2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

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.

Hypoxia inducible factor-1-alpha (HIF-1alpha) is related to both angiogenesis and inflammation in rheumatoid arthritis.

OBJECTIVES: Despite the important role of the transcription factor HIF-1alpha in angiogenesis and inflammation, only a few studies on HIF-1alpha expression have been performed in RA patients. The aim of the present study was to identify the layer in synovial tissue of RA patients where HIF1a is expressed and to find out whether HIF-1alpha expression is related to both angiogenesis and inflammation in synovium from RA patients. METHODS: A reproducible staining method for HIF-1alpha was developed. HIF-1alpha -positive cells were quantified in synovial tissue from patients with RA. As control we used synovial tissue from patients with osteoarthritis (OA). The number of HIF-1alpha-positive cells was compared with the number of blood vessels present and was correlated with the amount of inflammation. The amount of inflammation was determined by counting inflammatory cells, by estimating the proliferation marker Ki67 in inflamed tissue, and by using a recently published synovitis score which gives an accurate estimate of the amount of inflammation present. RESULTS: HIF-1alpha was expressed weakly in the lining layer and strongly in the sublining layer in RA synovial tissue. In contrast, HIF-1alpha was only weakly expressed in OA synovial tissue. The number of HIF-1alpha -positive cells correlated strongly with the number of blood vessels in RA synovial tissue and with inflammatory endothelial cell infiltration (blood vessels), cell proliferation (Ki67) and the synovitis score. CONCLUSIONS: HIF-1alpha expression is strongest in the sub-lining layer of RA synovium and is related to both angiogenesis and inflammation in synovium from RA patients. These results thus suggest that HIF-1alpha could serve as an important new therapeutic target in RA, targeting both angiogenesis and inflammation.

Interplay of co-inherited diseases can turn benign syndromes in a deadly combination: haemoglobinopathy and bilirubin transport disorder.

We present a case about a 25-year-old male patient suffering from a rare genetic disorder called Mizuho haemoglobin. He was admitted to the Intensive Care Unit with acute liver and renal failure. During admission he also developed a cardiac tamponade twice. Finally he received a liver transplantation. Hereafter the patient stabilised and his liver and renal functions improved. His symptoms could not be explained solely by his known disease. After searching the literature, similarities between his symptoms and a rare complication of sickle cell disease were found. Molecular diagnostics showed that the patient also suffered from Gilbert's syndrome. Due to his chronic haemolysis, symptoms of this other disease were masked. This stresses the importance of always looking for other causes if symptoms or changes cannot be explained by a known rare disorder.

Distinct phenotypes of infiltrating cells during acute and chronic lung rejection in human heart-lung transplants.

To differentiate between acute and chronic lung rejection in an early stage, phenotypes of infiltrating inflammatory cells were analyzed in 34 transbronchial biopsies (TBBs) of 24 patients after heart-lung transplantation. TBBs were taken during during acute lung rejection and chronic lung rejection, as diagnosed by clinical data and histopathological investigation. TBBs without rejection and normal lung tissue specimens served as controls. Distinct phenotypes of inflammatory cells were found in acute and chronic lung rejection. T cells were present both in acute and in chronic rejection, but did not differentiate between them. In contrast, B cells with antibody deposition were mainly present in chronic rejection and not in acute rejection. Activated macrophages were present only in acute rejection and not in chronic rejection. In nonrejecting lung transplants, perivascular infiltrating cells were virtually absent. In the biopsy specimen, vessels had to be available for analysis, because the cell phenotypes were best recognized in perivascular infiltrates. The analysis of specific phenotypes of inflammatory cells by immunohistochemistry supports the diagnosis of acute and chronic lung rejection, in particular in those cases in which TBB provides limited tissue without airways.

Azathioprine and prednisolone immunosuppression versus maintenance triple therapy including cyclosporine for orthotopic liver transplantation. A comparative biochemical and histologic study in one center.

