Journal: Blood

Article Title: KIT signaling is dispensable for human mast cell progenitor development

doi: 10.1182/blood-2017-03-773374

Figure Lengend Snippet: SCF is dispensable for mast cell progenitor survival in vitro. CD34+ progenitors were purified from buffy coats from healthy donors and analyzed with flow cytometry. (A-B) The representative gating strategy and the quantification of CD34+CD117int/hiFcεRI+ mast cell progenitors are shown (n = 9). The representative example in panel A is indicated with an open circle. The line in panel B represents the geometric mean. (C-G) CD34-enriched cells were cultured and analyzed by flow cytometry. (D) The fraction of CD117hiFcεRI+ pre–mast cells from panel C was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± standard error of the mean (SEM). (E) CD34+ progenitors were cultured with IL-3 and IL-6, and the fractions of CD34-expressing cells out of the CD117hiFcεRI+ mast cell population (days 3-7) are shown. The CD34 expression of CD117int/hiFcεRI+ cells is shown as the day 0 control. The bars represent the means ± SEM of 3 buffy coats. (F) The CD117 expression of CD34+ cells was analyzed on day 3. The red histogram indicates the cells cultured without cytokines. The blue line indicates the cells cultured with IL-3 and IL-6. The dashed line indicates the cells cultured with IL-3, IL-6, and SCF. One representative experiment out of 3 is shown. (G) The CD117 expression on CD34+ cells at day 3, shown in panel F, was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3 and IL-6 condition. The bars represent the means ± SEM of 3 buffy coats. (H-J) CD34+ cells were enriched from buffy coats, cultured, and analyzed by flow cytometry. The medium was supplemented with polyclonal goat IgG or anti-SCF neutralizing antibodies as indicated. (J) The integrin β7 expression was analyzed by flow cytometry before and after culture with IL-3, IL-6, and anti-SCF. The bars in panels I and J represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. The statistical analyses in panels D, E, G, and I were performed using 1-way ANOVA with Tukey’s multiple comparisons test. **Adjusted P < .01; ****adjusted P < .0001; ns = not significant. The unpaired 2-tailed Student t test was used for the statistical analysis in panel J. ****P < .0001.

Article Snippet: Polyclonal goat anti-human SCF or polyclonal goat IgG (5 μg/mL; both from PeproTech) was added to the culture medium in some experiments.

Techniques: In Vitro, Purification, Flow Cytometry, Cell Culture, Expressing, Control, Fluorescence

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Bronchial Secretory Immunoglobulin A Deficiency Correlates With Airway Inflammation and Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1164/rccm.201010-1629oc

Figure Lengend Snippet: Figure 7. Polymeric immunoglobulin re- ceptor (pIgR) expression and IgA transcytosis in air-liquid interface (ALI) cultured human bronchial epithelial cells (HBECs) grown with or without retinoic acid (RA). (A) Pseu- dostratified ciliated structure of bronchial epithelium and marked secretory compo- nent (SC)/pIgR expression in culture with RA added; squamous stratified structure of bronchial epithelium and absent SC/pIgR expression in culture grown without RA. Top row: hematoxylin and eosin (HE)–stained par- affin sections (original magnification 3400). Bottom row: immunohistochemistry of par- affin tissue sections with goat polyclonal anti–SC-pIgR antibody (original magnifica- tion 3400). (B) Western blot showing ex- pression of SC/pIgR in cell cultures with RA added and no pIgR expression in cells grown without RA. (C) PIGR mRNA expres- sion in RA1 and RA2 cultures (normalized to HPRT). (D) IgA and secretory IgA (SIgA) concentrations in apical washings of ALI cultured HBECs grown with or without RA. * P , 0.001 (compared with RA1 cul- tures). Mean 6 SEM is indicated for each concentration.

Article Snippet: Goat polyclonal antiSC/pIgR antibody (R&D Systems, Minneapolis, MN) was used for IHC and Western blotting.

