Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Comparison of serum Gd-IgA1 levels among four groups: J-HC, J-IgAN, G-HC, and G-IgAN The medians (quartile range) of Gd-IgA1 levels were 0.40 (0.23–0.52), 0.86 (0.55–1.25), 0.73 (0.38–1.17), and 1.29 (0.85–1.96), respectively. Gd-IgA1 levels differed significantly among the four groups (Kruskal-Wallis test, p < 0.001). Gd-IgA1 levels were significantly higher in J-IgAN, G-HC, and G-IgAN than in the reference group (J-HC) (Dunn’s correction, p < 0.001, p = 0.005, and p < 0.001, respectively). Gd-IgA1, galactose-deficient IgA1; J-HC, Japanese healthy control; J-IgAN, Japanese patients with IgAN; G-HC, Greek healthy control; G-IgAN, Greek patients with IgAN; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques:

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Comparison of desialylated IgA1 HR O -glycoforms between healthy controls (HCs) and patients with IgA nephropathy (IgAN) in Japanese and Greek cohorts (A) IgA1 HR O -glycoforms of the Japanese cohort. (B) IgA1 HR O -glycoforms of the Greek cohort. The medians of relative abundance (%) in each O -glycoform are represented by black bars. Relative abundance of IgA1 HR with 3GalNAc3Gal increased significantly, whereas that of 5GalNAc4Gal decreased significantly in J-IgAN compared with that in J-HC (Mann-Whitney test, p < 0.001 and Student’s t test, p = 0.040, respectively). In the Greek cohort, the relative abundance of IgA1 HR with 3GalNAc2Gal and 5GalNAc3Gal was significantly higher (Student’s t test, p = 0.008 and Mann-Whitney test, p = 0.043, respectively) and that of 6GalNAc4Gal and 6GalNAc5Gal was significantly lower in G-IgAN than in G-HC (Student’s t test, p = 0.006 and = 0.001, respectively). J-HC, Japanese-HCs; J-IgAN, Japanese patients with IgAN; G-HC, Greek HCs; G-IgAN, Greek patients with IgAN; ∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Representative mass spectra of the desialylated tryptic fragments of IgA1 HR O -glycoforms acquired from Japanese (A) and Greek (B) patients with IgAN The monoisotopic m/z value of the HR O -glycopeptide ions and the number of sugar moieties assigned are shown above the individual peaks. The HR O -glycoforms, the levels of which were higher in Japanese patients than in Greek patients, are represented by upward arrows above the individual peaks in the mass spectra of Japanese patients. The HR O -glycoforms, the levels of which were elevated in Greek patients than in Japanese patients, are represented by upward arrows in the mass spectra of the Greek patients. Comparison of two groups was performed using Student’s t test or Mann-Whitney test depending on whether the variables were distributed normally. ∗, 0.01

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: IgA1 glycoforms expressed based on a specific monosaccharide per HR (A) Relative abundance of IgA1 HR peptide with 3 GalNAc residues. This HR glycoform was higher in J-IgAN and G-IgAN than in the reference group (J-HC) based on Dunn’s multiple comparison test (p = 0.008 and p = 0.001, respectively). (B) Relative abundance of IgA1 HR peptide with 4 GalNAc residues. This HR glycoform was higher in G-IgAN than in J-HC (Dunnett’s correction p = 0.017). (C) Relative abundance of IgA1 HR peptide with 5 GalNAc residues. (D) Relative abundance of IgA1 HR peptide with 6 GalNAc residues. This HR glycoform was lower in G-IgAN than in J-HC (Dunnett’s correction p < 0.001). (E) Mean number of GalNAc per HR. The levels were lower in G-IgAN than in J-HC (Dunn’s correction p = 0.003. (F) Mean number of Gal per HR. (G) Mean number of Gd-glycan per HR. The data are shown in the scatter dot plot (with line drawn at the median). GalNAc, N -acetylgalactosamine; Gal, galactose; Gd-glycan, galactose-deficient-glycan; HR, hinge region; J-HC, Japanese healthy controls; J-IgAN, Japanese patients with IgAN; G-HC, Greek healthy controls; G-IgAN, Greek patients with IgAN. ∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques:

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet:

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: Software

Journal: Cell

Article Title: Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

doi: 10.1016/j.cell.2021.01.035

Figure Lengend Snippet:

Article Snippet: Five-fold serial dilutions of polyclonal macaque (Molecular Innovations) or human IgG, starting at a concentration of 1 μg/mL, were added to wells containing the coated goat anti-Human IgG λ and κ.

Techniques: Purification, Recombinant, Mass Spectrometry, Sequencing, Ligation, Luciferase, Enzyme-linked Immunospot, Plasmid Preparation, Software, Chromatography, Luminex, Expressing

Journal: Current research in biotechnology

Article Title: A point-of-care assay for alpha-1-acid glycoprotein as a diagnostic tool for rapid, mobile-based determination of inflammation

doi: 10.1016/j.crbiot.2019.09.002

Figure Lengend Snippet: (A) Representative images of test strips demonstrating varying T/C intensities at different concentrations of purified human AGP. (B) Representative images of negative control test strips demonstrating C line signal, but no T line signal. (C) Calibration curve of T/C obtained from the lateral flow assay against known concentrations of purified human AGP. Data are mean ± SEM.

Article Snippet: Antibodies included affinity purified goat anti-Human AGP (Lee Biosolutions, Maryland Heights, MO) and rabbit anti-goat IgG (Millipore Sigma, Burlington, MA).

Techniques: Purification, Negative Control, Lateral Flow Assay

Journal: Journal of Translational Medicine

Article Title: Eosinophils affect functions of in vitro-activated human CD3-CD4+ T cells

doi: 10.1186/1479-5876-11-112

Figure Lengend Snippet: Eosinophils enhance proliferation and CD25 expression of in vitro activated CD4 T-cells from healthy controls. Purified CD4 + T-cells from healthy subjects were activated with coated anti-CD3 and soluble anti-CD28 (1 μg/ml) antibodies for 48 hours, in absence and in presence of autologous IgA/anti-IgA activated eosinophils. Cells were then cultured for an additional 18 hours with H 3 -thymidine to assess proliferation (n=8) (2 a ), washed and stained with fluoro-conjugated antibodies for assessment of membrane CD25 expression by flow cytometry (n=6) (2 b ), or underwent brief re-stimulation with PMA and A23187 to assess intracellular cytokine expression (n=10) (2 c ).

Article Snippet: Eosinophils were resuspended in RPMI-FCS at 2×10 6 cells per ml, and activated in vitro for 18 hours (37°C, 5% CO 2 ) prior to co-incubation with CD4 T-cells, using human IgA (7,5 μg/ml)(Sigma-Aldrich, human IgA from colostrum), and affinity-purified goat anti-human IgA (10 μg/ml) (Gentaur).

Techniques: Expressing, In Vitro, Purification, Cell Culture, Staining, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Eosinophils affect functions of in vitro-activated human CD3-CD4+ T cells

doi: 10.1186/1479-5876-11-112

Figure Lengend Snippet: Eosinophils inhibit dendritic cell-induced activation of CD3 - CD4 + T cells in vitro. Purified CD3 - CD4 + T cells were cultured in presence of LPS-matured dendritic cells from healthy subjects for 5 days, in absence or in presence of IgA/anti-IgA-activated eosinophils. Proliferation was assessed on the basis of H 3 -thymidine incorporation (3 a ), IL-5 was measured in culture supernatants by ELISA (3 b ), and expression of activation markers CD25 and HLA-DR was assessed by flow cytometry (3 c ). Histograms represent mean proliferation (cpm) (3a, n=8) and IL-5 concentrations (pg/ml) (3b, n=6), and bars show the standard error of the mean for each condition. For cytometry studies (3 c ), histograms show CD25 and HLA-DR expression on CD3 - CD4 + T cells cultured alone (continuous fine line), with dendritic cells (dashed bold line), and with both dendritic cells and eosinophils (continuous bold line); results are representative of 3 independent experiments.

Article Snippet: Eosinophils were resuspended in RPMI-FCS at 2×10 6 cells per ml, and activated in vitro for 18 hours (37°C, 5% CO 2 ) prior to co-incubation with CD4 T-cells, using human IgA (7,5 μg/ml)(Sigma-Aldrich, human IgA from colostrum), and affinity-purified goat anti-human IgA (10 μg/ml) (Gentaur).

Techniques: Activation Assay, In Vitro, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry