Journal: bioRxiv

Article Title: Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

doi: 10.1101/2024.07.20.604410

Figure Lengend Snippet: C57BL/6 mice were infected i.n with IAV (X31 strain, either wild type or single cycle (S-FLU)) for 30 days. (A) Weight loss graph showing percentage of total body weight over time post infection (n=9) mice per group (naïve, WT-IAV and S-FLU). Data are combined from two independent experiments. (B) Histological analysis of inflammation in naïve, WT-IAV and S-FLU infected mice at day 30. Data are normally distributed and shown as mean +/- SEM. One way-ANOVA with Šidák’s multiple comparison, **P <0.01, ***P <0.001. (C) H&E staining showing airways (Aw), blood vessels (Bv) inflammatory foci/immune cell clusters (labelled with white stars) in naïve, WT-IAV and S-FLU infected mice at day 30. All images taken at 200x magnification, scale bar 200µm. Immunofluorescent staining shows the localization of CD4 T cells (green), B cells (B220+, red) and antigen presenting cells (MHCII, blue) in lung sections from naïve and IAV infected mice culled at day 30 post infection. All images taken at 200x magnification, scale bar 200µm. Airways (Aw), blood vessels (Bv) inflammatory foci/immune cell clusters (labelled with dashed white box). Positive cells are indicated by white arrows.

Article Snippet: For T cells, anti-CD4 APC-Alexa647 (RM4-5, ThermoFisher), anti-CD8 BUV805 (BD 53-6.7), anti-CD44 BUV395 (BD, IM7), anti-CD25 BV711 (Biolegend, PC61), anti-CD69 PE (BD, H1.2F3) anti-B220 eFluor 450 (RA3-6B2), anti-MHCII eFluor 450 (M5114) and anti- F480 eFluor 450 (BM8) all ThermoFisher.

Techniques: Infection, Comparison, Staining

Journal: bioRxiv

Article Title: Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

doi: 10.1101/2024.07.20.604410

Figure Lengend Snippet: (A) Schematic showing experimental design of IAV infection and in vitro re-challenge and co-culture. Lung CD45 negative cells were isolated from naïve or C57BL/6 mice infected 30 days earlier with IAV- WSN. After 24hours, the cells were infected with IAV-X31 and the cells examined by flow cytometry after a further 24hours or co-cultured with T cells isolated from the spleens of mice infected with IAV- X31 9 days earlier. (B) The percentage of CD44hi CD4 or CD8+ T cells that were CD25 or CD69 positive, normalized to the mean of the naïve- no infection control within each of the 2 experiments. CD4 data not normally distributed and tested via a ONE-WAY ANOVA and a Dunn’s multiple comparison test. CD8 data are normally distributed and tested via ONE-WAY ANOVA with Šidák’s multiple comparison test. Data are from two experiments with 4 mice within each naïve and IAV-memory group per experiment. (C) Representative FACS plots for IAV-NP for each cell type are shown for each group. Cells were gated based on live, single, CD45 negative populations that were CD31+ (endothelial cells), CD140+ (fibroblasts) and EpCAM1+ (epithelial cells). Numbers in plots indicate the percentages of cells that are IAV-NP+. Data are normally distributed and are from two combined experiments, with n=8 samples per timepoint. ONE-WAY ANOVA with Šidák’s multiple comparison test. (D) Cytokine levels were measured by Luminex assay in the supernatants of in vitro IAV infected and re-challenged groups taken 24hours after the in vitro infection. Data were normally distributed, are mean +/- SEM and analyzed by T test. Data combined from the same two experiments. In all graphs, each symbol represents a mouse, the horizontal line shows the mean and error bars are SEM and *:p<0.05,**: p<0.001,***: p <0.001, ****: p<0.0001.

Article Snippet: For T cells, anti-CD4 APC-Alexa647 (RM4-5, ThermoFisher), anti-CD8 BUV805 (BD 53-6.7), anti-CD44 BUV395 (BD, IM7), anti-CD25 BV711 (Biolegend, PC61), anti-CD69 PE (BD, H1.2F3) anti-B220 eFluor 450 (RA3-6B2), anti-MHCII eFluor 450 (M5114) and anti- F480 eFluor 450 (BM8) all ThermoFisher.

Techniques: Infection, In Vitro, Co-Culture Assay, Isolation, Flow Cytometry, Cell Culture, Control, Comparison, Luminex

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 1. Immunohistochemistry and immunofluorescence of coronary thrombus. (A) Aspirated material in a patient with STEMI due to total occlusion of coronary artery. (B) H&E staining of coronary artery thrombus. (C and D) Immunohistochemistry of coronary thrombus with MMP12 antibody. Arrows indicate examples of MMP12 positive cells. (E and F) Immunofluorescence staining of coronary thrombus with MMP12 antibody. Chartreuse: MMP12, Blue: Counterstain nucleus with DAPI. (E) Negative control. (F) Experimental group (magnification 400). Analysis of staining from 3 independent experiments showed similar results. STEMI=ST- segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 3. The plasma levels of MMP12 and TIMP1 in STEMI, SAP, and control groups. (A) Columns and lines show significant increase in MMP12 concentration but lower activity in STEMI compared with SAP group and normal coronary artery control group. (B) The STEMI group had significantly higher TIMP1 concentration than SAP group and control group. (C) The imbalance in MMP/TIMP ratio was observed in STEMI group compared with SAP control group. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. SAP=stable angina pectoris, STEMI=ST-segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Control, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 2. MMP12 was present in human coronary thrombi. (A) RT-PCR analysis of MMP12 gene fragment. Lane 1: DNA ladder; lanes 2–6: coronary thrombus; GAPDH blot (bottom panel) was included as loading control. (B) WB analysis of MMP12 expression in coronary artery thrombus by SDS–PAGE. Lane 1: molecular weight markers; lanes 2–6: WB products from coronary artery thrombus of STEMI-LTB; lane 7: recombinant human MMP12 with molecular weights of 45kDa. (C) The caseinolytic activity of MMP12 in coronary thrombi. Lane 1: molecular weight markers; lanes 2–4: zymography products from the coronary artery thrombus of STEMI patients; lane 5: zymography products from the coronary artery thrombus of STEMI patients incubated with antihuman MMP12 antibody. Lane 6: Recombinant human MMP12 with molecular weights of 45kDa. Analysis of the blots from 3 independent experiments showed similar results. LTB=large thrombus burden, RT-PCR=reverse transcription polymerase chain reaction, STEMI=ST-segment elevation myocardial infarction, WB=Western blot.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, SDS Page, Molecular Weight, Recombinant, Activity Assay, Zymography, Incubation, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 4. The plasma levels of MMP12 and TIMP1 in LTB and STB subgroups. (A) Columns and lines show significant increase in MMP12 concentration (P<.05) but lower activity (P>.05) in LTB compared with STB subgroup. (B) The LTB subgroup had significantly higher TIMP1 concentration than STB subgroup. (C) Although the difference did not reach statistical significance, the imbalance in MMP/TIMP ratio were higher in LTB than patients with STB. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. LTB=large thrombus burden, STB=small thrombus burden.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay, MANN-WHITNEY