Techniques: Expressing, Cell Culture, Staining, Immunohistochemistry, Western Blot, Concentration Assay

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Schematic representation of the full length HA protein of H9N2 (MsCon) virus. Precursor HA0 :1-560 amino acids (aa), HA1:19-338 aa, HA2: 339-560 aa, TM = transmembrane domain (525-547 aa) CT = cytosolic tail domain (548−560 aa). b Schematic representation of the soluble HA protein of H9N2 (MsCon) virus. The soluble H9HA was generated by removing the TM and CT domains (525-560 aa) and fusing the C-terminus of HA to 30 aa long trimerisation foldon sequence of the trimeric protein fibritin from T4 bacteriophage c His tag purification of the recombinant proteins. The expected sizes of the purified CD83 scFv antibody, rH9HA, and rH9HA-CD83 scFv are 30, 70, and 100 kDa respectively. Lane 1: control supernatant from the untransfected cells Lane 2: CD83 scFv Lane 3: rH9HA Lane 4: rH9HA-CD83 scFv. For the purification of the recombinant proteins, the harvested S2 cell culture supernatants containing recombinant protein bound to the metal ions (copper sulfate was used as an inducer of metallothionein promoter) were loaded onto uncharged “UNOsphere” resin derivatized with iminodiacetic acid functioning as a chelating ligand (Profinity ™ IMAC, Bio-Rad). Proteins were eluted with elution buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl and 50 mM imidazole. The purified proteins were analysed by 10% SDS-PAGE followed by Coomassie staining. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Generated, Sequencing, Purification, Recombinant, Cell Culture, SDS Page, Staining, Derivative Assay

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a BS3 cross-linking experiment for determining the native structure of H9HA ectodomain with foldon. Lane 1: rH9HA without BS3 Lane 2: rH9HA with 10 mM BS3 Lane 3: rH9HA-CD83 scFv without BS3 Lane 4: rH9HA-CD83 scFv with 10 mM BS3. About 15 µg of the recombinant protein was mixed with BS3 to a 10 mM final concentration and incubated for 1 h at room temperature. The cross-linking reaction was stopped by the addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM and incubated for 15 min at room temperature. After cross-linking, proteins were separated on 8% SDS-PAGE under reducing conditions, blotted, and analysed by western blot using anti-H9HA monoclonal antibody. M: monomer (70 kDa* 100 kDa^) D: dimer (140 kDa* 200 kDa^) T: Trimer (210 kDa* 300 kDa^) *rH9HA ^rH9HA-CD83 scFv. All blots were derived from the same experiment and were processed in parallel. b Haemagglutination assay to test the activity of recombinant H9HA with foldon to agglutinate chicken red blood cells. 1. rH9HA 2. rH9HA–CD83 scFv 3. Negative control (PBS). For the haemagglutination assay, a two-fold serial dilution of 35 μg of the recombinant HA proteins was carried out in 96-well plates. About 50 μl of 1% chicken RBCs was added. The plates were incubated at 4 °C for 1 h and the highest dilution of the protein causing the agglutination of the RBCs was noted.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Incubation, SDS Page, Western Blot, Derivative Assay, Hemagglutination Assay, Activity Assay, Negative Control, Serial Dilution, Agglutination

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Indirect ELISA for testing the activity of CD83 mAb b Indirect ELISA for testing the activity of CD83 scFv and rH9HA-CD83 scFv. Purified 8 μg of chicken CD83 ectodomain was coated onto each well of the ELISA plate, a two-fold serial dilution was carried out and the plate was incubated overnight for 4 °C. For detection, the plates were incubated with an equimolar concentration of purified CD83 scFv and rH9HA-CD83 scFv or 1 μg/ml of CD83 mAb. This was followed by incubation with goat anti-mouse HRP secondary antibody for ( a ) and HRP-conjugated anti-V5 secondary antibody for ( b ). The colorimetric detection was carried out by adding TMB substrate and absorbance at 450 nm was recorded.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Indirect ELISA, Activity Assay, Purification, Enzyme-linked Immunosorbent Assay, Serial Dilution, Incubation, Concentration Assay

