Journal: Frontiers in Pharmacology

Article Title: Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis

doi: 10.3389/fphar.2020.01098

Figure Lengend Snippet: Elevated levels of BPI present in plasma and BAL of people with CF. (A , B) LPS levels in CF plasma and BAL correlated with CF-ABLE score. Quantification of an association between variables was achieved by Spearman correlation. (C , D) Comparative analysis of BPI present in plasma or BAL of Phe508del homozygous PWCF (CF), healthy controls (HC) or NCFB patients was performed by ELISA. (C) BPI levels were significantly increased in plasma of CF compared to HC (n=22 and n=14 subjects per group, respectively, p=0.007, Mann Whitney U-test). (D) BAL levels of BPI were significantly increased in CF (n=5) compared to HC (n=4) or NCFB patients (n=5) (p<0.0001, One-way ANOVA, followed by Bonferroni post-hoc test for selected groups). (E) BAL samples from HC, CF, NCFB or COPD were subjected to SDS-PAGE and Western blot analysis for BPI. An immuno-band of increased intensity for BPI was detected in CF BAL samples (top panels). Lower panel, a control immunoblot to ensure BPI specificity. The blot was halved, with one half probed with secondary antibody only with no BPI immune-bands visible (↑ indicates where blot was cut). Human BPI (hBPI) was used as a positive control. All measurements are means ± SEM from biological replicates.

Article Snippet: Lipopolysaccharide (LPS) was quantified in plasma and BAL of PWCF ( ) using an LPS ELISA (Cusabio: catalog number CSB-E09945h).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, SDS Page, Western Blot, Control, Positive Control

Journal: Frontiers in Pharmacology

Article Title: Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis

doi: 10.3389/fphar.2020.01098

Figure Lengend Snippet: Increased levels of plasma autoantibodies and IgG-bound BPI in CF airway samples. (A) IgG autoantibodies against BPI were quantified in plasma of PWCF (CF, n=30) or healthy controls (HC, n=37). A significant increase in the titre of BPI autoantibodies were detected in CF (p<0.0001, Mann-Whitney U test). Positivity was set as 10 U/ml as indicated by the hatched line. All CF samples were positive for BPI autoantibodies. Increased circulating IgG BPI antibody level in CF individuals determined by ELISA. (B) Plasma samples from HC (n=38), CF (n=28) and CF individuals receiving ivacaftor treatment (n=10). No difference in levels of circulating anti-BPI IgG autoantibodies between CF and CF individuals receiving ivacaftor treatment (p=0.39). (C) Representative Coomassie blue stained SDS gel of purified IgG from CF BAL. Protein G Sepharose was used to isolate IgG from BAL of Phe508del homozygous PWCF. Starting BAL sample (St), unbound material (Un) or purified bound IgG (Bd) with or without DTT reduction are presented. Purified IgG (150 kDa, closed arrow), and the heavy (50 kDa) and light chains (25 kDa) of IgG are indicated (open arrows) (1 representative images of n = 5 biological repeats). (D) Protein G Sepharose was used to isolate IgG-BPI complexes from CF BAL. Reactions were analyzed by immunoblotting for BPI positivity using a mouse monoclonal anti-BPI antibody. Starting BAL sample (St), unbound material (Un) or IgG-bound BPI (Bd) are presented. Human BPI (hBPI) was used as a positive control (55 kDa). A control immunoblot to ensure BPI specificity omitted primary antibody (right hand panel), with no immune-band apparent. 3 representative images of n=5 biological repeats.

Article Snippet: Lipopolysaccharide (LPS) was quantified in plasma and BAL of PWCF ( ) using an LPS ELISA (Cusabio: catalog number CSB-E09945h).

Techniques: Clinical Proteomics, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Staining, SDS-Gel, Purification, Western Blot, Positive Control, Control