Cell-based BSEP trans-inhibition: A novel, non-invasive test for diagnosis of antibody-induced BSEP deficiency

Jan Stindt; Carola Dröge; Elke Lainka; Simone Kathemann; Eva-Doreen Pfister; Ulrich Baumann; Amelie Stalke; Enke Grabhorn; Mohammad Ali Shagrani; Yael Mozer-Glassberg; Jane Hartley; Marianne Wammers; Caroline Klindt; Paulina Philippski; Roman Liebe; Diran Herebian; Ertan Mayatepek; Thomas Berg; Anjona Schmidt-Choudhury; Constanze Wiek; Helmut Hanenberg; Tom Luedde; Verena Keitel

doi:10.1016/j.jhepr.2023.100690

Cell-based BSEP trans-inhibition: A novel, non-invasive test for diagnosis of antibody-induced BSEP deficiency

JHEP Rep. 2023 Feb 1;5(7):100690. doi: 10.1016/j.jhepr.2023.100690. eCollection 2023 Jul.

Authors

Jan Stindt¹, Carola Dröge^{1

2}, Elke Lainka³, Simone Kathemann³, Eva-Doreen Pfister⁴, Ulrich Baumann⁴, Amelie Stalke^{4

5}, Enke Grabhorn⁶, Mohammad Ali Shagrani⁷, Yael Mozer-Glassberg⁸, Jane Hartley⁹, Marianne Wammers¹, Caroline Klindt¹, Paulina Philippski², Roman Liebe¹, Diran Herebian¹⁰, Ertan Mayatepek¹⁰, Thomas Berg¹¹, Anjona Schmidt-Choudhury¹², Constanze Wiek¹³, Helmut Hanenberg^{13

14}, Tom Luedde¹, Verena Keitel^{1

2}

Affiliations

¹ Department of Gastroenterology, Hepatology and Infectious Diseases, Medical Faculty, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
² Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Magdeburg, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany.
³ Department of Pediatrics, Division of Gastroenterology and Hepatology, University Children's Hospital, Essen, Germany.
⁴ Pediatric Gastroenterology and Hepatology, Department for Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany.
⁵ Department of Human Genetics, Hannover Medical School, Hannover, Germany.
⁶ Pediatric Hepatology and Liver Transplantation, Transplantation Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
⁷ Department of Liver & SB Transplant & Hepatobiliary-Pancreatic Surgery, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia.
⁸ Institute of Gastroenterology, Nutrition and Liver Diseases, Schneider Children's Medical Centre of Israel, Petach Tikvah, Israel.
⁹ The Liver Unit Including Small Bowel Transplantation, Birmingham Women's and Children's Hospital, Birmingham, UK.
¹⁰ Department of General Pediatrics, Neonatology and Pediatric Cardiology, Medical Faculty, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
¹¹ Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany.
¹² Department of Pediatrics and Adolescent Medicine, Ruhr University Bochum, Bochum, Germany.
¹³ Department of Otorhinolaryngology, Heinrich Heine University School of Medicine, Düsseldorf, Germany.
¹⁴ Department of Pediatrics III, University Children's Hospital, University of Duisburg-Essen, Essen, Germany.

Abstract

Background & aims: Antibody-induced bile salt export pump deficiency (AIBD) is an acquired form of intrahepatic cholestasis, which may develop following orthotopic liver transplantation (OLT) for progressive familial intrahepatic cholestasis type 2 (PFIC-2). Approximately 8-33% of patients with PFIC-2 who underwent a transplant develop bile salt export pump (BSEP) antibodies, which trans-inhibit this bile salt transporter from the extracellular, biliary side. AIBD is diagnosed by demonstration of BSEP-reactive and BSEP-inhibitory antibodies in patient serum. We developed a cell-based test directly measuring BSEP trans-inhibition by antibodies in serum samples to confirm AIBD diagnosis.

Methods: Sera from healthy controls and cholestatic non-AIBD or AIBD cases were tested (1) for anticanalicular reactivity by immunofluorescence staining of human liver cryosections, (2) for anti-BSEP reactivity by immunofluorescence staining of human embryonic kidney 293 (HEK293) cells expressing BSEP-enhanced yellow fluorescent protein (EYFP) and immunodetection of BSEP-EYFP on Western blot, and (3) for BSEP trans-inhibition using HEK293 cells stably expressing Na⁺/taurocholate cotransporting polypeptide (NTCP)-mCherry and BSEP-EYFP. The trans-inhibition test uses [³H]-taurocholate as substrate and is divided into an uptake phase dominated by NTCP followed by BSEP-mediated export. For functional analysis, sera were bile salt depleted.

Results: We found BSEP trans-inhibition by seven sera containing anti-BSEP antibodies, but not by five cholestatic or nine control sera, all lacking BSEP reactivity. Prospective screening of a patient with PFIC-2 post OLT showed seroconversion to AIBD, and the novel test method allowed monitoring of treatment response. Notably, we identified a patient with PFIC-2 post OLT with anti-BSEP antibodies yet without BSEP trans-inhibition activity, in line with asymptomatic presentation at serum sampling.

Conclusions: Our cell-based assay is the first direct functional test for AIBD and allows confirmation of diagnosis as well as monitoring under therapy. We propose an updated workflow for AIBD diagnosis including this functional assay.

Impact and implications: Antibody-induced BSEP deficiency (AIBD) is a potentially serious complication that may affect patients with PFIC-2 after liver transplantation. To improve its early diagnosis and thus immediate treatment, we developed a novel functional assay to confirm AIBD diagnosis using a patient's serum and propose an updated diagnostic algorithm for AIBD.

Keywords: AIBD; Anti-BSEP antibody; Liver transplantation; Non-invasive diagnostic test; Progressive familial intrahepatic cholestasis type 2.