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Characterising the association of latency with α(1)-antitrypsin polymerisation using a novel monoclonal antibody.

Tan L, Perez J, Mela M, Miranda E, Burling KA, Rouhani FN, DeMeo DL, Haq I, Irving JA, Ordóñez A, Dickens JA, Brantly M, Marciniak SJ, Alexander GJ, Gooptu B, Lomas DA - Int. J. Biochem. Cell Biol. (2014)

Bottom Line: Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema.In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin.Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK; Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland.

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Distribution patterns of polymeric and latent α1-antitrypsin in liver tissues. Samples analysed in immunohistochemistry were from individuals who were not receiving augmentation therapy. (A) The typical location of polymers. Polymers (PiZZ) were most commonly observed at the periportal zone close to fibrous tissue (left, overview; right, magnified detail), suggesting a relationship between polymers and fibrosis. (B) Comparison of polymer and latent α1-antitrypsin staining. Serial slides (PiZZ) were stained using HRP–DAB, with the 2C1 (Pol) MAb, 1C12 (Lat) MAb, a non-specific generic mouse antibody (Ctrl, mouse IgG) and no primary antibody (Ctrl, no IgG). Images show an overview (left), magnified details of the positive signals (middle) and the control staining (right, separated by double lines). The presence of latent α1-antitrypsin in liver tissues was rare, whilst there were abundant hepatic polymers. (C) In addition to the HRP–DAB method, tissues were stained by the 2C1 MAb (and also 1C12 MAb, data not shown) and rabbit polyclonal antibody to co-localise the polymeric (Pol; green) and total α1-antitrypsin (Total; red), and examined by confocal microscopy. Exemplary image shows a moderate level of polymer staining in a PiZZ sample. (D) There is no staining in the control PiMM individual for polymers (left) or latent α1-antitrypsin (right). n = 30 individuals.
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fig0025: Distribution patterns of polymeric and latent α1-antitrypsin in liver tissues. Samples analysed in immunohistochemistry were from individuals who were not receiving augmentation therapy. (A) The typical location of polymers. Polymers (PiZZ) were most commonly observed at the periportal zone close to fibrous tissue (left, overview; right, magnified detail), suggesting a relationship between polymers and fibrosis. (B) Comparison of polymer and latent α1-antitrypsin staining. Serial slides (PiZZ) were stained using HRP–DAB, with the 2C1 (Pol) MAb, 1C12 (Lat) MAb, a non-specific generic mouse antibody (Ctrl, mouse IgG) and no primary antibody (Ctrl, no IgG). Images show an overview (left), magnified details of the positive signals (middle) and the control staining (right, separated by double lines). The presence of latent α1-antitrypsin in liver tissues was rare, whilst there were abundant hepatic polymers. (C) In addition to the HRP–DAB method, tissues were stained by the 2C1 MAb (and also 1C12 MAb, data not shown) and rabbit polyclonal antibody to co-localise the polymeric (Pol; green) and total α1-antitrypsin (Total; red), and examined by confocal microscopy. Exemplary image shows a moderate level of polymer staining in a PiZZ sample. (D) There is no staining in the control PiMM individual for polymers (left) or latent α1-antitrypsin (right). n = 30 individuals.

Mentions: Liver tissue samples from 30 individuals with five different α1-antitrypsin genotypes (Table 1) and two PiMM control samples were analysed by immunohistochemistry and confocal microscopy. None of these individuals were receiving α1-antitrypsin augmentation therapy. The staining showed that polymers were most commonly located in the periportal zone in close proximity to portal tract fibrous tissue, forming ring-like patterns (Fig. 5A). High power images revealed that hepatocytes containing the greatest concentration of polymers were almost always in close proximity to fibroblasts. Whilst polymer was detected in tissue samples from all individuals who were homozygous or heterozygous for the Z allele (Table 1), only five individuals (all PiZZ) showed positive signals for latent α1-antitrypsin. The latent signals, if present, were only found in PiZZ individuals with advanced liver fibrosis. Comparing the signal intensity and distribution, the latent signals were much more sparse than the polymer signals, as observed in the HRP–DAB staining of serial tissue slides (Fig. 5B). In confocal microscopy, co-localisation of the conformer-specific (polymeric or latent) signal was assessed with the non-selective signal of total α1-antitrypsin stained by a rabbit polyclonal antibody (Fig. 5C).


