Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 3 Interleukin-17F (IL-17F) production is decreased in colonic lamina propria (cLP) of Clec7a / mice. (a) Cytokine gene expression in cLP was examined by quantitative real-time reverse transcription PCR (qRT-PCR), and expression levels in Clec7a / mice are indicated relative to those in wild-type (WT) mice (n ¼ 3–8/group). (b,c) IL-17F þCD45 þ cell population in cLP layer from WT or Clec7a / mice was examined by flow cytometry. (n ¼ 4–5/group). (d) S100A8 þ and IL-17F þ cells in whole colonic cells from WT mice were examined by flow cytometry. Data are representatives of two independent experiments, and in a and c are expressed as mean±s.d. *Po0.05 vs. WT control.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Gene Expression, Reverse Transcription, Quantitative RT-PCR, Expressing, Flow Cytometry, Control

Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 6 Dectin-1 ligands are not detected in intestinal commensal microbiota. (a) Wild-type (WT) mouse fecal microbiota or Candida albicans (SC5314) were pretreated with non-immune goat or rabbit serum to block nonspecific binding. Then, they were treated with PE-labeled b-glucan- recognizing protein (Pi-BGRP) or the mutant form of the b-glucan recognition site of Pi-BGRP (Pi-BGRP.M), and PE þ population was determined by flow cytometry. (b) Thioglycollate-induced peritoneal macrophages (Mfs) from WT, Clec7a / , Myd88 / , or Clec7a / Myd88 / mice were cocultured with live Alcaligenes faecalis (multiplicity of infection (MOI): 1 or 10) for 24 h and the tumor necrosis factor-a (TNF-a) concentration in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA; n ¼ 3/group). (c) S100A8 expression in colonic epithelial cells (cECs) from A. faecalis monocolonized (A. faecalis), germ-free (GF), or specific-pathogen-free SPF mice was examined by flow cytometry (n ¼ 3–4/group). Data in b and c are representatives of two independent experiments and are expressed as mean±s.d.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Blocking Assay, Binding Assay, Labeling, Mutagenesis, Flow Cytometry, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: E-Selectin-Dependent Inflammation and Lipolysis in Adipose Tissue Exacerbate Steatosis-to-NASH Progression via S100A8/9

doi: 10.1016/j.jcmgh.2021.08.002

Figure Lengend Snippet: Antibodies

Article Snippet: S100A8/A9 (mouse) , 1:500 (staining) 1:5000 (WB) , Novus Biologicals.

Techniques: Staining

Journal: Biomolecules

Article Title: Evaluation of Plasma-Derived hsa_circ_003077 for Non-Invasive Diagnosis of Alzheimer’s Disease

doi: 10.3390/biom16030356

Figure Lengend Snippet: Plasma glial and receptor biomarkers in Alzheimer’s disease (AD) patients and cognitively healthy controls (HC). ( a ) GFAP, ( b ) NfL, ( c ) Tyro3, and ( d ) AXL levels were measured in plasma. Data are presented as mean ± SEM. Statistical comparisons were performed using the Mann–Whitney U test. Statistical significance is indicated as ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The Human Tyro3 DuoSet ELISA kit (Catalog No: DY8596-05) supplied by R&D Systems (Minneapolis, MN, USA) was used to measure Tyro3 levels, while the Human AXL DuoSet ELISA kit (Catalog No: DAXL00; R&D Systems, Minneapolis, MN, USA) was used to measure AXL levels.

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: Biomolecules

Article Title: Evaluation of Plasma-Derived hsa_circ_003077 for Non-Invasive Diagnosis of Alzheimer’s Disease

doi: 10.3390/biom16030356

Figure Lengend Snippet: Diagnostic performance of plasma biomarkers for differentiating Alzheimer’s disease (AD) from cognitively healthy controls (HC) based on ROC curve analysis. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of plasma biomarkers. The area under the ROC curve (AUC) was used to evaluate biomarker performance: 0.90–1.00, superior diagnostic efficacy; 0.80–0.89, good diagnostic efficacy; 0.70–0.79, moderate diagnostic efficacy; <0.70, poor/substandard diagnostic efficacy. Data are presented as mean ± SEM. Statistical comparisons were performed using the Mann–Whitney U test. hsa_circ_003077 exhibited the highest diagnostic accuracy (AUC = 0.90; 95% CI: 0.82–0.97), followed by NfL (AUC = 0.75) and pTau-217 (AUC = 0.77). AXL and GFAP showed lower diagnostic performance (AUC = 0.63 and 0.69, respectively), while pTau-181 and Tyro3 demonstrated substandard efficacy (AUC = 0.63 each).