Improvement of graft survival after orthotopic liver transplantation is often attributed to cyclosporine. In order to assess the effects on liver function and histology, we compared the results of conventional immunosuppression (azathioprine/prednisolone = group I) and a triple drug regimen, which included CsA (group II) during the first year after transplantation. Group I consisted of 33 patients; group II of 18 patients. Significant differences are present in favor of the CsA regimen with regard to transaminases and cholestatic parameters. Liver synthesis function was slightly better, though already very good under conventional immunosuppression. One week after transplantation, normal histology was not observed in group I, while 90% of the patients showed acute rejection. In group II, 53% of the patients showed normal histology; only 40% of the patients in group II showed acute rejection (P less than 0.0002). One year after transplantation, liver histology was normal in 57% of the conventionally treated patients and in 90% of the CsA-treated patients. Also, less rejection occurred in group II during the first year after transplantation. One-year graft survival was 67% in group I and in group II 75%, which is not statistically different. Creatinine clearance did not differ in both groups. However compared with pretransplantation creatinine clearances, kidney function in the CsA-treated patients decreased with approximately 20 ml/min. These results show that liver synthesis and liver function are better under the CsA-containing triple-drug-maintenance regimen, which is supported by the far better liver histology. Kidney function is reduced, even under low dose CsA treatment.

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.

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.

Predictive factors for portal fibrosis in pediatric liver transplant recipients.

BACKGROUND: Recent histopathological studies showed an unexpected high incidence of pathological changes in asymptomatic survivors after pediatric liver transplantation. The aim of this study was to analyze the occurrence of histological abnormalities, to assess the clinical significance, and to identify predictive factors for these pathological changes. METHODS: The first annual protocol graft biopsies of 84 consecutive liver transplants were analyzed and correlated with concomitant liver function tests. Identification of predictive factors for the histological abnormalities in the biopsies was performed by a multivariate logistic regression analysis. RESULTS: The incidence of portal fibrosis (PF) was 31%. Liver function tests showed except for the albumin level, an increase in the PF group compared with the group without PF. Mean values of alkaline phosphatase and direct bilirubin were 264 U/liter and 3 micromol/liter, respectively, in the normal group, and 435 U/liter and 23 micromol/liter, respectively, in the PF group (P=0.043 and 0.037). Eight of 19 univariantly tested variables were entered into a logistic regression model: cold ischemia time, preservation solution, type of allograft, cytomegalovirus recipient status, type of biliary reconstruction, biliary complications, graft complications, and rejection. A significant positive correlation with PF was found for cold ischemia time, biliary complications, and cytomegalovirus status. Acute rejection showed a negative correlation. CONCLUSIONS: The incidence of PF within 1 year post liver transplantation was 31%. This finding was accompanied by cholestatic liver function test abnormalities. Factors predisposing to PF were a prolonged cold ischemia time, biliary complications, and a positive cytomegalovirus recipient status. Acute rejection seemed to prevent for PF.

Acute rejection in human liver grafts: a comparative histologic study of cases maintained on azathioprine and prednisone versus cyclosporine A and low-dose steroids.

The morphology of acute rejection (AR) in biopsies of liver allografts obtained in the first 2 weeks after transplantation was analyzed. Material from patients maintained on azathioprine and prednisone (AZA; Groningen, The Netherlands) was compared with that of patients receiving cyclosporine A and prednisone (with or without azathioprine) in low doses (CSA; Minneapolis). Strict selection criteria were applied to exclude circulatory and biliary complications and viral infection in this early observation period after transplantation. Follow-up biopsies ranged from 3 weeks to 1 year after transplantation. Time zero biopsies and/or pretransplant biopsies served as baseline histology, Our data revealed an identical morphologic picture during AR early after transplantation in both patient groups, except for a more marked degree of venous endothelialitis and hepatocyte ballooning in the Minnesota material. The follow-up biopsies suggested a spontaneous resolution of these early rejection episodes without antirejection treatment in six of the ten AZA patients. No differences in the long-term survival rate between the CSA- and AZA-treated patients were observed.

Carcinoid in a horseshoe kidney. Morphology, immunohistochemistry, and cytogenetics.

Renal carcinoids are very rare neoplasms. We were able to culture and subsequently karyotype a carcinoid located in the isthmus of a horseshoe kidney, which revealed the following chromosomal pattern: 47,XX, + 13[8]/46,XX,t(13;14)(q31;q11.2)[5]/46,XX[2]. The DNA index was 1. Our results, compared with the sparse data from the literature, suggest that carcinoid of the kidney has no cytogenetic aberrations in common with carcinoids from other anatomical sites reported. On the other hand, numerical and structural aberrations of chromosome 13 seem to play a crucial role in the development of metanephric-derived renal tumors.

