Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC

Nobuyoshi Fukushima, Greg Nalbandian, Judith A Van de Water, Kandra White, Aftab A. Ansari, Patrick S Leung, Thomas Kenny, Shizuo G. Kamita, Bruce D. Hammock, Ross L. Coppel, Freida Stevenson, Hiromi Ishibashi, M. Eric Gershwin

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Abstract

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pxruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Cα constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further rupport of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.

Original languageEnglish (US)
Pages (from-to)1383-1392
Number of pages10
JournalHepatology
Volume36
Issue number6
DOIs
StatePublished - Dec 1 2002

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Biliary Liver Cirrhosis
Autoantibodies
Immunoglobulin A
Baculoviridae
Polymeric Immunoglobulin Receptors
Epithelial Cells
Monoclonal Antibodies
Transcytosis
Sf9 Cells
Antibodies
Immunoblotting
Sodium Dodecyl Sulfate
Autoimmune Diseases
Insects
Canidae
Liver Diseases
Polyacrylamide Gel Electrophoresis
Mitochondria
Oxidoreductases
Mucous Membrane

ASJC Scopus subject areas

  • Hepatology

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Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC. / Fukushima, Nobuyoshi; Nalbandian, Greg; Van de Water, Judith A; White, Kandra; Ansari, Aftab A.; Leung, Patrick S; Kenny, Thomas; Kamita, Shizuo G.; Hammock, Bruce D.; Coppel, Ross L.; Stevenson, Freida; Ishibashi, Hiromi; Gershwin, M. Eric.

In: Hepatology, Vol. 36, No. 6, 01.12.2002, p. 1383-1392.

Research output: Contribution to journalArticle

Fukushima, N, Nalbandian, G, Van de Water, JA, White, K, Ansari, AA, Leung, PS, Kenny, T, Kamita, SG, Hammock, BD, Coppel, RL, Stevenson, F, Ishibashi, H & Gershwin, ME 2002, 'Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC', Hepatology, vol. 36, no. 6, pp. 1383-1392. https://doi.org/10.1053/jhep.2002.37140
Fukushima, Nobuyoshi ; Nalbandian, Greg ; Van de Water, Judith A ; White, Kandra ; Ansari, Aftab A. ; Leung, Patrick S ; Kenny, Thomas ; Kamita, Shizuo G. ; Hammock, Bruce D. ; Coppel, Ross L. ; Stevenson, Freida ; Ishibashi, Hiromi ; Gershwin, M. Eric. / Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC. In: Hepatology. 2002 ; Vol. 36, No. 6. pp. 1383-1392.
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abstract = "Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pxruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Cα constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6{\%} nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further rupport of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.",
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AU - Ansari, Aftab A.

AU - Leung, Patrick S

AU - Kenny, Thomas

AU - Kamita, Shizuo G.

AU - Hammock, Bruce D.

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AU - Stevenson, Freida

AU - Ishibashi, Hiromi

AU - Gershwin, M. Eric

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AB - Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pxruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Cα constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further rupport of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.

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