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Cytokines and chemokine mRNA levels in splenocytes analysed using qRT-PCR. b IFNγ protein level analysed using ELISA. For a : splenocytes were isolated from the spleen of 3-week-old SPF chickens using Histopaque 1083 and stimulated with 10 μg of CD83 scFv/rH9HA/rH9HA-CD83 scFv for 5, 22, and 30 h in vitro. Stimulated splenocytes were harvested for RNA extraction and expression levels of the respective cytokines and chemokine were measured by qRT-PCR. Data were calculated using 2 −∆∆CT approach ( n -fold change compared to the media only control group) and reported as values normalised to the expression level of a housekeeping gene RPLPO1. For b : supernatants from the stimulated splenocytes were analysed for the presence of IFNγ by ELISA. Data are represented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. *** p < 0.001 ** p < 0.01 * p < 0.05. The data represent three independent experiments.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, In Vitro, RNA Extraction, Expressing

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: Groups of 7 days old chickens ( n = 8) were immunised with 2.8, 28, and 49 µg of recombinant rH9HA-CD83 scFv equivalent to 2, 20, and 35 µg of recombinant rH9HA (equimolar concentration). Boost vaccination was given after 7 days post primary vaccination. The chickens were bled on day 6, 14, 21, and 28 posts primary vaccination. Antibody titres in sera were measured by haemagglutinin inhibition (HI) assay. The highest dilution of serum inhibiting the agglutination of RBCs by H9N2 virus (UDL01/08) was recorded. Data are presented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001 *** p < 0.001 ** p < 0.01 * p < 0.05. ppv post primary vaccination.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Inhibition, HI Assay, Agglutination

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: Groups of 7-day-old chickens ( n = 7) were immunised with 35 µg of recombinant rH9HA-CD83 scFv equivalent to 25 µg of recombinant rH9HA (equimolar concentration). The second vaccine dose as boost was given on day 7 post primary vaccination. The chickens were challenged with 100 μl of 1 × 10 7 pfu/ml H9N2 virus (UDL 01/2008) after 7 days post boost vaccination. Some of the vaccinated and control birds were left unchallenged to serve as contacts. Swabs were taken from the buccal cavity until day 7 post infection and the amount of live virus particles recovered from swabs was titrated via plaque assays and presented as plaque-forming units (PFU)/ml. a Shedding profiles of each chicken from the buccal cavity at different days post infection are indicated by a single coloured dot. b The average shedding profile from the buccal cavity per group for directly infected birds. c The average shedding profile from buccal cavity per group for contact birds. Data are presented as mean ± SD for ( a ) and analysed by one-way ANOVA followed by Tukey’s multiple comparison test for ( a ) and by unpaired t-test for ( b, c ). For ( b ) the asterisks represent significant difference between rH9HA and rH9HA-CD83 scFv (direct) groups. **** p < 0.0001 *** p < 0.001 ** p < 0.01 * p < 0.05. pi post infection.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Infection

Journal: The Journal of Physiology

Article Title: Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission

doi: 10.1113/JP275437

Figure Lengend Snippet: Antibodies

Article Snippet: A list of the primary antibodies is given in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Primary antibody Source Host Working dilution Anti‐human PDGFRα R&D Systems, Minneapolis, MN, USA Goat 1:100 Anti‐human SCFR (Kit) R&D Systems Goat 1:100 Anti‐nNOS (used with anti‐Kit and anti‐PDGFRα) Santa Cruz Biotechnology Inc., Dallas, TX, USA Rabbit 1:500 Anti‐nNOS (used with anti‐VIP) Gift from Dr Piers Emson, Molecular Science Group, Cambridge, UK Sheep 1:500 Anti‐SK3 Alomone Labs, Jerusalem, Israel Rabbit 1:500 Anti‐VIP Immunostar Antibodies, Hudson, WI, USA Rabbit 1:500 Open in a separate window Antibodies Imaging Cryosections were examined using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Techniques: