Journal: Allergy

Article Title: Acetylcholine From Solitary Chemosensory Cell, Not Neuron, Regulates Basal Cell Fate Driving Eosinophilic Chronic Rhinosinusitis With Nasal Polyps

doi: 10.1111/all.70048

Figure Lengend Snippet: Increased Ach levels in eCRSwNP, predominantly derived from SCC. (A) Histogram showing relative mRNA expression of ChAT and VAchT in bulk RNA‐seq. (B) Histogram showing relative mRNA expression of ChAT and VAchT in control, neCRSwNP, and eCRSwNP tissues through qRT‐PCR analysis ( n = 6 per group). (C) Immunohistochemical staining showing ChAT expression in tissues from control, neCRSwNP, and eCRSwNP groups. (D‐E) Histogram showing ChAT immunoreactivity and positive staining areas in each group of tissues ( n = 8 per group). (F) IF staining of ChAT (red) and the neuronal marker βIII Tublin (green) in control and eCRSwNP tissues. (G) Histogram showing CHAT + βIII Tublin + cell numbers in subepithelium ( n = 8 per group). (H) IF staining of ChAT (red) and the SCC marker GNAT3 (green) in control and eCRSwNP tissues. (I) Histogram showing CHAT + GNAT3 + cell numbers ( n = 8 per group). (J) IF staining showing co‐localization of ChAT (red) with CC marker FOXJ1 (green), GC marker MUC5AC (red), BC marker P63 (green), and SCC marker GNAT3 (green) in ALI cultures. (K) Histogram showing Ach concentrations in the basolateral medium of ALI cultures under the following conditions: Untreated, α‐NETA‐treated, and IL‐4/IL‐5/IL‐13 treatment with or without α‐NETA, as measured by ELISA ( n = 3 per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Depending on the experimental condition, the cultures were additionally treated with PBS, 10 μM α‐NETA (HY‐138097, MCE, Monmouth Junction, NJ, USA), 5 ng/mL IL‐4 (HY‐ P70445 , MCE) with or without 10 μM α‐NETA, 5 ng/mL IL‐5 (HY‐ P70612 , MCE) with or without 10 μM α‐NETA, 5 ng/mL IL‐13 (HY‐ P70568 , MCE) with or without 10 μM α‐NETA, or 5 mM acetylcholine chloride (HY‐ P70445 , MCE) with or without 100 nM tiotropium bromide (TB, HY‐17360, MCE).

Techniques: Derivative Assay, Expressing, RNA Sequencing, Control, Quantitative RT-PCR, Immunohistochemical staining, Staining, Marker, Enzyme-linked Immunosorbent Assay

Journal: Thyroid

Article Title: A Truncating Germline Mutation of TINF2 in Individuals with Thyroid Cancer or Melanoma Results in Longer Telomeres

doi: 10.1089/thy.2019.0156

Figure Lengend Snippet: Analyses of the mutant TINF2. (A) Schematic of the mutant and the wild-type TINF2 protein structures. TBM, the TERF1 binding motif; DC cluster, the region with TINF2 mutations in the patients with short telomere syndromes. The numbers indicate the positions of the amino acids in TINF2. (B) Quantitative RT-PCRs were performed with RNAs from lymphoblastoid cell lines established from individuals as indicated. GAPDH was used as an internal control. Mut, individuals with the mutant TINF2; Wt, individuals with the wild-type TINF2. n, sample size; solid dots, individuals from one family. (C) Co-immunoprecipitation assay and Western blot. HEK293T cells were transfected with the indicated plasmid DNAs (lanes 1–5). Co-immunoprecipitations (Myc IP) with protein extracts from transfected HEK293T cells and normal HEK293T cells (lane 6) were analyzed by Western blotting. The positions of the full length and the truncated TINF2 are marked. GAPDH serves as the loading control (Input). DC, dyskeratosis congenital; PCR, polymerase chain reaction; TBM, TERF1 binding motif.

Article Snippet: Making expression constructs, cell culture, and transfection Expression plasmid DNA constructs of TINF2 and TERF1 were purchased from Origen (Cat No.: RC214419 and RG217957, respectively; OriGene Technologies, Inc.).

Techniques: Mutagenesis, Binding Assay, Control, Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 1. Differential expression and phosphoryla tion of STAT1 in eosinophils from allergic and healthy non-allergic donors. (A) Purified eosino phils from allergic (n = 10) and healthy donors (n = 12) were fixed and permeabilized, stained with the respective isotype control or specific antibodies against total-STAT1 and phospho-STAT1 and measured by flow cytometry. Data were analyzed as fold increase in fluorescence intensity with respect to the isotype control and reported as phospho-STAT1/ total-STAT1 (mean ± SEM). ** P < 0.01; Student’s t-test. (B-D) Purified eosinophils from healthy donors were pretreated with (B) vehicle (n = 7), (C) bar icitinib (BARI, 200 nM, n = 6) or (D) tofacitinib (TOFA, 200 nM, n = 6) for 30 min at 37 ◦C, stimu lated with vehicle or IL-5 (300 pM) for 10 min at 37 ◦C, fixed and permeabilized, stained with isotype control or a phospho-STAT1 antibody and analyzed by flow cytometry. Data are expressed as fold in crease in fluorescence intensity with respect to the isotype control (mean ± SEM). * P < 0.05; Student’s t-test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Quantitative Proteomics, Purification, Staining, Control, Flow Cytometry, Fluorescence

