Journal: Cell reports

Article Title: Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

doi: 10.1016/j.celrep.2022.111775

Figure Lengend Snippet: (A) Targeting strategy for the SERPINA1 (AAT) locus. (B) Schematic of directed differentiation protocol for generating iHeps. (C) Representative flow cytometry plots of fixed, permeabilized ZZ, MZ, and MM iHeps. (D) MFI of intracellular AAT protein in ZZ, MZ and MM iHeps. (E and F) Immunostaining of ZZ, MZ, and MM iHeps for AAT, 2C1, and HNF4α; scale bar, 10 μm. (G and H) Secreted total (G) AAT and (H) ZAAT protein in ZZ, MZ, and MM iHep supernatants. (I) Assay of anti-neutrophil elastase inhibition in concentrated iHep supernatants. (J) Representative quantification of AAT secretion kinetics using 35 S-Met/Cys-labeled AAT protein from intracellular protein lysates and supernatants. (K) Kinetic of aggregated AAT-labeled cell lysate and supernatants from (J). n = 3 independent experiments from each of the syngeneic backgrounds (D and G–I). n = 1 independent experiment from each of the syngeneic backgrounds (K). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test using MZ as control. Neutrophil elastase inhibition ZZ versus MZ **p < 0.01, ZZ versus MM ### p < 0.001, MZ versus MM $$$ p < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Mouse monoclonal anti-AAT polymers (clone 2C1) , Hycult Biotech , HM2289.

Techniques: Flow Cytometry, Immunostaining, Inhibition, Labeling

Journal: Cell reports

Article Title: Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

doi: 10.1016/j.celrep.2022.111775

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-AAT polymers (clone 2C1) , Hycult Biotech , HM2289.

Techniques: Recombinant, Electron Microscopy, Lysis, Lactate Assay, Pyruvate Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Sequencing, Mutagenesis, Software, Modification

Journal: Biomolecules

Article Title: The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients

doi: 10.3390/biom12030380

Figure Lengend Snippet: Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.

Article Snippet: Membranes were blocked for 1 h with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4 °C with specific primary antibodies: mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), or mouse monoclonal anti-AAT polymer antibody (LG96, 1:500).

Techniques: Diffusion-based Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients

doi: 10.3390/biom12030380

Figure Lengend Snippet: Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 ( A ) and 2C1 ( B )(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.

Article Snippet: Membranes were blocked for 1 h with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4 °C with specific primary antibodies: mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), or mouse monoclonal anti-AAT polymer antibody (LG96, 1:500).

Techniques: Western Blot, Purification, Isolation, Positive Control