Annette S. H. Gouw

Nomenclature of the finer branches of the biliary tree: Canals, ductules, and ductular reactions in human livers

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field. (HEPATOLOGY 2004; 39:1739–1745.)

Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

Ductular reactions in human liver: Diversity at the interface

Interest in hepatic ductular reactions (DRs) has risen in recent years because of a greater appreciation of their potential roles in regeneration, fibrogenesis, and carcinogenesis. However, confusion exists because there is significant, but often unappreciated diversity at the tissue, cellular, and subcellular levels in DRs of different diseases and stages of disease. DRs are encountered in virtually all liver disorders in which there is organ‐wide liver damage and cell loss, but are also present in focal lesions such as focal nodular hyperplasia and adenoma. Moreover, diverse DR phenotypes can be present within any single disease entity, and are shaped by the etiology and evolution of the disease. Although much remains to be clarified, recent studies suggest that the diversity of appearances of the DRs are likely to reflect the differing signals at the anatomic, cellular, and molecular levels driving the proliferative response. These appear to determine the relative proportions of transit‐amplifying cells, the degree of hepatocytic or cholangiocytic differentiation, and their relationships with stromal, vascular, and inflammatory components. The molecular signaling pathways governing these regenerative fate decisions closely replicate those found in human and other vertebrate embryos and more generally in stem cell niches throughout the body. Like the latter, complex interactions with matrix as well as mesenchymal and inflammatory cells, vessels, and innervation are likely to be of fundamental importance. Embracing systems/tissue biological approaches to exploring DRs, in addition to more traditional cellular and molecular biological techniques, will further enhance our understanding and, thereby, we believe potentiate new therapeutic possibilities. (HEPATOLOGY 2011)

Ex vivo Normothermic Machine Perfusion and Viability Testing of Discarded Human Donor Livers

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well‐preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Graft fibrosis after pediatric liver transplantation: Ten years of follow‐up

Previously we reported the presence of portal fibrosis in 31% (n = 84) of the grafts in protocol biopsies 1 year after pediatric liver transplantation (LTx). To assess the natural history of graft fibrosis after pediatric liver transplantation, we extended the analysis of graft histology in follow‐up protocol biopsy specimens obtained 5 and 10 years after transplantation. We correlated histological results with clinical parameters at the time of LTx and during follow‐up, to allow identification of risk factors for the development of fibrosis. From 1 year to 5 years after LTx, the prevalence of fibrosis increased from 31% to 65% (n = 66) but remained stable thereafter (at 10 years, 69%, n = 55). At 10 years after LTx, however, the percentage of patients with severe fibrosis had increased from 10% (at 5 years) to 29%. Of the 69% of children without fibrosis at 1 year post‐transplantation, 64% (n = 39) had developed some degree of fibrosis at 10 years. Fibrosis was strongly related to transplant‐related factors such as prolonged cold ischemia time, young age at the time of transplantation, high donor/recipient age ratio, and the use of partial grafts (P < 0.05). Fibrosis was not significantly related to rejection, chronic hepatitis, or the nature of the immunosuppressive therapy. Conclusion: Biopsies after pediatric LTx show that most grafts developed fibrosis within 5 years. At 10 years after LTx, the graft fibrosis had progressed to severe fibrosis in at least 25% of the patients. Development of fibrosis, starting either before or after the first year post‐LTx, was strongly related to transplant‐related factors, indicating the importance of these factors to long‐term graft prognosis. (HEPATOLOGY 2008.)

Nonanastomotic biliary strictures after liver transplantation, part 2: Management, outcome, and risk factors for disease progression

Nonanastomotic biliary strictures (NAS) after orthotopic liver transplantation (OLT) are associated with high retransplant rates. The aim of the present study was to describe the treatment of and identify risk factors for radiological progression of bile duct abnormalities, recurrent cholangitis, biliary cirrhosis, and retransplantation in patients with NAS. We retrospectively studied 81 cases of NAS. Strictures were classified according to severity and location. Management of strictures was recorded. Possible prognostic factors for bacterial cholangitis, radiological progression of strictures, development of severe fibrosis/cirrhosis, graft survival, and patient survival were evaluated. Median follow‐up after OLT was 7.9 years. NAS were most prevalent in the extrahepatic bile duct. Twenty‐eight patients (35%) underwent some kind of interventional treatment, leading to a marked improvement in biochemistry. Progression of disease was noted in 68% of cases with radiological follow‐up. Radiological progression was more prevalent in patients with early NAS and one or more episodes of bacterial cholangitis. Recurrent bacterial cholangitis (>3 episodes) was more prevalent in patients with a hepaticojejunostomy. Severe fibrosis or cirrhosis developed in 23 cases, especially in cases with biliary abnormalities in the periphery of the liver. Graft survival, but not patient survival, was influenced by the presence of NAS. Thirteen patients (16%) were retransplanted for NAS. In conclusion, especially patients with a hepaticojejunostomy, those with an early diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree, are at risk for progressive disease with severe outcome. Liver Transpl 13:725–732, 2007.

EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS AND REPLACEMENT OF DONOR CELLS BY RECIPIENT ONES IN HUMAN-LIVER GRAFTS

The disappearance of certain cell populations of donor origin and their replacement by recipient-specific cells constitutes a possible explanation for the relatively mild course of acute rejection despite lack of MHC compatibility in human orthotopic liver transplantation (OLT). In the present report, graft biopsies of 12 OLT patients from a total of 42 patients were studied for expression of MHC antigens after transplantation using monoclonal antibodies to HLA-ABC and HLA-DR. The patients were selected based upon donor-recipient mismatching for HLA-A2, B7, Drw52, or DQw1. Monoclonal antibodies to these 4 polymorphic HLA antigens and monoclonal antibodies to HLA-ABC and -DR were applied to frozen tissue sections and visualized using an immunoperoxidase technique. Expression of HLA-ABC and -DR on, respectively, hepatocytes and bile duct epithelium were observed in posttransplant graft conditions such as viral infections, cholangitis, and acute rejection. However, no specific pattern of MHC antigen distribution was observed for these various pathological graft conditions. Disappearance of DR-positive Kupffer cells of donor origin and immigration of recipient ones was encountered in the early posttransplant biopsies. This Kupffer cell replacement coincided with a reversible episode of acute rejection. The disappearance of highly immunogenic cellular components as HLA-DR positive Kupffer cells of graft origin may be one of the mechanisms contributing to the mild rejection response observed in human liver transplantation.

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.

RESPIRATORY VIRAL-INFECTIONS AGGRAVATE AIRWAY DAMAGE CAUSED BY CHRONIC REJECTION IN RAT LUNG ALLOGRAFTS

Airway damage resulting in bronchiolitis obliterans occurs frequently in patients after heart-lung and lung transplantation. Generally, chronic rejection is assumed to be the most important cause of bronchiolitis obliterans. However, viral infections might also be potential causes of airway damage after lung transplantation. In the present study, we investigated whether viral infections could induce airway damage in rat lung transplants in the absence or presence of chronic rejection. We compared the histopathology of the airways in 3 groups of rats: (1) nontransplanted LEW lungs, (2) LEW-to-LEW syngeneic lung transplants, and (3) BN-to-LEW allogeneic lung transplants. Nontransplanted and transplanted rats were treated with CsA to induce permanent graft acceptance of the allografts. Six months after transplantation, 4 noninfected rats of each group were killed for histological investigation (another 4 noninfected allografted rats were killed 56 days later). The remaining 16 rats in each group were infected with Sendai virus (parainfluenza type 1) intratracheally. These rats were killed for histological investigation 4, 7, 21, and 56 days after infection. In the lungs of the noninfected rats of the nontransplanted and syngeneically transplanted groups, airway changes were absent. After viral infection in these lungs, mild inflammation developed in the airways that was transient and completely resolved by day 56 after infection. In contrast, in the allogeneically transplanted lungs the viral infection caused severe and permanent damage of the airways. In the bronchioles and the large airways throughout the allogeneic lung transplants, inflammation with epithelial necrosis and formation of granulation tissue was present. On day 56 after infection, the bronchioles showed scarring in the submucosa and obliteration of the lumen, typical features of bronchiolitis obliterans. This study shows that a respiratory viral infection aggravates the airway damage in rat lung allografts with chronic rejection. The findings suggest that viral infections and chronic rejection play a synergistic role in the development of bronchiolitis obliterans after human heart-lung and lung transplantation: the virus infection may stimulate chronic rejection and rejection may hamper the local defense against the virus.