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Background: Vascularized composite allograft transplantation is a treatment option for complex tissue injuries; however, ischemia reperfusion injury and high acute rejection rates remain a challenge. Hypothermic machine perfusion using acellular storage perfusate is a potential solution. This study evaluated the University of Wisconsin Kidney Preservation Solution-1 (KPS-1) compared with normal saline (NS) for preservation of donor rat hindlimbs subjected to 24 h of ex vivo perfusion cold storage. Methods: Hindlimbs were subjected to 24-h perfusion cold storage with heparinized KPS-1 (nâ =â 6) or heparinized NS (nâ =â 6). Flow, resistance, and pH were measured continuously. At the end of the 24-h period, tissue was collected for histological analysis of edema and apoptosis. Results: KPS-1 perfused limbs showed significantly less edema than the NS group, as evidenced by lower limb weight gain (Pâ <â 0.001) and less interfascicular space (Pâ <â 0.001). KPS-perfused muscle had significantly less cell death than NS-perfused muscle based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (Pâ <â 0.001) and cleaved caspase-3 staining (Pâ =â 0.045). During hypothermic machine perfusion, a significant decrease in pH over time was detected in both groups, with a significantly greater decline in pH in the KPS-1 group than in the NS group. There were no significant differences overall and over time in flow rate or vascular resistance between the KPS and NS groups. Conclusions: Perfusion with KPS-1 can successfully extend vascularized composite allograft perfusion cold storage for 24 h in a rat hindlimb model without significant edema or cell death.
RESUMO
BACKGROUND AND AIMS: The etiology of biliary atresia (BA) is not known and is likely multifactorial, including a genetic predisposition, a viral or environmental trigger, an aberrant autoimmune response targeting cholangiocytes, and unique susceptibilities of the neonatal bile ducts to injury. Damaged cholangiocytes may express neo self-antigens and elicit autoreactive T-cell-mediated inflammation and B-cell production of autoantibodies. The aim of this study was to discover autoantibodies in BA that correlated with outcomes. APPROACH AND RESULTS: An autoantigen microarray encompassing approximately 9,500 autoantigens was used to screen for serum immunoglobulin M (IgM) and immunoglobulin G (IgG) autoantibodies in patients with BA or other liver disease controls. Validation of candidate autoantibodies by enzyme-linked immunosorbent assay on a second cohort of subjects (6-12 months following Kasai portoenterostomy) and correlations of autoantibodies with outcomes were performed (serum bilirubin levels and need for liver transplant in first 2 years of life). Mean anti-chitinase 3-like 1 (CHI3L1), anti-delta-like ligand (DLL-4), and antisurfactant protein D (SFTPD) IgM autoantibodies in BA were significantly higher compared with controls, and IgM autoantibody levels positively correlated with worse outcomes. Immunofluorescence revealed cholangiocyte-predominant expression of CHI3L1, DLL-4, and SFTPD. The humoral autoantibody response was associated with C3d complement activation and T-cell autoimmunity, based on detection of cholangiocyte-predominant C3d co-staining and peripheral blood autoreactive T cells specific to CHI3L1, DLL-4 and SFTPD, respectively. CONCLUSIONS: BA is associated with cholangiocyte-predominant IgM autoantibodies in the first year after Kasai portoenterostomy. Anti-CHI3L1, anti-DLL-4, and anti-SFTPD IgM autoantibody correlations with worse outcomes and the detection of C3d on cholangioctyes and antigen-specific autoreactive T cells suggest that autoimmunity plays a role in the ongoing bile duct injury and progression of disease.