Journal: ImmunoHorizons

Article Title: IL-17RA–Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection

doi: 10.4049/immunohorizons.2300078

Figure Lengend Snippet: IL-17 signaling impacts expression of genes that contribute to innate barrier function and restricts chronic inflammation. ( A ) Differential abundance of genes in the stomach at 3 mo after H. pylori infection in Il17ra −/− mice compared with C57BL/6 mice using the nCounter Immunology NanoString panel. The table represents genes that are lower in expression in Il17ra −/− with a log 10 adjusted p value of >2 and a log 2 fold change of greater than −1.25. On the volcano plot, these genes are in purple. The multiplex RNA hybridization assay was performed on six mice per genotype. ( B ) Gastric murine organoids from C57BL/6 mice (gastroids) were stimulated with 50 ng/ml IL-17A, IL-17F, and the heterodimer IL-17A/F. RNA was then extracted from these gastroids and qPCR assays of genes associated with epithelial cell responses including Cxcl1 , Nox1 , and Pigr were measured. Gapdh was used as an endogenous control, and unstimulated gastroids were pooled and used as a reference sample. The data are representative of three independent experiments. Error bars represent ± SEM. One-way ANOVA with a Dunnett multiple comparison test was used to determine significance. * p < 0.05, **** p < 0.0001, compared with unstimulated.

Article Snippet: Murine TaqMan gene expression assays included the following: Gapdh (Mm99999915_g1), Il17a (Mm00439619_m1), Il21 (Mm00517640_m1), Cd19 (Mm00515420_m1), Pigr (Mm00465049_m1) Nox1 (Mm00549170_m1), Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), S100a8 (Mm00496696_g1), S100a9 (Mm00656925_m1), Epcam (Mm00493214_m1), and Il17ra (Mm00493214_m1).

Techniques: Expressing, Infection, Multiplex Assay, Hybridization, Control, Comparison

Journal: ImmunoHorizons

Article Title: IL-17RA–Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection

doi: 10.4049/immunohorizons.2300078

Figure Lengend Snippet: H. pylori infection of Il17ra ΔGI-epi mice results in the upregulation of proinflammatory genes. ( A ) Differential expression of a number of inflammatory genes was determined using the NanoString immunology panel (Rosalind analysis) from RNA isolated from stomachs of Il17ra fl/fl versus Il17ra ΔGI-Epi at 3 mo postinfection. Loss of the Il17ra in epithelial cells leads to enrichment of pathways involved in cell trafficking and T lymphocyte and B lymphocyte activation (complete listing of differentially expressed genes is in ). In this panel, two genes were significantly less abundant in Il17ra ΔGI-Epi compared with controls, Il17ra , and Pigr. ( B ) Real-time qPCR was performed to confirm differential gene expression of key proinflammatory genes. Data are representative of three independent experiments; individual values from each sample are shown with the SEM for each group. An unpaired t test was performed to assess statistical significance. ** p < 0.01, *** p < 0.001.

Article Snippet: Murine TaqMan gene expression assays included the following: Gapdh (Mm99999915_g1), Il17a (Mm00439619_m1), Il21 (Mm00517640_m1), Cd19 (Mm00515420_m1), Pigr (Mm00465049_m1) Nox1 (Mm00549170_m1), Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), S100a8 (Mm00496696_g1), S100a9 (Mm00656925_m1), Epcam (Mm00493214_m1), and Il17ra (Mm00493214_m1).

Techniques: Infection, Quantitative Proteomics, Isolation, Activation Assay, Gene Expression

Journal: ImmunoHorizons

Article Title: IL-17RA–Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection

doi: 10.4049/immunohorizons.2300078

Figure Lengend Snippet: Despite reduced expression of Pigr in Il17ra ΔGI-epi mice after H. pylori infection, IgA levels increase in the gastric mucosa. ( A and B ) Real-time qPCR analysis of Pigr expression was performed on RNA isolated from gastric tissue, and levels of IgA were determined by ELISA at (A) 1 mo postinfection and (B) 3 mo postinfection. Data are representative a minimum of two experiments. Real-time qPCR data are expressed in relative units using Gapdh as the housekeeping gene and uninfected gastric RNA as the calibrator sample. Individual values from each sample are shown with the SEM for each group. An unpaired t test was performed to assess statistical significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Murine TaqMan gene expression assays included the following: Gapdh (Mm99999915_g1), Il17a (Mm00439619_m1), Il21 (Mm00517640_m1), Cd19 (Mm00515420_m1), Pigr (Mm00465049_m1) Nox1 (Mm00549170_m1), Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), S100a8 (Mm00496696_g1), S100a9 (Mm00656925_m1), Epcam (Mm00493214_m1), and Il17ra (Mm00493214_m1).

Techniques: Expressing, Infection, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 4. POPG inhibits LPS-induced MAPK and IB phosphorylation and MKP-1 expression. A, POPG liposomes (200 g/ml) were added to monolayer cultures of differentiated U937 cells that received either no treat- ment or 10 ng/ml LPS. After incubating for the indicated times, cells were lysed using buffer containing detergent, protease inhibitors, and phospha- tase inhibitors. Aliquots with 15 g of protein from lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The amount of phosphorylation was detected using phospho-specific antibodies to p38 MAPK, p42/p44 ERK, p46-p54 JNK, and phosphorylated IB. To determine equal loading of proteins between samples, the membranes were probed with rabbit polyclonal p46 JNK, p42/p44 ERK, p38 MAPK, and IB antibodies. The expression of MKP-1 was detected with a polyclonal MKP-1 antibody. B, POPC and DPPG liposomes (200 g/ml) were compared with POPG lipo- somes (200 g/ml) as antagonists of LPS activation of cells using the same conditions as described for panel A. The time of analysis following LPS expo- sure for p38, ERK, and JNK was 30 min, and that for IB and MKP-1 was 60 min.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Expressing, Liposomes, SDS Page, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 9. CD14 binds to solid phase lipids. A, affinity-purified preparations (2 g each) of the extracellular domains of CD14 (sCD14) and TLR4 (sTLR4) and the full-length MD-2 were analyzed by gel electrophoresis under reduc- ing and denaturing conditions. The electrophoretic gels were stained with Coomassie Blue. mol. mass st., molecular mass standards. B, phospholipids (1.25 nmol) in 20 l of ethanol were placed onto microtiter wells, and the solvent was evaporated. Nonspecific binding was blocked with 20 mM Tris buffer(pH7.4)containing0.15 MNaCl,5mMCaCl2(intheupperpanel),or2mM EGTA (in the lower panel) and 5% (w/v) bovine serum albumin (buffer A). Varying concentrations of human CD14 in buffer A were added and incu- bated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using anti-CD14 monoclonal antibody as described under “Experimental Pro- cedures.” The data shown are the means S.E. from three separate experi- ments with duplicate samples in each experiment.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Affinity Purification, Nucleic Acid Electrophoresis, Staining, Solvent, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 10. PG Inhibits CD14 binding to solid phase LPS. A, various types of PG were coated onto microtiter plates and incubated with CD14 (1 g/ml) at 37 °C for 1 h. The binding of CD14 to PG was detected using anti-CD14 mono- clonal antibody, and the ELISA-based absorbance of CD14 bound to POPG was defined as 100%. Types of PG shown on the graph are: dilauroylphos- phatidylglycerol (DLPG), DMPG, DPPG, and 16:0/18:1 POPG. B, LPS (2 g) in 20 l of ethanol was placed onto microtiter wells, and the solvent was evaporated. After blocking the nonspecific binding with buffer A, the mix- ture of CD14 (1 g/ml) and phospholipid liposomes (20 g/ml) in buffer A, which was preincubated at 37 °C for 1 h, was added and incubated at 37 °C for 1 h. The binding of CD14 to LPS was detected using anti-CD14 mono- clonal antibody. The ELISA-based absorbance of CD14 bound to LPS was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, **, p 0.01, when compared with LPS-CD14 binding in the absence of phospholipids.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Solvent, Blocking Assay, Liposomes

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE11.MonoclonalantibodiesspecificfortheLPSbindingsiteinhibit CD14 interaction with POPG and PI. POPG (A) or PI (B) were coated onto microtiter plates. After blocking the nonspecific binding with buffer A, the mixture of CD14 (1 g/ml) and monoclonal antibodies or isotype control IgG (50g/ml)inbufferA,whichwaspreincubatedat37 °Cfor1h,wasaddedand incubated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of CD14 bound to phospholipid was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, when compared with CD14 binding in the absence of monoclonal antibody. mIG, mouse Ig. C, CD14 (2 g) was coated onto microtiter plates, and nonspecific binding was blocked with buffer A. Monoclonal antibodies or isotype control IgG (50 g/ml) in buffer A were added and incubated at 37 °C for 1 h. The CD14 was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of solid phase CD14 alone was defined as 100%.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Blocking Assay, Binding Assay, Bioprocessing, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Differentially expressed genes in progenitor vs. effector club cells a .

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Most commonly enriched genes in each club cell subcluster.

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques: Expressing, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Nonsmoker primary small airway epithelium (SAE) basal cells were cultured in air–liquid interface (ALI) culture for 28 days. a Morphology of the cultures at day 7 and 28. Shown are cross sections of the ALI culture, hematoxylin and eosin stain. b , c Multicolor immunofluorescence assessment of club cell subtypes. All club cells were identified using SCGB1A1 marker (green); effector club cells were further identified by the co-expression of MUC5B (panel b red) or SCGB3A1 (panel c red). DAPI identifies the nucleus of all cells. The number of days after establishment of the air liquid interface is noted. Green arrowheads mark cells that are SCGB1A1 + club cells that lack other markers. Orange arrowheads mark cells that are positive for SCGB1A1 and either MUC5B or SCGB3A1. Red arrowheads mark cells that are positive for MUC5B or SCGB3A1, but express little if any SCGB1A1. Scale bars are 50 μm. d Gene expression on ALI over time. TaqMan probes for genes enriched in effector club cells were assessed by qPCR and normalized to an 18S rRNA control. Probes were tested at days 0, 7, 14, and 28 of SAE nonsmoker cells differentiated in ALI. Error bars represent standard deviation among three different ALI cultures. Genes assessed for expression include MUC5B, SCGB1A1, SLPI, PIGR, and LYZ.

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques: Cell Culture, H&E Stain, Immunofluorescence, Marker, Expressing, Gene Expression, Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Smoking-related reprogramming of small airway epithelium club cells.

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Numbers of club cells in smokers vs. nonsmokers. Club cells from nonsmoker and smoker human small airway epithelium (SAE) cytopreps, identified by SCGB1A1 + immunostaining and absence of KRT5, were quantified and compared against total cells, quantified by DAPI staining, per cytoprep. Three samples of each phenotype were evaluated by a blinded observer, with over 500 total cells per sample; plots show mean ± standard error. b Proportion of club cells exhibiting characteristics of subclusters 1 (progenitor), 2 (proliferative), and 3 (effector) in the population of club cells derived from nonsmokers and smokers. c Effect of cigarette smoke extract (CSE) exposure on differentiation of small airway epithelial club cells. Exposure of basal cells differentiated on air liquid interface (ALI) to cigarette smoke extract (3% Marlboro Red) led to a decrease in defense-related transcript in cells: CYP1A1, positive control demonstrating exposure to cigarette smoke extract; MUC5B; PIGR; SLPI; LYZ; and MUC1. Values are expressed as normalized expression relative to 18S rRNA. Each point represents one well of an experiment done in triplicate; plot shows mean ± standard deviation; p values are from a two-sided unequal variance Student’s t -test. All data is from ALI day 28.