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 2. Baricitinib and tofacitinib similarly inhibit eosinophil differentiation and JAK1/2 activation. Bone marrow cells were collected from the femurs and tibiae of three female BALB/c mice and cultured at 106/mL in media supplemented with 100 ng/mL SCF and 100 ng/mL FLT3L from day 0 to day 4. On day 4, media were replaced with media containing 10 ng/mL IL-5 supplemented with or without baricitinib (BARI, 200 nM) or tofacitinib (TOFA, 200 nM). (A) Total cells, (B) Siglec F positive cells and (C) CCR3 positive cells were enumerated by flow cytometry on the indicated days. On day 12 (D) JAK1 and (E) JAK2 phosphorylation was measured by flow cytometric intracellular staining. Data are shown as mean ± SEM from 3 independent experiments, ** P < 0.01, *** P < 0.005; (A-C) two-way ANOVA and (D and E) one- way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Phospho-proteomics, Staining

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 3. Effects of baricitinib and tofacitinib on eosinophil migratory responsiveness. (A) Purified eosinophils were seeded on L-lysine coated chamber slides and pretreated with baricitinib (200 nM), tofacitinib (200 nM) or vehicle for 30 min at room temperature. F-actin polymerization was induced by stimulation with IL- 5 (1 nM) at 37 ◦C for 15 min. Adherent cells were fixed, permeabilized and blocked prior to F-actin staining with Phalloidin-Texas Red. Slides were mounted with mounting media with DAPI and fluorescence images were taken by an Olympus IX70 microscope connected with a Hamamatsu ORCA-ER digital camera. Olympus xcellence® imaging analysis software 1.1 was used for acquiring images. Representative pictures of 4 independent experiments are shown. (B-E) Purified eosinophils (n = 4–16) were pretreated with vehicle, baricitinib (BARI, 200 nM or 500 nM) (B and D) or tofacitinib (TOFA, 200 nM or 500 nM) (C and E) for 30 min at 37 ◦C and were allowed to migrate towards eosinophil chemoattractants: IL-5 300 pM, complement 5a 10 nM, CCL11 10 nM and PGD2 30 nM for 60 min in a 48-well micro- Boyden chamber. Migrated cells were enumerated by flow cytometry (BD FACSCanto II, high flow-rate for 30 s). Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005; Student’s t-test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Staining, Fluorescence, Microscopy, Imaging, Software, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 4. Effects of baricitinib and tofacitinib on neutrophil chemotaxis. PMNL (n = 14) were pre treated with vehicle, baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 30 min at 37 ◦C and were allowed to migrate towards neutrophil chemo attractants: IL-8 (10 nM) and complement 5a (10 nM) for 60 min in a 48-well micro-Boyden chamber. Migrated cells were enumerated by flow cytometry (BD FACSCanto II, high flow-rate for 30 min). Data are shown as mean ± SEM. * P < 0.05; Student’s t- test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Chemotaxis Assay, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 5. Effects of baricitinib and tofacitinib on eosinophil ROS production. Purified eosinophils were pretreated with vehicle, baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 30 min at 37 ◦C and ROS production was induced by IL-5 (A, n = 4–8), complement 5a (B, n = 6), CCL11 (C, n = 5–8) and PGD2 (D, n = 7–9). ROS production was detected by flow cytometry using a dihydrorhodamin-123 based assay. Data are expressed as geometric mean fluorescence intensity (MFI) and shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005; two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Flow Cytometry, Fluorescence

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 6. Effects of baricitinib and tofacitinib on eosinophil phagocytosis and degranulation. Purified eosinophils were pretreated with vehicle, baricitinib (500 nM and 2.5 µM) (A, n = 4) or tofacitinib (500 nM and 2.5 µM) (B, n = 4) for 30 min at 37 ◦C, incubated with opsonized Escherichia coli-FITC and analyzed by flow cytometry. Phagocytosis is presented as the percentage of E. coli-FITC-positive eosinophils. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01; One-way ANOVA. (C) Purified eosinophils were pretreated with vehicle, baricitinib or tofacitinib (500 nM) for 30 min at 37 ◦C and then stimulated with complement 5a for 30 min at 37 ◦C. The release of EPO activity into the supernatant was determined by photometry. Data were expressed as a percentage of the vehicle response (unstimulated) and are shown as the mean ± SEM; n = 14. Two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Incubation, Flow Cytometry, Activity Assay

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 7. Effects of baricitinib and tofacitinib on eosinophil apoptosis. Purified eosinophils were cultured in vehicle or IL-5 100 pM-treated medium, supplemented with or without baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 18 h. Apoptosis and necrosis were detected by flow cytometry using FITC-annexin-V/ propidium iodide (PI). Annexin/PI double-negative cells were identified as live cells (A), annexin positive/PI negative cells as early apoptotic cells (B), annexin/PI double-positive cells as late apoptotic cells and annexin negative/PI positive cells as necrotic cells (D). Data are expressed as % of total cells and shown as mean ± SEM of 11 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005; two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Cell Culture, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 8. Effects of baricitinib and tofacitinib on HDM-induced lung inflammation and airway hyperresponsiveness. (A) On day 1, eight-week-old female BALB/c mice were sensitized intranasally with 1 µg HDM and challenged by intranasal application of 10 µg HDM per day from day 7 to 12. From day 8 to 14 mice received intragastric applications of tofacitinib (TOFA, 50 µmol, twice a day) and baricitinib (BARI, 50 µmol, once a day). (B) Gating strategy for lung single cell suspensions. After single cell and CD45pos gating, eosinophils were identified as CD11cneg/Siglec Fpos and alveolar macrophages as CD11cpos/Siglec Fpos cells. CD11c/ Siglec F double negative cells were characterized as CD11bpos/Ly6Gpos neutrophils, CD11bpos/Ly6Gneg monocytes and FSC/SSClow CD11bneg/Ly6Gneg lymphocytes. On day 15, (C) lungs, (D) spleens, and (E) bone marrow were taken from six mice per group and inflammatory cells were analyzed by flow cytometry: Total CD45pos

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Single Cell, Flow Cytometry