Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

BACKGROUND: Antibodies to a cytosolic soluble liver antigen (SLA) are specifically detected in patients with autoimmune hepatitis (AIH). The target of anti-SLA has been identified as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP((Ser)Sec)), through the screening of cDNA libraries. A recent report questioned the identity of tRNP((Ser)Sec )as the real SLA antigen. The latter study identified α-enolase as a major anti-SLA target, through proteomic analysis. METHODS: In an attempt to explain the observed discrepancy we have investigated reactivity of SLA positive sera against α-enolase and tRNP((Ser)Sec )using rat and primate liver homogenate and the recombinant antigens. Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA antibodies were detected by an inhibition ELISA and confirmed by immunoblot using human liver homogenate. Autoantibody reactivity was further evaluated using preparations of primate and rat liver homogenates. Anti-α-enolase antibody reactivity has been tested by immunoblot using recombinant α-enolase. An affinity purified goat polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP((Ser)Sec )antibody reactivity was detected by ELISA or dot blot using recombinant tRNP((Ser)Sec )antigen. RESULTS AND DISCUSSION: The affinity purified IgG antibody directed to human α-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP((Ser)Sec )antibody serum gave a single band of ~50 kDa in both liver preparations. All but one anti-SLA antibody positive sera reacted with a ~50 kDa but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP((Ser)Sec )protein. None of the anti-SLA negative sera reacted with tRNP((Ser)Sec). Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre-incubation of anti-SLA positive sera with tRNP((Ser)Sec )completely abolished the 50 kDa band. The findings of the present study indicate that α-enolase and tRNP((Ser)Sec )are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP((Ser)Sec )– but not anti-α-enolase – correlates with anti-SLA antibody reactivity. CONCLUSION: Our findings indicate that tRNP((Ser)Sec )is the most likely target of anti-SLA

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use. </jats:p