Journal: The Journal of Allergy and Clinical Immunology

Article Title: Bronchial mucosal IFN-α/β and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations

doi: 10.1016/j.jaci.2018.04.003

Figure Lengend Snippet: Bronchial epithelial IFN-α and IFN-β protein staining is deficient in asthmatic patients in vivo . Immunohistochemistry-stained cells are seen as yellow/brown positivity. A-D, A control subject at baseline shows weak (nongoblet) epithelial staining (arrowheads) for IFN-α (Fig 1, A ) and IFN-β (Fig 1, B ), and an asthmatic patient at baseline demonstrates faint staining (arrowheads) in some epithelial cells for IFN-α (Fig 1, C ) and IFN-β (Fig 1, D ). E, Negative control (normal sheep IgG as primary antibody) shows an absence of signal ( internal scale bar = 20 μm for all). F and G, Dot graphs show scores of epithelial staining intensity for IFN-α (Fig 1, F ) and IFN-β (Fig 1, G ) in bronchial biopsy specimens of control subjects and asthmatic patients at baseline and day 4 and week 6 after infection. Triangles show individual scores, and horizontal bars show median values (Wilcoxon matched-pairs test and Mann-Whitney U test).

Article Snippet: The following panel of antibodies was applied to tissue sections: sheep polyclonal antibodies to human IFN-α (31100-1) and IFN-β (31400-1; PBL Biomedical Laboratories, Piscataway, NJ), a mouse mAb to human TLR3 (IMG-315A; Imgenex, San Diego, Calif), and goat polyclonal antibodies to human RIG-I (sc-48929) and MDA5 (sc-48031; Santa Cruz Biotechnology, Dallas, Tex).

Techniques: Staining, In Vivo, Immunohistochemistry, Negative Control, Infection, MANN-WHITNEY

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Bronchial mucosal IFN-α/β and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations

doi: 10.1016/j.jaci.2018.04.003

Figure Lengend Snippet: Weaker epithelial IFN-α or IFN-β staining at day 4 after infection is associated with greater viral load and clinical illness severity during rhinovirus infection and with greater baseline serum IgE levels. In all subjects taken together, correlations between nasal viral load and scores of epithelial IFN-α positivity at day 4 (A) ; between total cold ( B and C ) and total chest ( D and E ) symptom scores (summed daily scores on days 0-14) after the RV16 infection period and scores of epithelial IFN-α and IFN-β at day 4, respectively; between PC 10 histamine at day 6 and scores of epithelial IFN-α (F) and IFN-β (G) at day 4, IFN-β at baseline (H) and IFN-β at week 6 (I) , respectively; between maximum decrease in PEF (as a percentage) on days 0 to 14 after infection and epithelial IFN-β at day 4 (J) ; between maximum decrease in FEV 1 (as a percentage) and scores of epithelial IFN-α (K) and IFN-β (L) positivity at day 4, respectively; between baseline serum IgE and scores of epithelial IFN-α (M) and IFN-β (N) positivity at day 4, respectively, are shown. Solid circles , Asthmatic patients; open circles , control subjects. Spearman rank correlation was used.

Article Snippet: The following panel of antibodies was applied to tissue sections: sheep polyclonal antibodies to human IFN-α (31100-1) and IFN-β (31400-1; PBL Biomedical Laboratories, Piscataway, NJ), a mouse mAb to human TLR3 (IMG-315A; Imgenex, San Diego, Calif), and goat polyclonal antibodies to human RIG-I (sc-48929) and MDA5 (sc-48031; Santa Cruz Biotechnology, Dallas, Tex).

Techniques: Staining, Infection

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Bronchial mucosal IFN-α/β and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations

doi: 10.1016/j.jaci.2018.04.003

Figure Lengend Snippet: In asthmatic patients only, there is a trend toward correlations between sputum viral load and scores of epithelial IFN-α positivity at day 4 (A) and between total cold (B) and total chest (C) symptom scores (summed daily scores on days 0-14) after the RV16 infection period and scores of epithelial IFN-β at day 4, respectively (Spearman rank correlation).

Article Snippet: The following panel of antibodies was applied to tissue sections: sheep polyclonal antibodies to human IFN-α (31100-1) and IFN-β (31400-1; PBL Biomedical Laboratories, Piscataway, NJ), a mouse mAb to human TLR3 (IMG-315A; Imgenex, San Diego, Calif), and goat polyclonal antibodies to human RIG-I (sc-48929) and MDA5 (sc-48031; Santa Cruz Biotechnology, Dallas, Tex).

Techniques: Infection

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Bronchial mucosal IFN-α/β and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations

doi: 10.1016/j.jaci.2018.04.003

Figure Lengend Snippet: Numbers of subepithelial TLR3, MDA5, and RIG-I protein–expressing inflammatory cells in asthmatic patients are induced in response to rhinovirus infection in vivo . Counts for subepithelial IFN-α + (A) , IFN-β + (B) , TLR3 + (C) , MDA5 + (D) , and RIG-I + (E) cells in bronchial biopsy specimens of control subjects and asthmatic patients at baseline and day 4 and week 6 after infection are shown. Data are expressed as numbers of positive cells per square millimeter of subepithelium. Triangles show individual counts, and horizontal bars show median values (Wilcoxon matched-pairs test and Mann-Whitney U test).

Article Snippet: The following panel of antibodies was applied to tissue sections: sheep polyclonal antibodies to human IFN-α (31100-1) and IFN-β (31400-1; PBL Biomedical Laboratories, Piscataway, NJ), a mouse mAb to human TLR3 (IMG-315A; Imgenex, San Diego, Calif), and goat polyclonal antibodies to human RIG-I (sc-48929) and MDA5 (sc-48031; Santa Cruz Biotechnology, Dallas, Tex).

Techniques: Expressing, Infection, In Vivo, MANN-WHITNEY

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Bronchial mucosal IFN-α/β and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations

doi: 10.1016/j.jaci.2018.04.003

Figure Lengend Snippet: Numbers of subepithelial type I interferon–producing monocytes/macrophages, but not neutrophils, are deficient during rhinovirus infection in asthmatic patients. Results of double-immunofluorescence staining to demonstrate colocalization of neutrophils and CD68 + monocytes/macrophages (green) with IFN-α and IFN-β (red) in a bronchial biopsy specimen from an asthmatic patient at day 4 after infection are shown. Elastase-positive neutrophils ( A and D ) and CD68 + monocytes/macrophages ( G and J ) are shown by using fluorescein isothiocyanate green fluorescence, and IFN-α ( B and H ) and IFN-β ( E and K ) immunopositivity are shown by using Texas Red fluorescence. Coexpression is seen as yellow fluorescence in each case in neutrophils/IFN-α (C) and neutrophils/IFN-β (F) . However, there are faint or no yellow fluorescence double-labeled cells for CD68/IFN-α (I) and CD68/IFN-β (L) . Internal scale bars = 10 μm for all. Nuclei are counterstained blue with 4′-6-diamidino-2-pheynlindole dihydrochloride. Counting data of double-immunofluorescence staining are shown in graphs of percentages of neutrophils (M) and CD68 + monocytes/macrophages coexpressing (N) IFN-α and IFN-β. Triangles show individual percentages, and horizontal bars show median values (Mann-Whitney U test).

Article Snippet: The following panel of antibodies was applied to tissue sections: sheep polyclonal antibodies to human IFN-α (31100-1) and IFN-β (31400-1; PBL Biomedical Laboratories, Piscataway, NJ), a mouse mAb to human TLR3 (IMG-315A; Imgenex, San Diego, Calif), and goat polyclonal antibodies to human RIG-I (sc-48929) and MDA5 (sc-48031; Santa Cruz Biotechnology, Dallas, Tex).

Techniques: Infection, Double Immunofluorescence Staining, Fluorescence, Labeling, MANN-WHITNEY

Journal: Proteomics

Article Title: Mass Spectrometric Immunoassay for Quantitative Determination of Transthyretin and its Variants

doi: 10.1002/pmic.201100023

Figure Lengend Snippet: (a) Representative transthyretin standards mass spectra, and (b) Standard curve generated with the transthyretin (TTR) mass spectrometric immunoassay by using beta-lactoglobulin (BL) as an internal reference standard.

Article Snippet: Rabbit anti-human polyclonal antibody to beta-lactoglobulin (BL) was obtained from GeneTex (Irvine, CA, Cat. No. GTX77272, 1 mg/mL).

Techniques: Generated

Journal: Endocrine Connections

Article Title: The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors

doi: 10.1530/EC-13-0025

Figure Lengend Snippet: β-Catenin staining localization distribution in the different study groups.