Article Snippet: The Human Tyro3 DuoSet ELISA kit (Catalog No: DY8596-05) supplied by R&D Systems (Minneapolis, MN, USA) was used to measure Tyro3 levels, while the Human AXL DuoSet ELISA kit (Catalog No: DAXL00; R&D Systems, Minneapolis, MN, USA) was used to measure AXL levels.

Techniques: Diagnostic Assay, Clinical Proteomics, Generated, Biomarker Discovery, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Elevated Levels of Neutrophil Activated Proteins, Alpha-Defensins (DEFA1), Calprotectin (S100A8/A9) and Myeloperoxidase (MPO) Are Associated With Disease Severity in COVID-19 Patients

doi: 10.3389/fcimb.2021.751232

Figure Lengend Snippet: Details of the COVID-19 patients examined at presentation.

Article Snippet: Levels of alpha defensins, DEFA1 and S100A8/A9 in serum and plasma were quantified using commercially available ELISA kits, Neutrophil DEFA1 (USCN, Wuhan) & quantikine ELISA for human S100A8/A9 Heterodimer (R & D systems, Minneapolis) as per manufacturer’s protocols.

Techniques:

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 2. Effects of crisaborole on neutrophil infiltration in atopic dermatitis mouse model. (A–F) Skin was dissected from mice on day 8 after EtOH (A, E, F), MC903 (MC: B, E, F), MC903+vehicle (MC+VH: C, E, F), or MC903+crisaborole treatment (MC+Cris: D, E, F). Skin sections were immunostained with an antibody for PGP9.5 and Ly6G to visualize nerve fibres (green) and neutrophils (red), respectively. Dotted lines indicate the dermal-epidermal junction. White arrows indicate neutrophils contact with nerve fibres. (E) Scale bar indicates 20 µm. Total number of neutrophils in the epidermis was quantified in EtOH (white bars), MC (green bars), MC+VH (dark grey bars), and MC+Cris mice (blue bars). Error bars are SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, significant difference (1-way analysis of variance (ANOVA) followed by Tukey test, n = 5/group). (F) As in (E) for epidermal neutrophils in contact with nerves. (G and H) Skin sections were immunostained with an antibody for Ly6G and S100A8 or S100A9.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques:

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 3. S100A8/A9 facilitates pruritogen- or algogen-evoked scratching and wiping behaviours in mice. (A) S100A8/A9 (1, 10, 100 ng) or vehicle (phosphate-buffered saline; PBS) was intradermally injected into the nape of the neck. Following the injection, scratch bouts were counted over a 30-min period. Error bars are standard error of mean (SEM) n = 6–8. (B, C) S100A8/A9 (1, 10 ng) or vehicle was intradermally injected into the nape of the neck with either histamine (54 nmol: B) or chloroquine (97 nmol: C). Error bars are SEM n = 6–8. *p < 0.05, **p < 0.01, ****p < 0.0001, significant difference from the vehicle-treated group. One-way analysis of variance (ANOVA) followed by the post hoc Tukey test. F (2, 19)=18.60, p < 0.0001 for histamine. F (2, 15)=6.353, p = 0.01 for chloroquine. (D–I) S100A8/A9 (10 ng) or vehicle was intradermally injected into the cheek with either histamine (54 nmol: D, G), chloroquine (97 nmol: E, H), or capsaicin (33 nmol: F, I). Error bars are SEM n = 5–6. *p < 0.05, ***p < 0.001, ****p < 0.0001, significant difference from the vehicle-treated group. Two-tailed unpaired t-test.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques: Saline, Injection, Two Tailed Test

