Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: Characterisation of hexameric-Fc binding. ( A ) The binding of fluorescently tagged γ1 hexameric-Fc and IVIg to PBMC subsets was analysed by flow cytometry. Shown is 1 representative donor out of 3 independent donors. ( B ) Flow cytometric analysis of FcγR expression on PBMCs. Cells from the same donor as in ( A ) were stained with FcγR antibodies to analyse FcγRI, FcγRII and FcγRIII expression on PBMC subsets. ( C ) Binding of hexameric-Fc to HEK293 cells transfected with indicated FcγR constructs. Data shows the mean of 3 independent experiments + /- SEM. ( D ) Representative SPR traces showing the binding of γ4-hexameric-Fc and IgG4 to FcγRIIa. Hexameric-Fc was titrated in a two-fold dilution series from 1μM to 7.8 nM. IgG4 was titrated in a two-fold dilution series between 50μM and 0.39 μM.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: Binding Assay, Flow Cytometry, Expressing, Staining, Transfection, Construct

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vitro disruption of FcγR function. ( A ) Macrophages were incubated with hexameric-Fc s or IvIg at 75 µg/ml for 1 hour. Cells were then washed and incubated for the indicated period. Cells were then labelled with fluorescently-conjugated hexameric-Fc. Data shows means from three donors ± SEM. ( B ) Pre-incubation with hexameric-Fc inhibits phagocytosis for 72hrs. Human monocyte-derived macrophages were incubated with the indicated hexameric-Fcs or IVIg at 75 µg/ml or 300 µg/ml for 1 hour, washed and cultured for 1 to 72 hours before co-culture with autologous CFSE-labelled B cell targets in the presence of 0.1 μg/ml anti-CD20. The disappearance of target cells was measured by flow cytometry after 18hrs and plotted as % inhibition of total antibody-dependent phagocytosis. Data are the mean ± SEM for three donors.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vitro, Disruption, Incubation, Derivative Assay, Cell Culture, Co-Culture Assay, Flow Cytometry, Inhibition

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vivo effects of hexameric-Fc. ( A ) 125 I γ1 Hexameric-Fc was administered to mice at 0.5 mg/kg, 2 mg/kg or 10 mg/kg. At indicated timepoints, plasma was collected from three mice per timepoint and concentration of protein bound radioactivity in plasma determined by direct measurement in a gamma counter. Values were corrected to calculate μg-equivalents of hexameric-Fc per mL of plasma. ( B ) γ4eng F234L F296Y hexameric-Fc administered to cynomolgus monkeys by IV bolus at 1 dose of 2 mg/kg. Concentrations of hexameric-Fc and smaller and larger human Fc-containing moieties were detected in plasma by mass spectroscopy. n of 3 animals, ± SEM. ( C ) Binding of γ1-hexameric-Fc to immobilised recombinant FcRn was investigated by SPR. Hexameric-Fc was titrated in a two-fold dilution series from 2.5μM to 39 nM. ( D ) To assess hexameric-Fc mediated FcγR blockade in cynomolgus monkeys, whole blood samples were collected after a 30 mg/kg IV dose of γ4eng F234L F296Y hexameric-Fc. Surface labelling of samples was carried out to identify monocytes (CD14 + ) and occupancy of FcγRs assessed using a AF647-conjugated γ4eng F234L F296Y prior to analysis by flow-cytometry. 3 animals, ± SEM. ( E ) To assess the effect of hexameric-Fc in an ITP model, 10 mg/kg hexameric-Fc was administered to mice IV, at the timepoints indicated, prior to addition of anti-CD41 (MWReg30) to induce platelet depletion. Whole blood samples were taken immediately prior to and 24 hour post anti-CD41 in order to determine platelet numbers. n = 6 mice per group, graph shows mean ± SEM, *=p < 0.05, ***=p < 0.01, by ANOVA and Dunnetts multiple comparison test.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vivo, Clinical Proteomics, Concentration Assay, Radioactivity, Mass Spectrometry, Binding Assay, Recombinant, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection

doi: 10.1101/2021.03.29.437516

Figure Lengend Snippet: SARS-CoV-2 infected mothers with male fetuses demonstrate reduced placental transfer of SARS-CoV-2 antibodies compared to those with female fetuses. Dot plots showing relative Spike-, RBD-, S1-, S2-, and N-specific maternal blood (M) and cord blood (C) titers of (A) IgG2, (B), IgG3, (C) FcRn, (D) FCGR2A, (E) FCGR2B, (F) FCGR3A, and (G) FCGR3B. Females are shown in light orange while males are shown in dark orange with black border. Wilcoxon matched pairs signed rank test was performed to determine significance. *p<0.05, **p<0.01, ***p<0.0001.

