Dhawan Lab, Institute of Liver Studies
King's College London
Suppression of the innate immune response using cytoptopic therapeutic agents to prevent early cell loss following hepatocyte transplantation
Charlotte Lee1, Ragai R. Mitry1, Paradzai Chitongo2, Simon Walker1, Raquel Fernandez Dacosta1, Celine Filippi1, Sharon Lehec1, Richard A. Smith 3, Anil Dhawan1, Emer Fitzpatrick1.
1Dhawan Lab, Institute of Liver Studies, King's College London, London, United Kingdom; 2Viapath, King's College Hospital, London, United Kingdom; 3MRC Centre for Transplantation, King's College London, London, United Kingdom
Introduction: Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in patients with liver-based metabolic disease or acute liver failure. Long-term success is significantly hindered by early cell loss shortly after transplantation. This is partly due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in activation of complement and coagulation pathways and destruction of transplanted cells. A novel cytotopic therapeutic agent, thrombalexin (PTL004a), a direct inhibitor of thrombin enzyme activity that binds to cell surfaces, has shown local anticoagulant effects and may also affect the complement cascade. We set out to investigate the potential of thrombalexin to overcome the IBMIR in the context of hepatocyte transplantation.
Methods: Primary human hepatocytes were treated with thrombalexin (1-5µM) for 20 min at 4°C. Immunofluorescence microscopy and FACS were used to measure fluorescein-conjugated thrombalexin binding to the lipid membrane. Effects of thrombalexin on hepatocyte viability and function were determined by MTT, albumin and urea assays. A Chandler loop model using heparin-coated PVC tubing was incubated at 37°C and rotated at 24rpm to mimic portal vein blood flow. This was used to analyse activation of the IBMIR, comparing 5x106 untreated hepatocytes with 5µM thrombalexin treated hepatocytes when in contact with ABO-matched blood. Samples were taken at 0, 15 and 30 min following perfusion and full blood count measured on a haematology analyser. C3a and TAT concentrations were measured using ELISA’s. Thrombin time (TT) and activated partial thromboplastin time (APTT) were measured to further determine the effect on the coagulation of human plasma. Image analysis software was used to analyse the differences in fibrin structure between clots formed in the loops from images taken by scanning electron microscopy (SEM).
Results: Thrombalexin (1-5µM) bound readily to the phospholipid membrane of more than 80% of hepatocytes for 24h (n=3). Viability, urea and albumin production of hepatocytes was not significantly affected at any concentration of thrombalexin (n=6; 1-5µM, P>0.05). The Chandler loop model showed 5x106 hepatocytes elicited a significant drop in platelet count (50 x109 cell/L ± 45) compared to the control (191 x109 cell/L ± 18)(n=3, P<0.05) after 30 min. 5µM thrombalexin treated hepatocytes showed no significant difference in platelet count (127 x109 cell/L ± 50)(n=3, P>0.05) compared to the control, suggesting thrombalexin inhibited coagulation activation. Hepatocytes significantly increased C3a (794 ng/ml ± 6) and TAT (6.8ng/ml ± 2.7) concentrations compared to the control (480ng/ml ± 61) and (4.25 ± 0.8) respectively (n=3, P<0.001 and P<0.05). TT and APTT significantly increased in plasma containing hepatocytes treated with 5µM thrombalexin (73.6 ± 20.8 and 42.1 ±2.9) compared to the control (20.2 ± .078 and 33.6 ± 1.6)(n=6, P<0.01), further suggesting inhibition of coagulation. SEM images revealed thrombalexin treated hepatocytes produced clots with a significant increase in mean pore area (35.7µM2 ± 4.6) compared to clots containing hepatocytes only (16.4 µM2 ± 1.4)(P<0.01).
Conclusion: Results suggest treating hepatocytes with thrombalexin before transplantation may provide local inhibition of coagulation, improving cell engraftment and the success of the technique.
NIHR Biomedical Research Centre at Guys and St Thomas' Foundation Trust.
11:00 - 12:30
|Hepatocyte Transplantation||Suppression of the innate immune response using cytoptopic therapeutic agents to prevent early cell loss following hepatocyte transplantation||Room 111-112|