Journal: medRxiv

Article Title: Anomalous epithelial variations and ectopic inflammatory response in chronic obstructive pulmonary disease

doi: 10.1101/2020.12.03.20242412

Figure Lengend Snippet: A. Representative images immunostained with CCL2, TNC, and CXCL8 with AT2 cell markers (SFTPC or ABCA3) in COPD and never-smoker lung sections. B . Violin plots for COL4A1, COL4A2, and COL4A3 in AT1 clusters. C. Violin plots for FKBP5 in selected cell types. a) present dataset; b) combined publicly-available datasets.

Article Snippet: Antibodies and reagents were as follows: anti-PD-L1 (rabbit, 1:1000, #ab205921, Abcam, Cambridge, UK); anti-TNC (rabbit, 1:100, #HPA004823, Sigma-Aldrich); anti-CCL2 (rabbit, 1:100, #HPA019163, Sigma-Aldrich); anti-RAGE (rabbit, 1:1000, #ab216329, Abcam); anti-SFTPC (rabbit, 1:1000, #HPA010928, Sigma-Aldrich); anti-ABCA3 (mouse, 1:1000, #WMAB-ABCA3-17, Seven Hills Bioreagents, OH, USA); anti- macrophage inflammatory protein 3 alpha (rabbit, 1:1000, #ab224188, Abcam); anti- CXCL1 (mouse, 1:100, #MAB275, R&D Systems, MN, USA); anti-CXCL8 (mouse, 1:100, #MAB208, R&D Systems); and Hoechst 33242 (1:1000, #H342, Sigma-Aldrich).

Techniques:

Journal: medRxiv

Article Title: Anomalous epithelial variations and ectopic inflammatory response in chronic obstructive pulmonary disease

doi: 10.1101/2020.12.03.20242412

Figure Lengend Snippet: A. 100% stacked bar charts of the percentage of cell populations for AT1 subtype clusters, AT2 subtype clusters, and basal lineage clusters. B. Bar charts displaying the percentages of subpopulations such as AT1-B, AT2-A, AT2-B, club, goblet, and basal clusters across patient states. C. a UMAP plot of epithelial cells focusing on the AT2-C cluster (left). The cell population of the AT2-C cluster (right). The y-axis represents the ratio of cells in the AT2-C cluster across patient states. Brackets () represent the percentage of cells of the AT2-C cluster based on epithelial cells in each of the patient states. D. Violin plots of representative inflammatory-related genes displaying the AT2-C (iAT2) cluster. E. Violin plots for CXCL1 and CXCL8 in AT2 cells in combined publicly-available datasets. F. Representative images immunostained with CD274 (PD-L1), CXCL1 and CXCL20 with AT2 cell markers (SFTPC or ABCA3) in COPD and never-smoker lung sections. Scale bars, 25 μm (left), 12.5 μm (right).

Article Snippet: Antibodies and reagents were as follows: anti-PD-L1 (rabbit, 1:1000, #ab205921, Abcam, Cambridge, UK); anti-TNC (rabbit, 1:100, #HPA004823, Sigma-Aldrich); anti-CCL2 (rabbit, 1:100, #HPA019163, Sigma-Aldrich); anti-RAGE (rabbit, 1:1000, #ab216329, Abcam); anti-SFTPC (rabbit, 1:1000, #HPA010928, Sigma-Aldrich); anti-ABCA3 (mouse, 1:1000, #WMAB-ABCA3-17, Seven Hills Bioreagents, OH, USA); anti- macrophage inflammatory protein 3 alpha (rabbit, 1:1000, #ab224188, Abcam); anti- CXCL1 (mouse, 1:100, #MAB275, R&D Systems, MN, USA); anti-CXCL8 (mouse, 1:100, #MAB208, R&D Systems); and Hoechst 33242 (1:1000, #H342, Sigma-Aldrich).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: Production of C. difficile spores, toxins, and host responses to infection. (A) Colony counts of spores recovered after heat treatment, and total cells in the cell-associated C. difficile fraction (infected cell lysates) in the 2D epithelial model. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. (B) ELISA for C. difficile toxins A and B shows increased toxin production after extended infection. Toxins were measured from medium obtained from the apical compartment containing uninfected cell layers incubated for 24 or 48 h (Control) or cells infected with C. difficile for 3, 6, 24, or 48 h. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) ELISA for human IL-8 indicates increased IL-8 production at 24 and 48 h p.i. IL-8 was measured in medium obtained from the basolateral compartment containing uninfected cell layers incubated for 3, 6, 24, or 48 h (Control) or cells infected with C. difficile for 3–48 h. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001 as determined by one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: C. difficile spores and toxin production, and host response to infection in the 3D-VDC model. (A) Colony counts of spores and total cells in the host cell-associated C. difficile fraction (infected cell lysates). Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. (B) Toxin A and B levels from apical compartment supernatants as determined by ELISA in the 3D model. Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) Human IL-8 levels in supernatants from the basolateral compartments with uninfected cells incubated for 24 h or with cells infected with C. difficile for 3 and 24 h, as determined by ELISA. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, ** p < 0.01, as determined by the one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 5. sRAGE and anti-RAGE inhibit CXCL8- and TNF-- but not ATRA-induced type X collagen expression. Primary normal human knee articular chondrocytes (donor ages, 29–62) (5 105 cells/six-well dish) were stimulated with CXCL8 (A), ATRA (B), or TNF- (C) in the presence or absence of 1 g/ml sRAGE or 20 g/ml anti-RAGE. SDS- PAGE and Western blotting analysis for type X collagen were performed on cell lysates at 5 days in culture. These data are representative of results from six normal human donors.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 3. RAGE expression and inducible S100A11 secretion in cul- tured human articular chondrocytes. A, RAGE expression. First-passage human knee articular chondrocytes (5 105 cells/six-well dish) from six normal donors (age range, 29–62), were prepared as described in Mate- rials and Methods, were studied for RAGE expression by flow cytometric analysis. The panels, taken from studies of one normal donor representative of all the donors, depict binding of isotype control IgG (open histogram) and additionally of anti-RAGE IgG without stimulation or in response to CXCL8, TNF-, and ATRA for 24 h (solid histograms). In normal chon- drocytes, constitutive RAGE expression was detected in 59 9% of cells, and there were no significant changes in the proportions of RAGE-express- ing chondrocytes in response to CXCL8, TNF-, or ATRA, as seen in the representative results from the single donor demonstrated here. B, S100A11 expression and multimerization in chondrocytes. SDS-PAGE/ Western blotting analysis was performed on 1 g of human recombinant S100A11 isolated from transfected 293 cells as described in Materials and Methods (left side of figure), and on 30-g aliquots of protein precipitated from conditioned medium of first-passage normal human knee chondro- cytes (right side of figure) (donor ages, 29–62). The chondrocytes were prepared under the same conditions as for A above, and stimulated for 24 h by addition of S100A11, CXCL8, TNF-, or ATRA, at which time con- ditioned media were studied. Results are representative of those from six different normal human donors, as in A.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, Binding Assay, Control, SDS Page, Western Blot, Recombinant, Isolation, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 7. Schematic of RAGE signaling in the induction of chondro- cyte hypertrophy. This schematic depicts the induction by CXCL8 and TNF- of secretion of S100A11 (and likely other chondrocyte-expressed RAGE ligands not limited to calgranulins). The paradigm further depicts subsequent RAGE signaling critically transduced by p38 MAPK pathway activation as a central event in chondrocyte hypertrophy associated with low-grade inflammation in the OA joint articular cartilage.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Activation Assay