Article Snippet: Samples were then incubated in primary antibodies diluted in 5% bovine serum albumin (BSA) for 1.5h at room temperature (Placental Alkaline Phosphatase (PLAP – ab212383) - 1:1000, Neonatal Fc Receptor (FcRn – ab193148) – 1:100, CD16 (CD16 – Leica NCL-L-CD16) – 1:100), CD32 (R&D AF1330) – 10ug/ml, CD64 (Origene TA506331)-1:100.

Techniques: Infection

Journal: bioRxiv

Article Title: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection

doi: 10.1101/2021.03.29.437516

Figure Lengend Snippet: Maternal and cord blood titers of HA and PTN in SARS-CoV-2 infected and non-infected mothers. Dot plots showing relative hemagglutinin (HA)- and pertussis (PTN)-specific maternal blood (M) and cord blood (C) titers of (A) IgG1, (B) IgG2, (C) IgG3, (D) FcRn, (E) FCGR2A, (F) FCGR2B, (G) FCGR3A, and (H) FCGR3B. SARS-CoV-2 - females are shown in light blue, SARS-CoV-2 + females are shown in light orange, SARS-CoV-2 – males are shown in dark blue, and SARS-CoV-2 + males are shown in dark orange. Y-axis units for all plots are PBS-corrected mean fluorescence intensity (MFI). Wilcoxon matched-pairs signed rank test was performed to determine significance. * p < 0.05, † p<0.10.

Article Snippet: Samples were then incubated in primary antibodies diluted in 5% bovine serum albumin (BSA) for 1.5h at room temperature (Placental Alkaline Phosphatase (PLAP – ab212383) - 1:1000, Neonatal Fc Receptor (FcRn – ab193148) – 1:100, CD16 (CD16 – Leica NCL-L-CD16) – 1:100), CD32 (R&D AF1330) – 10ug/ml, CD64 (Origene TA506331)-1:100.

Techniques: Infection, Fluorescence

Journal: bioRxiv

Article Title: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection

doi: 10.1101/2021.03.29.437516

Figure Lengend Snippet: (A-C) qPCR analyses of fetal male or fetal female expression of ( A ) FCGR3B, ( B ) FCGR2A, and ( C ) FCGR2B in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ . (D) Representative immunoblots and quantification of FCγR2 in female or male placental biopsies from mothers testing negative (blue) or positive (orange) for SARS-CoV-2. (E) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR2 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (F) Box-and-whisker plots showing FCγR2/FcRn co-localization in placental villi. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. * p<0.05.

Article Snippet: Samples were then incubated in primary antibodies diluted in 5% bovine serum albumin (BSA) for 1.5h at room temperature (Placental Alkaline Phosphatase (PLAP – ab212383) - 1:1000, Neonatal Fc Receptor (FcRn – ab193148) – 1:100, CD16 (CD16 – Leica NCL-L-CD16) – 1:100), CD32 (R&D AF1330) – 10ug/ml, CD64 (Origene TA506331)-1:100.

Techniques: Expressing, Western Blot, Staining, Marker, Whisker Assay

Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet:

Article Snippet: The rat anti-mouse Ly-6G (Biolegend #127601) and rabbit monoclonal anti-mouse CD68/SR-D1 clone 2449D (R&D Systems #MAB101141) were diluted 1:1000, and the goat anti-human polyclonal anti-perilipin (Abcam #ab61682) was diluted 1:200.

Techniques: RNAscope, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Reporter Gene Assay, Plasmid Preparation, Software