Evidence for Recipient-Derived Cells in Peribiliary Glands and Biliary Epithelium of the Large Donor Bile Ducts After Liver Transplantation.

INTRODUCTION: Chimerism after orthotopic liver transplantation (OLT) has largely been investigated in intrahepatic cellular constituents. However, little is known about chimerism in the extrahepatic and large intrahepatic bile ducts. Our aim was to evaluate the presence and extent of chimerism after OLT in the peribiliary glands (PBG) and the luminal epithelium of the large donor bile ducts. METHODS: For this study, we examined six extrahepatic and large intrahepatic bile ducts from livers that were re-transplanted. In all cases there was a sex-mismatch between donor and recipient (female donor organ and male recipient), which allowed to discriminate between donor- and recipient-derived cells. Specimens from female to female transplants were used as negative controls and male to male transplants as positive controls. Fluorescence in situ hybridization (FISH) for Y and X chromosomes was performed and the percentage of XY positive cells was determined among biliary epithelial cells. Immunohistochemistry was used to correlate chimerism with histological features. RESULTS: Cholangiocellular chimerism in all studied specimens ranged from 14 to 52%. The degree of chimerism was not associated with biliary damage. Marked chimerism was present at 5 days post-OLT. Ki-67-positivity was detected in 1-8% of the epithelial cells at the time of liver re-transplantation, and this correlated inversely with the degree of chimerism. CONCLUSION: Recipient-derived cholangiocytes are present in the large bile ducts of the donor liver after OLT. The presence of chimerism in the large bile ducts suggests that recipient-derived cells may play a role in biliary regeneration following ischemia-induced injury during OLT.

Normothermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor livers.

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non-DCD livers. DCD and non-DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma-glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP-preserved livers compared to SCS-preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2-fold higher in NMP-preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP-preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non-DCD and DCD livers, respectively) compared with SCS-preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non-DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP-preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994-1005 2016 AASLD.

High peak alanine aminotransferase determines extra risk for nonanastomotic biliary strictures after liver transplantation with donation after circulatory death.

Orthotopic liver transplantation (OLT) with donation after circulatory death (DCD) often leads to a higher first week peak alanine aminotransferase (ALT) and a higher rate of biliary nonanastomotic strictures (NAS) as compared to donation after brain death (DBD). This retrospective study was to evaluate whether an association exists between peak ALT and the development of NAS in OLT with livers from DBD (n = 399) or DCD (n = 97) from two transplantation centers. Optimal cutoff value of peak ALT for risk of development of NAS post-DCD-OLT was 1300 IU/l. The 4-year cumulative incidence of NAS after DCD-OLT was 49.5% in patients with a high ALT peak post-OLT, compared with 11.3% in patients with a low ALT peak. (P < 0.001). No relation between peak ALT and NAS was observed after DBD-OLT. Multivariate analysis revealed peak ALT ≥1300 IU/l [adjusted hazard ratio (aHR) = 3.71, confidence interval (CI) (1.26-10.91)] and donor age [aHR = 1.04, CI 1.00-1.07] to be independently associated with development of NAS post-DCD-OLT. A peak ALT of <1300 IU/l carries a risk for NAS similar to DBD-OLT. Thus, in DCD-OLT, but not in DBD-OLT, peak ALT discriminates patients at high or low risk for NAS.

Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.

BACKGROUND: Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model. METHODS: After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system. RESULTS: After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024). CONCLUSIONS: Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts.

Duct-to-duct reconstruction in liver transplantation for primary sclerosing cholangitis is associated with fewer biliary complications in comparison with hepaticojejunostomy.

There is no consensus on the preferred type of biliary reconstruction for patients undergoing orthotopic liver transplantation (OLT) for primary sclerosing cholangitis (PSC). The aim of this study was to compare long-term outcomes after OLT for PSC using either duct-to-duct anastomosis or Roux-en-Y hepaticojejunostomy for biliary reconstruction. In a consecutive series of 98 adult patients undergoing OLT for PSC, 45 underwent duct-to-duct reconstruction, and 53 underwent Roux-en-Y biliary reconstruction. The median follow-up was 8.2 years (interquartile range = 3.9-14.5 years). The outcomes of the 2 groups were compared. There were no significant differences in patient demographics or general surgical variables between the groups. The overall patient and graft survival rates were similar for the 2 groups. The incidence of biliary strictures and biliary leakage within the first year after transplantation did not differ between the 2 groups. However, significantly more patients in the Roux-en-Y group suffered at least 1 episode of cholangitis within the first year (9% in the duct-to-duct group versus 25% in the Roux-en-Y group, P = 0.04). In addition, Roux-en-Y reconstruction was associated with a significantly higher rate of late-onset (>1 year after transplantation) nonanastomotic biliary strictures (NAS) in comparison with duct-to-duct reconstruction (24% versus 7% at 5 years and 30% versus 7% at 10 years, P = 0.01). In conclusion, duct-to-duct biliary reconstruction in patients with PSC is associated with lower rates of posttransplant cholangitis and late-onset NAS in comparison with Roux-en-Y hepaticojejunostomy. If technically and anatomically feasible, duct-to-duct anastomosis can be performed safely in patients undergoing OLT for PSC.

Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.

BACKGROUND & AIMS: Although regeneration of intrahepatic bile ducts has been extensively studied and intrahepatic progenitor cells have been identified, few studies have focussed on the extrahepatic bile duct (EHBD). We hypothesized that local progenitor cells are present within the EHBD of humans. Human EHBD specimens (n = 17) were included in this study. METHODS: Specimens of normal EHBD tissue were obtained from healthy donor livers (n = 6), mildly injured EHBD from patients with cholangitis (n = 6) and severely injured EHBD from patients with ischaemic type biliary lesions (n = 5). Double immunostaining for K19 and the proliferation marker Ki-67 was performed to identify and localize proliferating cells. In addition, immunofluorescent doublestaining using antibodies against K19 and c-Kit was performed to identify and localize cholangiocytes co-expressing putative progenitor cell markers. RESULTS: In normal EHBD, few Ki-67(+) cells were detected, whereas large numbers of Ki-67(+) were found in the diseased EHBD. In EHBD affected by cholangitis, Ki-67(+) cells were mainly located in the basal layer of the lumen. EHBD specimens from patients with ischaemic type biliary lesions displayed histological signs of epithelial cell loss and large numbers of Ki-67(+) cells were observed in the peribiliary glands. C-Kit expression was localized throughout the EHBD wall and immunofluorescent doublestaining identified a few K19(+) /c-Kit(+) cells in the luminal epithelium of the EHBD as well as in the peribiliary glands. CONCLUSIONS: These findings support the hypothesis that progenitor cells exist in the EHBD and that the peribiliary glands can be considered a local progenitor cell niche in the human EHBD.

Protection of bile ducts in liver transplantation: looking beyond ischemia.

Biliary complications, especially nonanastomotic biliary strictures (NAS), are a major cause of morbidity after orthotopic liver transplantation. Of all donor and recipient characteristics known to increase the risk of developing NAS, the role of prolonged ischemia times is most extensively described in the literature. However, there is increasing evidence that several other, non-ischemia-related factors play a critical role in the pathogenesis of NAS as well. The clinical presentation of NAS may vary considerably among liver transplant recipients, including large variations in time of occurrence, and in location and severity of the strictures. Additional underlying causes such as bile salt toxicity and immune-mediated injury are believed to explain the wide spectrum of biliary strictures after orthotopic liver transplantation. Current and emerging insight in the pathogenesis of NAS and potential targets to reduce biliary injury and preserve bile ducts are discussed in this overview.