Long-term follow-up after liver transplantation for erythropoietic protoporphyria.

OBJECTIVE: Erythropoietic protoporphyria (EPP) is an inherited disorder of haem synthesis, causing excess of protoporphyrin in blood, skin, liver and other organs. Protoporphyrin causes rapidly progressive liver failure in a minority of EPP patients. Long-term follow-up after liver transplantation for EPP is poorly documented. DESIGN: Two EPP patients were followed for 7 years after liver transplantation. Porphyrin levels were monitored and serial liver biopsies were taken. RESULTS: After transplantation, serum protoporphyrin levels remained elevated. In one patient, long periods with normal liver tests, low protoporphyrin levels and the absence of photosensitivity were followed by episodes of cholestasis and elevated protoporphyrin levels in blood, faeces and liver tissue. These episodes could be managed successfully with blood transfusions and changes in medication. The simultaneous rise of protoporphyrin concentration in both blood and faeces in this patient argues for increased protoporphyrin production as the cause of liver cell injury. The other patient acquired hepatitis B infection during the transplantation. From 3 months onwards she had continuously elevated liver tests, cholestasis, elevated protoporphyrin levels in blood, faeces and liver tissue, and photosensitivity. In this case, cholestasis and impaired protoporphyrin excretion may have played an important role in the persistent liver injury. Sequential liver biopsies of both patients showed various degrees of liver injury related to variations of the hepatic protoporphyrin concentrations. Eight and six months respectively after liver transplantation the livers of both patients showed fibrosis and hepatocellular protoporphyrin accumulation. CONCLUSIONS: The main cause of liver damage in EPP is overproduction of protoporphyrin in the bone marrow. Liver transplantation must be considered as symptomatic therapy with a high-risk for recurrent disease.

Immunocytology of body cavity fluids. MOC-31, a monoclonal antibody discriminating between mesothelial and epithelial cells.

This study was designed to assess whether monoclonal antibody MOC-31, which recognizes a membrane glycoprotein of 40-kd molecular weight present on epithelial cells and not on mesothelial cells, is a useful adjunct in the differential diagnosis of reactive pleural or ascitic fluids and adenocarcinoma. A panel of antibodies against carcinoembryonic antigen, epithelial membrane antigen, vimentin (antivimentin), keratin 18 (RGE-53), and cytokeratins of several molecular weights (AE1/AE3) was employed for comparison. Ninety-eight cases were selected based on the availability of pleural and peritoneal biopsy specimens for histologic confirmation. All cases with adenocarcinoma stained MOC-31 positive, including three cases that had not been identified by morphologic criteria. All cases of reactive mesothelial hyperplasia were negative, as well as five cases of malignant mesothelioma. One case of squamous cell carcinoma was also negative. Anti-carcinoembryonic antigen only identified half of the carcinoma cases, and anti-epithelial membrane antigen and both anticytokeratin antibodies were positive in the majority of tumor cells as well as in the mesothelial cells. This study indicates that monoclonal antibody MOC-31 is a highly sensitive and reliable reagent in the differential diagnosis between mesothelial and epithelial cells.

Antinucleair antibody (ANA) positivity caused by paraneoplastic antibodies due to abundant p53 expression in early hepatic carcinoma.

A 51-year-old patient is described who presented with locomotor pain and highly significant positive ANA due to p53 antibodies, which appeared to be associated with primary hepatic carcinoma.

Cigarette smoking and human papillomavirus in patients with reported cervical cytological abnormality.

OBJECTIVE: To assess the relation between two risk factors for cervical neoplasia: smoking and infection with oncogenic human papillomavirus. It has been suggested that smoking causes a local immunological defect, which could facilitate the infection and persistence of human papillomavirus. DESIGN: Cross sectional epidemiological study. Completion of a structured questionnaire by the patients, analysis of cervical scrapes for human papillomavirus, and morphological examination of biopsy specimens. SETTING: Outpatient gynaecological clinic. SUBJECTS: 181 women with a report of cervical cytological abnormality. MAIN OUTCOME MEASURES: Prevalence of infection with oncogenic human papillomavirus and smoking habits. RESULTS: Oncogenic human papillomavirus was found in the cervix of 26 (41%) of the 63 women who did not smoke, 22 (58%) of the 38 who smoked 1-10 cigarettes a day, 28 (61%) of the 46 who smoked 11-20 cigarettes a day, and 26 (76%) of the 34 who smoked > or = 21 cigarettes a day. The prevalence of the virus thus increased in accordance with the number of cigarettes smoked (p = 0.001). This relation remained after adjustment for age at first intercourse and lifetime number of sexual partners. Of the 63 non-smokers, 23 had previously smoked at least 10 cigarettes a day at some time. Of these 23 women, 14 (61%) had oncogenic human papillomavirus in their cervix. Of the 40 women who had never smoked at least 10 cigarettes a day, 12 (30%) had the virus. The prevalence of oncogenic human papillomavirus in non-smokers therefore depended on previous smoking habits (p = 0.03). CONCLUSION: The dose dependent effect of cigarette smoking on the occurrence of oncogenic human papillomavirus favours a causal relation between these risk factors for cervical neoplasia.

Experimental autologous substitute vascular graft for transplantation surgery.

Vascular complications in liver transplantation are a major cause of graft failure and mortality. The aim of the study was to create autologous vascular graft without risk of rejection. Posterior rectus fascia sheath lined with peritoneum was used for iliac artery replacement in seven mongrel dogs. The patency was followed by palpation and Doppler ultrasound. The grafts were removed after one month. Five grafts remained patent. The Doppler showed good, relatively increased flow (median flow rate: 383 cm/sec) after one month in all of the cases. Slight increase in diameter was present in all cases. By microscopy the five patent grafts showed viable morphology, fibroblasts, smooth muscle cells and thin fibrin layer in the wall. The grafts were lined partially with a neoendothelial monolayer and a thin fibrin layer. In conclusion, this graft presents an acceptable patency rate and low thrombogenicity, and could be useful in transplantation. Further investigations are needed to study the effect of immunosuppression and rejection on long-term morphology and patency of the grafts.

[Two sisters with progressive familial intrahepatic cholestasis]. / Twee zusjes met progressieve familiaire intrahepatische cholestase.

A few months after birth two sisters aged 5 and 9 years had developed cholestasis and abnormal liver functions with symptoms including itching and jaundice. The younger sister also developed rickets and clotting disorders. On clinical, biochemical and immunohistopathological grounds the diagnosis of 'progressive familial intrahepatic cholestasis (PFIC) type 2' was made. Medical treatment was not succesfull in reducing symptoms. An ileocolonic bypass in the younger child was not effective. Subsequently, both patients underwent partial external biliary diversion (PEBD). Except for a period of intermittent itching in the younger child, both patients remained free of symptoms 2 years after PEBD. In cases where an early diagnosis is made, PEBD could delay or even prevent the necessity of liver transplantation.

Hepatitis B-associated liver cirrhosis as an indication for liver transplantation.

Fourteen HBsAg-positive patients received a liver transplant in Groningen. Two were HBeAg-positive and 12 HBeAg-negative. No anti-HBs immunoglobulin was given at the time. Both HBeAg-positive and 9 of 12 of the HBeAg-negative patients became HBsAg-positive again after transplantation. Virus titers were tested in eight patients. Two HBeAg-negative patients were HBV-DNA-negative at transplantation and are still HBV-DNA-negative one-and-half-years after transplantation, both by the branched DNA hybridization technique and by PCR (cut-off values 0.7 x 10(6) and 10(3) HBV genomes/ml, respectively). One patient who had a low HBV-DNA titer at transplantation remained PCR-positive thereafter, but became HBsAg-negative. All other patients were HBV-DNA-positive and had a recurrence that rapidly led to high HBV titers. The liver histology was characterized by fibrosis and cirrhosis, centrilobular cholestasis and high expression of HBsAg and HBcAg, but with little inflammatory infiltrate. We conclude from these results that without anti-HBs immunoglobulin prophylaxis there is a high rate of HBV recurrence after transplantation. The current policy is that patients who test negative in the HBV-DNA dot-blot assay (< 10(7) genomes/ml) are transplantation candidates and are treated with high-dose anti-HBs immunoglobulin after transplantation. HBV-DNA-positive patients (> 10(7) genomes/ml) remain poor candidates for liver transplantation, even with anti-HBs immunoprophylaxis.