Article Snippet: The sections were incubated overnight at 4 °C with the appropriate diluted primary antibodies: rabbit anti-human MABs to p53 (453M-94; 1:100; Cell Marque, Rocklin, CA, USA), p21 (421M-14; 1:50; Cell Marque), p27 (427M-94; 1:500; Cell Marque), cyclin D1 (271R-14; 1:500; Cell Marque), Ki-67 (27R-14; 1:100; Cell Marque), and E-cadherin (246R-14; 1:200; Cell Marque) and rabbit anti-human polyclonal antibodies to β-catenin (424A-14; 1:500; Cell Marque).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: IFNB-iPSCs express functional IFN-β. IFNB-iPSCs and K7-iPSCs were cultured in parallel and used for RNA isolation; the preparation of cell extracts; and the collection of cell culture supernatants. ( a ) IFNB-iPSCs display an increased expression of IFNB as compared with the parental K7-iPSC line. RNAs isolated from IFNB-iPSCs and K7-iPSCs were subjected to RT-PCR; IFNB expression values were normalized relative to GAPDH , and fold changes were calculated as relative IFNB mRNA levels in IFNB-iPSCs relative to K7-iPSCs (2 −∆∆Cq ). Dotted line, fold change = 2 (significance threshold). Data are shown as boxes and whiskers with minimal and maximal values (K7-iPSCs, LA8-iPSCs, and LC8-iPSCs, summarized results of at least 3 independent experiments; LE4-iPSCs, one experiment, technical replicates). Note that LA8-iPSCs and LC8-iPSCs bear a homozygous IFNB insertion, whereas LE4-iPSCs are heterozygous. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( b , c ) IFNB-iPSCs express IFN-β at the protein level. ( b ) IFNB-iPSCs and K7-iPSCs were pelleted, frozen, and analyzed in a Western blot. The graph shows the relative densitometric values of IFN-β normalized to β-actin. ( c ) The supernatants were collected from IFNB-iPSCs and K7-iPSCs and analyzed using ELISA (the results of two independent experiments; in each of them, K7-iPSCs, LA8-iPSCs, and LC8-iPSCs were cultured in parallel and independently of cells of another experiment). ( d ) The supernatants of IFNB-iPSCs exhibit IFN-β-like functional activity. The supernatants were obtained from IFNB-iPSC and K7-iPSC cultures and were added to THP-1 macrophage-like cells pre-activated with PMA. Control THP-1 cells were cultured in the absence of iPSC supernatants. THP-1 RNA was isolated from all cultures, and the expressions of ISGs were analyzed in RT-qPCR using RPL27 as a housekeeping gene. Data are shown as boxes and whiskers with minimal and maximal values and individual points. The representative results of one out of two experiments are presented. Numbers on the graphs show the FDRs (Benjamini, Krieger, and Yekutieli correction for multiple comparisons). FDRs < 0.05 were considered as significant; for the comparison of IFNB-iPSCs and K7-iPSCs, FDRs > 0.05 are also shown. The differences between LA8-iPSCs and LC8-iPSCs were insignificant. PMA, phorbol 12-myristate-13-acetate.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Functional Assay, Cell Culture, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: Exogenous IFN-β disrupts the expression of ectoderm-associated genes in differentiating parental K7-iPSCs. K7-iPSCs were subjected to spontaneous ( a ) or directed ( b – d ) differentiation in the absence or in the presence of recombinant human IFN-β; at the end of the differentiation, the expression of ectoderm-associated markers was analyzed using RT-PCR (housekeeping gene, RPL27 ) and compared with that observed in original K7-iPSCs. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( a ) Changes in the expression of ectoderm-associated genes in spontaneously differentiated EBs. Gene expression was analyzed on day 20. Data are shown as boxes and whiskers with minimal and maximal values. The summarized results of two independent experiments are shown. ( b ) Light microscopy of K7-iPSCs differentiating in the absence or in the presence of IFN-β. Made on differentiation days 1 and 7. ( c ) Changes in the expression of ectoderm-associated genes following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium. Gene expression was analyzed on day 7 (as recommended by the manufacturer). Data are shown as boxes and whiskers with minimal and maximal values. The representative results of one out of two experiments are presented. ( d ) Changes in the expression of the IFNB gene following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium (the representative results of one out of two experiments).

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Light Microscopy