Characterising the association of latency with α(1)-antitrypsin polymerisation using a novel monoclonal antibody.

Tan L, Perez J, Mela M, Miranda E, Burling KA, Rouhani FN, DeMeo DL, Haq I, Irving JA, Ordóñez A, Dickens JA, Brantly M, Marciniak SJ, Alexander GJ, Gooptu B, Lomas DA - Int. J. Biochem. Cell Biol. (2014)

Distribution patterns of polymeric and latent α1-antitrypsin in liver tissues. Samples analysed in immunohistochemistry were from individuals who were not receiving augmentation therapy. (A) The typical location of polymers. Polymers (PiZZ) were most commonly observed at the periportal zone close to fibrous tissue (left, overview; right, magnified detail), suggesting a relationship between polymers and fibrosis. (B) Comparison of polymer and latent α1-antitrypsin staining. Serial slides (PiZZ) were stained using HRP–DAB, with the 2C1 (Pol) MAb, 1C12 (Lat) MAb, a non-specific generic mouse antibody (Ctrl, mouse IgG) and no primary antibody (Ctrl, no IgG). Images show an overview (left), magnified details of the positive signals (middle) and the control staining (right, separated by double lines). The presence of latent α1-antitrypsin in liver tissues was rare, whilst there were abundant hepatic polymers. (C) In addition to the HRP–DAB method, tissues were stained by the 2C1 MAb (and also 1C12 MAb, data not shown) and rabbit polyclonal antibody to co-localise the polymeric (Pol; green) and total α1-antitrypsin (Total; red), and examined by confocal microscopy. Exemplary image shows a moderate level of polymer staining in a PiZZ sample. (D) There is no staining in the control PiMM individual for polymers (left) or latent α1-antitrypsin (right). n = 30 individuals.
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fig0025: Distribution patterns of polymeric and latent α1-antitrypsin in liver tissues. Samples analysed in immunohistochemistry were from individuals who were not receiving augmentation therapy. (A) The typical location of polymers. Polymers (PiZZ) were most commonly observed at the periportal zone close to fibrous tissue (left, overview; right, magnified detail), suggesting a relationship between polymers and fibrosis. (B) Comparison of polymer and latent α1-antitrypsin staining. Serial slides (PiZZ) were stained using HRP–DAB, with the 2C1 (Pol) MAb, 1C12 (Lat) MAb, a non-specific generic mouse antibody (Ctrl, mouse IgG) and no primary antibody (Ctrl, no IgG). Images show an overview (left), magnified details of the positive signals (middle) and the control staining (right, separated by double lines). The presence of latent α1-antitrypsin in liver tissues was rare, whilst there were abundant hepatic polymers. (C) In addition to the HRP–DAB method, tissues were stained by the 2C1 MAb (and also 1C12 MAb, data not shown) and rabbit polyclonal antibody to co-localise the polymeric (Pol; green) and total α1-antitrypsin (Total; red), and examined by confocal microscopy. Exemplary image shows a moderate level of polymer staining in a PiZZ sample. (D) There is no staining in the control PiMM individual for polymers (left) or latent α1-antitrypsin (right). n = 30 individuals.
Mentions: Liver tissue samples from 30 individuals with five different α1-antitrypsin genotypes (Table 1) and two PiMM control samples were analysed by immunohistochemistry and confocal microscopy. None of these individuals were receiving α1-antitrypsin augmentation therapy. The staining showed that polymers were most commonly located in the periportal zone in close proximity to portal tract fibrous tissue, forming ring-like patterns (Fig. 5A). High power images revealed that hepatocytes containing the greatest concentration of polymers were almost always in close proximity to fibroblasts. Whilst polymer was detected in tissue samples from all individuals who were homozygous or heterozygous for the Z allele (Table 1), only five individuals (all PiZZ) showed positive signals for latent α1-antitrypsin. The latent signals, if present, were only found in PiZZ individuals with advanced liver fibrosis. Comparing the signal intensity and distribution, the latent signals were much more sparse than the polymer signals, as observed in the HRP–DAB staining of serial tissue slides (Fig. 5B). In confocal microscopy, co-localisation of the conformer-specific (polymeric or latent) signal was assessed with the non-selective signal of total α1-antitrypsin stained by a rabbit polyclonal antibody (Fig. 5C).

Bottom Line: Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema.In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin.Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK; Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland.

Show MeSH
Related in: MedlinePlus