Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Human Milk-derived Peptide Drives Rapid Regulation of Macrophage Inflammation Responses in the Neonatal Intestine

doi: 10.1016/j.jcmgh.2024.101420

Figure Lengend Snippet: CASB 135-150 mitigates the pro-inflammatory response in intestinal macrophages. ( A ) The neonatal rats were gavaged with either saline or saline supplementation with human milk EVs. After a 2-hour period, the ileums were collected for immunofluorescence analysis of IBA1, β-casein, and DAPI. n = 3. ( B–C ) Intestinal lamina propria lymphocytes were isolated from NEC animals that received CASB 135-150 or CASB 135-150 -Mut treatment (20 mg/kg/day for 4 days). CD45+F4/80+CD11b/c+ macrophages were gated for analysis of TNF-α by flow cytometry ( B ) and the percentage of TNF-α+ cells quantification, n = 3 ( C ). ( D–F ) BMDMs were pre-treated with CASB 135-150 at the indicated concentrations for 1 hour, followed by stimulation with LPS (10 ng/ml) for 6 hours. Subsequently, the levels of inflammation factors were assessed using qRT-PCR, GAPDH as a reference gene, n = 3. ( G–H ) BMDMs were pre-treated with CASB 135-150 at a concentration of 50 μM, followed by LPS (10 ng/ml) treatment for 24 hours. Afterward, the cells were collected for flow cytometry analysis of CD86 and CD206 ( G ) and quantification ( H ). n = 3 Data are presented as mean ± SD. Statistical significance was determined using 1-way ANOVA with Tukey’s post hoc test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: We then centrifuged and discarded the supernatant, added 100 μL of fixation and permeabilization solution (554714, BD), resuspended the cells, incubated at 4 °C in the dark for 40 minutes, centrifuged and discarded the supernatant, added 150 μL of permeabilization wash buffer (554714, BD), centrifuged and discarded the supernatant, then added the diluted Alexa Fluor 700 anti-TNF-α antibody (NBP1-19532AF700, Novus Biologicals, 1:100 dilution), resuspended the cells, and incubated in the dark for 60 minutes.

Techniques: Saline, Immunofluorescence, Isolation, Flow Cytometry, Quantitative RT-PCR, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Human Milk-derived Peptide Drives Rapid Regulation of Macrophage Inflammation Responses in the Neonatal Intestine

doi: 10.1016/j.jcmgh.2024.101420

Figure Lengend Snippet: FHL2 overexpression amplifies the anti-inflammatory effects controlled by CASB 135-150 treatment. ( A–B ) RAW264.7 cells were overexpressed with FHL2 for 48 hours, followed by pre-treatment with CASB 135-150 for 1 hour and subsequent exposure to LPS for 6 hours. The production of Tnf-α ( A ) and IL-6 ( B ) was assessed using qPCR. GAPDH as a reference gene. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: We then centrifuged and discarded the supernatant, added 100 μL of fixation and permeabilization solution (554714, BD), resuspended the cells, incubated at 4 °C in the dark for 40 minutes, centrifuged and discarded the supernatant, added 150 μL of permeabilization wash buffer (554714, BD), centrifuged and discarded the supernatant, then added the diluted Alexa Fluor 700 anti-TNF-α antibody (NBP1-19532AF700, Novus Biologicals, 1:100 dilution), resuspended the cells, and incubated in the dark for 60 minutes.

Techniques: Over Expression

Journal: Cancers

Article Title: Response to FEC Chemotherapy and Oncolytic HSV-1 Is Associated with Macrophage Polarization and Increased Expression of S100A8/A9 in Triple Negative Breast Cancer

doi: 10.3390/cancers13215590

Figure Lengend Snippet: FEC + oHSV-1 therapy increases S100A8A9 in the peripheral blood. C57/Bl6 mice bearing E0771 subcutaneous tumors were treated with either PBS ( n = 5), FEC ( n = 5), oHSV-1 ( n = 5), or FEC + oHSV-1 ( n = 10). Blood was taken 10 days after the start of treatment for analysis via flow cytometry. ( A ) Bar plots showing the frequencies of S100A8/A9 + cells in circulating PBMCs. Dots are representative of individual mice. Error bars are representative of the standard deviation. ANOVA test was used for statistical analysis. ( B ) Line graphs illustrating the mean frequency of macrophages expressing S100A8/A9, monocytes expressing S100A8/A9 and MDSCs expressing S100A8/A9 in the peripheral blood over multiple days. Error bars are representative of standard deviation. ( C ) Representative flow histograms showing the expression of S100A8/A9 on macrophages, monocytes, and MDSCs in the peripheral blood of mice treated with FEC + oHSV-1 therapy. ( D ) Bar plots showing the frequencies of S100A8/A9+ cells in circulating M1-like macrophages. Dots are representative of individual mice. Error bars are representative of the standard deviation. ANOVA test was used for statistical analysis.

Article Snippet: The PBMCs were treated with Fc block (anti-CD16/CD32; BD Biosciences (Mississauga, Ont., Canada), #553141) and surface stained antibodies for FVS (BD Biosciences, #564406), CD19 (Fisher Scientific, #14-019-482), B220 (BD Biosciences, #563894), CD4 (BD Biosciences, #561830), CD8 (BD Biosciences, #563046), CD11b (BD Biosciences, #553311), Ly6C (BD Biosciences, #553104), Ly6G (BD Biosciences, #560602), F4/80 (BD Biosciences, #743282), and S100A8/A9 (Novus Biologicals, #NBP2-47667AF700).

