed to further differentiate in ESGRO Complete Basal medium with 30 ng/ml human recombinant ActivinA (ActA, R&D systems).

Techniques: Knock-In, Expressing, Knock-Out, Quantitative RT-PCR, Immunofluorescence, Staining, RNA Sequencing Assay, Dispersion

Journal: Frontiers in Pharmacology

Article Title: Biotechnologically Produced Lavandula angustifolia Mill. Extract Rich in Rosmarinic Acid Resolves Psoriasis-Related Inflammation Through Janus Kinase/Signal Transducer and Activator of Transcription Signaling

doi: 10.3389/fphar.2021.680168

Figure Lengend Snippet: Schematic representation of the proposed mechanisms of action of Lavandula angustifolia extract (LV) and rosmarinic acid (RA) in psoriasis-like inflammation model in human keratinocytes. HaCaT cells exposed to IFN-γ/IL-17A/IL-22 respond with activation of JAK2/STAT1 and PI3K/AKT signaling pathways. Activation of JAK2 upon cytokine stimulation leads to STAT1 activation and its subsequent phosphorylation. Following nuclear translocation the phosphorylated STAT1 induces transcriptional activation of psoriasis-related inflammatory genes ( e.g., IL6 , CCL20 , CCL2 ). Activated PI3K/AKT axis in psoriatic keratinocytes correlates with induction of hyperproliferation and aggravation of the inflammatory milieu. In the present investigation, both RA and LV inhibited JAK2 and diminished STAT1 phosphorylation, hence, preventing inflammatory activation. Additionally, the LV extract disrupted PI3K/AKT signaling which could contribute to decrease in proliferation rate in activated keratinocytes. Taken together, the obtained data suggest that LV and RA possess inhibitory effect on psoriasis-related inflammation in human keratinocytes.

Article Snippet: The recombinant human IL-17A (#ENZ-PRT188) and IL-22 (#ENZ-PRT250) were purchased from Enzo Life Sciences AG (Lausen, Switzerland) and IFN-γ (#10067-IF) was obtained from R&D Systems (Minneapolis, MN, United States).

Techniques: Activation Assay, Translocation Assay

Journal: Frontiers in immunology

Article Title: Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

doi: 10.3389/fimmu.2022.919854

Figure Lengend Snippet: FIGURE 9 | IL-21 rescued CD40L and anti-IgM induced cell death in human primary B cells. (A) Representative graphs of CFSE labelling cells showed no differentiation was induced by any stimulatory factors in 3 days. (B) Representative graphs showed that IL-21 could suppress cell death induced by CD40L and anti- IgM in terms of increased number of DAPI- cells. (C) Representative graphs showed that IL-21 was able to induce CD38+ B cell differentiation both in the absence and presence of CD40L.

Article Snippet: Cells were then seeded into 96-well round bottom plate at the density of 2E5 cells/100μl, and then challenged with 50 ng/ml IL-21 and 1 μg/ml CD40L (#6420-CL/CF, R&D Systems, Minneapolis, USA) or 2 μg/ml anti-IgM.

Techniques: Cell Differentiation