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 4. Effects of S100A8/A9 on pruritogen- or algogen-induced calcium response in mouse dorsal root ganglion (DRG) neurones. (A) Time-course graph of histamine-induced calcium response in DRG neurones. DRG neurones were treated with vehicle (black line) or S100A8/A9 (blue line) with a subsequent challenge with histamine (100 μM). (B) As in (A) for chloroquine (100 μM). (C) As in (A) for capsaicin (1 μM). (D) Averaged area under the curve of the calcium responses to histamine. DRG neurones were treated with vehicle (black column) or S100A8/A9 (blue column). Error bars are SEM n = 19–27. ***p < 0.001, a significant difference from the vehicle group. 2-tailed unpaired t-test. (E) As in (D) for chloroquine. n = 11–22. (F) As in (D) for capsaicin. n = 13–14. (G) Proportions of histamine-, chloroquine-, or capsaicin-responsive DRG neurones pre-treated with vehicle (black column) or S100A8/A9 (blue column). *p < 0.05. Fisher exact test. n = 69–128.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques:

Journal: Aging Cell

Article Title: Single Cell Sequencing Identifies Distinct Cellular Alterations in Impaired Aged and Diabetic Wounds

doi: 10.1111/acel.70217

Figure Lengend Snippet: Neutrophils in diabetic and NG‐Aged oral mucosal wounds exhibit functional differences. (A) Representative immunofluorescent images showing myeloperoxidase (MPO) expression in diabetic and NG‐Aged conditions in 7 day wounds. Vertical yellow lines indicate wound edges. Yellow box highlights the region of interest (ROI). (A′) Bar graph depicts MPO‐positive cells per mm 2 ( n = 6). (B) Jitterplot of inflammation‐associated genes that are differentially expressed in neutrophils in the NG‐Aged group versus diabetic group. Genes that are significantly upregulated ( p adj value < 0.05) are highlighted in red. The size of the dot depicts the percent of cells expressing the gene. (C) Bar graph of pathways significantly enriched in neutrophils in the NG‐Aged group compared to the diabetic counterpart. (D) UMAP plot of neutrophil clusters. (D′) Dotplot of differentially expressed genes in neutrophils. Size of the dot depicts the percent of cells expressing the gene. (D′) UMAP of single cell expression of S100a8 , S100a9 , Cxcr2 , and Ccl6 in the neutrophil from NG‐Aged group. Black dotted line circumscribes cluster 1. (E) Immunofluorescent images showing MPO (green) and S100a8 (red) positive cells across conditions. Dotted vertical lines represent the wound edge; yellow box highlights the ROI; yellow arrows point to MPO + S100a8 + double‐positive cells. (E′) Quantification of MPO + S100a8 + double‐positive cells per mm 2 ( n = 6). (F) RNAScope of Cxcr2 gene expression in diabetic and NG‐Aged conditions in 7 day wounds. Dotted vertical lines indicate wound edges; yellow box highlights the ROI; yellow arrows point to Ccr2 + cells. (F′) Quantification of MPO + Cxcr2 + double positive cells per μm 2 ( n = 6). (G) Bar graph of pathways significantly enriched in neutrophils in the diabetic group compared to the NG‐Aged counterpart. (H) Jitterplot of cathepsin genes differentially expressed in neutrophils from diabetic versus NG‐Aged wounds. Genes that are significantly upregulated are highlighted in red. (I) Monocle3‐pseudotime trajectories of neutrophils projected in UMAP. (I′) Co‐expression of cathepsin and inflammatory genes visualized in neutrophil UMAP. (J) Multiplexed RNAScope in situ hybridization of Ctsz transcripts localized to MPO + neutrophils. (J′) Percent MPO + Ctsz + double‐positive cells per total number of cells. (J″) Quantification of Ctsz + dots per MPO + cells. Low magnification scale bar: 250 μm and 25 μm in high magnification images (A) 100 μm and 10 μm in (D). Bar graphs (A′ and D′) represent mean ± SEM, p ‐values were calculated using unpaired two‐tailed Student's t ‐test for pairwise comparison between diabetic and NG‐Aged groups. p values are shown numerically; statistical significance was defined as p < 0.05.

Article Snippet: Slides were incubated with the MPO (1 μg/mL dilution, Bio‐Techne, #AF3667) and S100a8 (6.25 ng/mL, Cell Signaling, #47310) or Ctsc (2 μg/mL, R&D Systems, #AF1034‐SP) antibodies at 4°C overnight.

Techniques: Functional Assay, Expressing, RNAscope, Gene Expression, In Situ Hybridization, Two Tailed Test, Comparison