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques: Immunostaining, Staining, Derivative Assay, Positive Control, Expressing, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Immunofluorescence analysis of cytopreps containing club cells (SCGB1A1 + ) expressing the effector club cell genes PIGR or MUC5B. White arrowheads indicate dual-labeled cells. White arrows indicate SCGB1A1 + club cells that lack significant expression of a PIGR or MUC5B. Black arrows mark examples of SCGB1A1-negative non-club cells in the cytoprep. Bar = 20 µm. b Total number of dual labeled (SCGB1A1 + PIGR + or SCGB1A1 + MUC5B + ) cells, or SCGB1A1 single labeled cells were quantified by a blinded observer in nonsmokers and compared with smokers. Plot shows mean of three experiments ± standard error; p values are from a two-sided unequal variance Student’s t -test.

Article Snippet: To assess the timing of expression of club cell-specific markers, the following probes (ThermoFisher Scientific) were utilized, including: 18S RNA (4310893E), MUC5B (Hs00861588_m1), SCGB1A1 (Hs00171092_m1), SCGB3A1 (Hs00369360_g1), LYZ (Hs00426232_m1), PIGR (Hs00922561_m1) and SLPI (Hs00268204_m1).

Techniques: Immunofluorescence, Expressing, Labeling

Journal: mAbs

Article Title: Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment

doi: 10.1080/19420862.2021.1987180

Figure Lengend Snippet: Surface Biolayer Interferometry-based binding of mAbs and bifunctional molecules to spike glycoprotein and to pIgR . Antibodies and antigens are indicated in the graphs. Graphs represent magnitude of response (nm) over time. Association and dissociation are displayed along with fitted curves

Article Snippet: Primary antibodies against pIgR (R&D Systems, MAB27171) and VHH domains (Genscript, A01995) were diluted to 5 mg/mL final concentration in PBST supplemented with 10% FBS and 500 mL was applied to the apical and basolateral chamber for 2 h at room temperature.

Techniques: Binding Assay

Journal: mAbs

Article Title: Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment

doi: 10.1080/19420862.2021.1987180

Figure Lengend Snippet: Bifunctional molecules Display Specific Functional Activity Against SARS-CoV-2 . (a) Functional competition ability is plotted vs. antibody concentration. Antibodies are indicated in the graph. (b) PBMC-mediated ADCC of MDCK-pIgR cells is plotted as green area per well (normalized to 0 h) vs concentration of bifunctional molecule (nM). Antibodies are indicated in the legend

Article Snippet: Primary antibodies against pIgR (R&D Systems, MAB27171) and VHH domains (Genscript, A01995) were diluted to 5 mg/mL final concentration in PBST supplemented with 10% FBS and 500 mL was applied to the apical and basolateral chamber for 2 h at room temperature.

Techniques: Functional Assay, Activity Assay, Concentration Assay

Journal: mAbs

Article Title: Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment

doi: 10.1080/19420862.2021.1987180

Figure Lengend Snippet: Transcytosis in lung microtissues . A) Transcytosis of bifunctional molecules in EpiAirway tissue model in 24 h post application. For each sample, 20 mg of protein was added to the basolateral well, and after 24 hr, the mucosal surface was washed and the levels of transcytosed antibodies were quantified. Levels are shown in total micrograms transcytosed in a 24 h period. Error bars represent standard error and are representative of at least 2 independent experiments. B-C) Confocal image showing the human EpiAirway microtissue for CV19B307 (b) and CV19B290 (c). Staining shows: blue (nuclei), green (anti-VHH), and red (pIgR). Scale bars shows 50 mm (insets) and 100 mm (main image)

Article Snippet: Primary antibodies against pIgR (R&D Systems, MAB27171) and VHH domains (Genscript, A01995) were diluted to 5 mg/mL final concentration in PBST supplemented with 10% FBS and 500 mL was applied to the apical and basolateral chamber for 2 h at room temperature.

Techniques: Staining