Diagnosis of de novo autoimmune hepatitis (AIH) after orthotopic liver transplantation (OLT) is challenging especially in the absence of hyper-
De novo autoimmune hepatitis (AIH) after orthotopic liver transplantation (OLT) should be suspected in any unexplained graft dysfunction, in both children and adults transplanted for an indication different from autoimmune liver diseases [
Differential diagnosis with the far more common graft rejection and viral hepatitis is challenging in spite of the diagnostic criteria for de novo AIH proposed in 2006 by the Banff working group [
Both typical and atypical circulating autoantibodies have been found to be associated with de novo AIH. Among the typical ones [
Here, we report for the first time in a child with de novo AIH cytochrome P450 2C19 (CYP-2C19) as a hepatocellular autoantigen of new liver microsomal (LM) autoantibodies. LM against CYP-2C19 are different from the previously described LM autoantibodies directed against CYP-1A2, considered hallmark of AIH in children affected by Type 1 Autoimmune Polyglandular Syndrome (APS 1) [
The case is a 9-year-old boy who developed evidence for de novo AIH, seven years after OLT for biliary atresia. Controls included 50 healthy subjects and 66 nontransplanted patients affected by idiopathic autoimmune liver diseases: 11 with AH-1 (ANA and/or SMA positives), 11 with AH-2 (LKM-1 and/or LC-1 positives), 2 with AH-3 (SLA positives), 26 with Primary Biliary Cholangitis (PBC, AMA positives), and 16 with APS-1 (one LM positive, five LKM positives).
Rat kidney, liver, and stomach tissue sections were used for ANA, SMA, LKM, and AMA detection (SCIMEDX, Denville, New Jersey, USA), and human epithelial type 2 (HEp-2) cells were used for ANA, AMA, and anti-actin antibodies detection (MarDx diagnostics, Carlsbad, California, USA). Briefly, slides were incubated in moist chamber for 30 minutes with serum serial dilutions (from 1 : 40 to 1 : 1280) at room temperature (RT). Then they were washed twice for 5 minutes in phosphate buffer saline (PBS) solution, incubated at RT for 30 minutes with FITC-conjugated goat anti-human sera (IgA + IgG + IgM, Medic, Castello di Pavone, TO, Italy), diluted 1 : 100 in PBS, washed twice as before, and read with a fluorescence microscope (Olympus BX51).
IB experiments were carried out using both rat and human liver tissues. Human liver was obtained during liver transplantation from a reduced size donor liver whose material in excess would be anyway discarded. Microsomal, mitochondrial, and soluble protein fractions from rat and human liver were prepared by differential centrifugations and ultracentrifugations as described previously [
Baculovirus insect cell-expressed human hepatic recombinant cytochrome P450s and UGT1 were purchased from Gentest Corp. (Woburn, MA, USA) [
A 9-year-old boy developed de novo AIH 7 years after liver transplantation for biliary atresia. He was under treatment with cyclosporine (CyA) (trough blood levels of 151
The patient serum was negative for typical ANA, SMA, and LKM autoantibodies associated with AIH. IF analysis showed a novel pattern, involving prevalently the pericentral zone and extending to the mid-lobule zone of the liver (Figure
(a-b) Indirect immunofluorescence (IF) results using the patient serum diluted at 1 : 640 in phosphate buffered saline and overlaid on rat liver (a) and kidney (b) sections (photomicrograph; lens 10x). For immune staining, a goat anti-human Ig FITC-conjugate antiserum was used at 1 : 100 dilution. The new LM antibody staining pattern involved mainly hepatocytes of the pericentral zone ((a), white arrow points to the central vein) and extended to the mid-lobule zone of the liver ((a), white circle, yellow arrows indicate the surrounding negative zone) while sparing the renal proximal tubular cells ((b), white arrows). (c) Immunoblotting results using human liver microsomal proteins separated in PAGE. Lane 1, LKM positive control serum from a patient affected by type 2 idiopathic AIH, main protein band recognized at ~50 kD; lane 2, LM positive serum from the reported case affected by de novo AIH, main protein band recognized at >51 kD; lane 3, atypical LKM positive control serum from a post-OLT patient, no protein band recognized. Markers on the left indicate the molecular weight standards in kilodaltons (kD). (d) Immunoblotting results using six recombinant cytochromes P450. Lanes from the left to the right: CYP-2D6, CYP-2C9, CYP-2C19, CYP-1A2, CYP-2A6, and CYP-3A4. (A) Results observed using the LM positive serum from the reported case affected by de novo AIH, autoantigen recognized: CYP-2C19. (B) Results observed using a LKM positive control serum from a patient affected by type 2 idiopathic AIH, autoantigen recognized: CYP-2D6. Markers on the left indicate the molecular weight standards in kilodaltons (kD).
While no reactivity was observed in experiments with rat liver proteins (data not shown), a positive reaction with a protein band was observed above the 51 kD molecular weight standard in IB experiments performed with the microsomal fraction of human liver (Figure
In order to identify the main target antigen of the novel LM antibodies observed in IF, we performed IB analysis using several commercially available human recombinant CYPs and UGTs (BD Gentest,
Atypical LKM positive sera of our previously described post-OLT patients [
CYP-2C19 was then tested as a potential antigen in 24 control sera from patients with idiopathic AIH, 26 patients with PBC, 16 patients with APS-1, and 50 healthy controls. None of these sera showed a positive reaction with the CYP-2C19 (data not shown).