Techniques: Flow Cytometry, Standard Deviation, Expressing

Journal: Cancers

Article Title: Response to FEC Chemotherapy and Oncolytic HSV-1 Is Associated with Macrophage Polarization and Increased Expression of S100A8/A9 in Triple Negative Breast Cancer

doi: 10.3390/cancers13215590

Figure Lengend Snippet: S100A8 and S100A9 are independent predictors of response to FEC therapy in breast cancer patients. S100A8 and S100A9 mRNA expression was compared between responders ( n = 84) versus non-responders ( n = 219) to FEC treatment in a cohort of all breast cancer patients (S100A8: AUC = 0.71, p < 0.0001; S100A9: AUC = 0.626, p = 0.0014). Response defined as complete pathological response versus residual disease after completing therapy.

Article Snippet: The PBMCs were treated with Fc block (anti-CD16/CD32; BD Biosciences (Mississauga, Ont., Canada), #553141) and surface stained antibodies for FVS (BD Biosciences, #564406), CD19 (Fisher Scientific, #14-019-482), B220 (BD Biosciences, #563894), CD4 (BD Biosciences, #561830), CD8 (BD Biosciences, #563046), CD11b (BD Biosciences, #553311), Ly6C (BD Biosciences, #553104), Ly6G (BD Biosciences, #560602), F4/80 (BD Biosciences, #743282), and S100A8/A9 (Novus Biologicals, #NBP2-47667AF700).

Techniques: Expressing

Journal: bioRxiv

Article Title: Alteration of skin fibroblast steady state contributes to healing outcomes

doi: 10.1101/2024.12.06.627278

Figure Lengend Snippet: (A) Diagram of dermal fibroblast isolation and sequencing process; (B) Cell cluster UMAP plot; (B) Cell type distribution in each genotype; (D) Dot plot of selected marker genes for each cell cluster; (E) Feature plots of representative markers for Schwann cell development within Sox10+ population; (F) Histogram of Sox10-700 intensity of unstained and stained dermal fibroblasts and quantification of Sox10+ cells in WT and TSP2KO groups (n = 3, Unpaired T test, two-tailed, *, p<0.05).

Article Snippet: The following primary antibodies were used for flow cytometry, IHC or IF: Sox10 Antibody [Alexa Fluor 700] (1:200, Novus Biologics, NBP2-59621AF700), anti-Neurofilament H (NFH) (1:200, EMD Millipore, AB1989), anti-Vimentin (1:500, EMD Millipore, AB5733), TGFB3 Rabbit pAb (1:250, ABclonal, A8460), Sox10 Rabbit pAb (1:100, ABclonal, A15100), β-catenin (IF: 1:20, R&D system, AF1329).

Techniques: Isolation, Sequencing, Marker, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Alteration of skin fibroblast steady state contributes to healing outcomes

doi: 10.1101/2024.12.06.627278

Figure Lengend Snippet: (A) Immunofluorescence staining of NFH, Sox10, TGFb3 with Vimentin in D7 WT and TSP2KO mouse skin wounds. Quantification of (B) number of nerve bundles (NBs) in the center, (C) diameter of NBs (µm), (D) Sox10+ area/HPF (%), and (E) Tgfb3+ area/HPF (%) across the whole wounds (dashed circles indicate nerve bundles, n = 3-4, Unpaired T-test, two-tailed, *, p<0.05, **, p<0.01).

Article Snippet: The following primary antibodies were used for flow cytometry, IHC or IF: Sox10 Antibody [Alexa Fluor 700] (1:200, Novus Biologics, NBP2-59621AF700), anti-Neurofilament H (NFH) (1:200, EMD Millipore, AB1989), anti-Vimentin (1:500, EMD Millipore, AB5733), TGFB3 Rabbit pAb (1:250, ABclonal, A8460), Sox10 Rabbit pAb (1:100, ABclonal, A15100), β-catenin (IF: 1:20, R&D system, AF1329).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP gating strategy and in vitro results. (A) Transmission electron microscopy images of EP. (B) The EP gate was defined using 0.5 µm and 1.53 µm calibration beads. EP population shift was observed after staining with ASGR1 antibodies. (C) In vitro , TNF-α induced human hepatocyte–derived EP. Primary human hepatocytes following liver resection were isolated, cultured overnight, and stimulated with 10 and 20 ng/ml TNF-α. EP surface antigens were stained, and absolute AnnV + , CD130 + , Cx43 + , ASGR1 + EP were analyzed. The ordinary one-way ANOVA with Tukey’s post hoc test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Staining, Derivative Assay, Isolation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Time course of EP after LT (n = 11). (A) Total EP (/µl) (B) AnnV + , CD130 + , CD31 + EP (/µl) (C) Cx43 + , ASGR1 + , MDR3 + EP (/µl) were stained and analyzed. The Wilcoxon matched-pairs signed ranked test was used. The plots are indicated by the median, and all error bars indicate the interquartile range (IQR). A single asterisk indicates significance at p < 0.05 (D) Patients at risk for acute rejection. EP dynamics from preoperative state to POD 1 of the total, AnnV + , CD130 + , Cx43 + , ASGR1 + , MDR3 + , CD31 + EP (/µl) and their fold change were analyzed. Absolute EP were non-normally distributed; the two-tailed Mann-Whitney U test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP profiles during histology-proven ACR. EP were determined in the blood plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. EP surface antigens (A) AnnV + , (B) CD130 + , (C) Cx43 + , (D) ASGR1 + , (E) MDR3 + , (F) CD31 + EP were stained, and relative EP (%) were analyzed. (G) Example FSC-A (forward scatter) histograms of MDR3 + EP (%) (H) demonstrating the highest receiver operating characteristic (ROC) of all single antigens. One-way analysis of variance followed by Tukey’s post hoc test was used. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Staining