The precise mechanism underlying de novo AIH is still obscure, and controversy exists about autoimmunity versus alloreactivity for explaining this immune-mediated graft dysfunction that can arise after an allogenic organ transplantation [
Circulating autoantibodies are a common finding after OLT especially in children [
Our case had most of the features of de novo AIH, including increasing serum
To the best of our knowledge, LM in children has hitherto been described only in the context of APS1-related AIH [
Studies investigating LM and LKC autoantibodies with more than one immunological assay in patients affected by AIH of different etiologies. Studies are ordered by year of publication.
Reference | Year | Diagnosis | Ab | Assay: IF on |
Assay other than IF | Target Ag (kD) | Age of patients |
---|---|---|---|---|---|---|---|
Bourdi et al. [ |
1990 | AIH-drug (dihydralazine induced) | LM | Liver [ |
IB, rat [−] |
CYP-1A2 |
Adult |
|
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Sacher et al. [ |
1990 | AIH-APS-1 | LM | Liver [ |
IB, human [ |
CYP-1A2 |
Pediatric |
|
|||||||
Clemente et al. [ |
1997 | AIH-APS-1 | LM | Liver [ |
IB, rat [−] |
CYP-1A2 |
Pediatric |
|
|||||||
Gebre-Medhin et al. [ |
1997 | AIH-APS-1 | LM | Liver [ |
IB, human [ |
CYP-1A2 |
Pediatric |
|
|||||||
Obermayer-Straub [ |
2001 | AIH-APS-1 | LM | Liver [ |
IB human CYPs | CYP 1A2 | Adult and pediatric |
|
|||||||
Aguilera et al. [ |
2001 | Post-OLT AIH | LKC | Liver [ |
Human liver |
GSTT1 |
Adult |
|
|||||||
Salcedo et al. [ |
2002 | Post-OLT AIH | LKC | Liver [ |
ID, rat [+] |
Cytosol Ag not studied | Adult |
|
|||||||
Huguet et al. [ |
2007 | Post-OLT AIH | LKC |
Liver [ |
2D-IB |
Cytosol Ag = GSTT1 |
Adult |
|
|||||||
Meloni et al. [ |
2012 | AIH-APS-1 | LM | Liver [+] |
IB human CYPs | CYP1A2 | Pediatric |
|
|||||||
Clemente et al. | Present study | Post-OLT AIH | LM | Liver [+] |
IB, rat [−] |
CYP-2C19 |
Pediatric |
Ag, antigen; AIH, autoimmune hepatitis; AIH-drug, drug-induced AIH; AIH-APS-1, Type 1 Polyglandular Syndrome associated AIH; CYP, cytochrome P450; GSTT1, glutathione-S-transferase T1; IB, immunoblotting; 2D-IB, two-dimensional immunoblotting; ID, immunodiffusion; IF, immunofluorescence; kD, kilodaltons of Ag molecular weight; LKC, liver kidney cytosol antibody; LM, liver microsomal antibody; LKM, liver and kidney microsomal antibody; post-OLT AIH, postorthotopic liver transplantation “de novo” AIH; Pts, patients.
Similar to what has happened in the CYP-1A2 identification, in the present study, CYP-2C19 could be identified as LM target antigen when IB was performed with human liver microsomes, although the IF was performed on rodent tissue. This apparent discrepancy of LM antibodies has been explained by differences existing between the rat and human CYPs’ structure [
As the target antigen is an enzyme of the cytochrome P450 family, the novel LM antibodies detected in our case are unlikely donor specific antibodies (DSA). DSA react either to donor lymphocytes or to a panel of HLA antigen targets; their production derives from a mismatch in the liver expressed proteins [
A post-OLT graft dysfunction mimicking de novo AIH has also been described when GSTT1 is an alloantigen becoming a
Since about 3% of Caucasians lack CYP-2C19 entirely [
Several human liver cytochrome P450s have been reported as autoantigens of the liver/kidney or liver microsomal (LKM or LM) autoantibodies associated with chronic hepatitis of different etiologies, with very little overlap [
In spite of the continuous progress in discovering new autoantibodies and new autoantigens, etiology and pathogenesis are clear only for the drug-induced forms of AIH. By binding covalently to its specific metabolizing enzyme, the drug creates a new molecule that acts as a neoantigen and triggers the autoimmune response in genetically predisposed individuals [
The present study is the first report on novel LM autoantibodies directed against CYP-2C19 in a child with de novo AIH. Correct information on human versus rat tissue antigens tested by methods other than IF for antibodies characterization may have significant implications for the correct diagnosis and management of patients followed up after OLT.
We were, however, not able to clarify whether the novel LM autoantibodies here described are marker of autoimmune reactions against the “self” or they derived from alloimmune reactions against the “non-self” after OLT. This study represents a pathfinder on this topic and warrants exploration in more extensive future studies.
All the patients and healthy subjects enrolled in this study provided informed consent.
All the authors have declared that no conflicts of interest, real, potential, or perceived, exist.
The first author, Dr. Maria Grazia Clemente, wrote the first draft of the manuscript.
This work is partially supported by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Ministry for the University and Scientific and Technological Research) Rome, Italy, and from the Assessorato Igiene, Sanità e Assistenza Sociale (Councillorship for Hygiene, Health and Social Welfare of the Autonomous Region of Sardinia), Cagliari, Italy.