Isolated hepatocytes versus hepatocyte spheroids: in vitro culture of rat hepatocytes

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 +/- 4.4%), while in G2 it was 72.1 +/- 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.     

Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 x 10(9) hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean +/- SD bilirubin level was 530 +/- 38 micromol/L before and 359 +/- 46 micromol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.     

C-erb-B2 (HER2/neu) expression in synovial sarcoma of the head and neck

BACKGROUND: Synovial sarcoma is a malignant mesenchymal tumor composed of varying proportions of spindle and epithelial cell components. Because of the histologic and immunohistochemical similarity of synovial sarcoma to epithelial carcinomas, we hypothesized that the human epithelial growth factor receptor 2 (C-erb-B2, also termed HER2/neu) may contribute to the tumor phenotype and provide a new therapeutic target for this soft tissue tumor. METHODS: Three head and neck, one chest wall, and seven extremity synovial sarcomas were evaluated for C-erb-B2 (HER2/neu) expression by immunohistochemistry, Western immunoblotting, and fluorescence in situ hybridization (FISH). RESULTS: The head and neck cases demonstrated immunohistochemically strong positive staining, whereas tumors from other anatomic locations showed neither positive nor cytoplasmic restricted staining. Antigen-targeted antibody therapy (trastuzumab) was initiated in two patients. CONCLUSIONS: These results demonstrate that C-erb-B2 (HER2/neu) may play a role in the tumorigenesis of synovial sarcoma; and, therefore, antigrowth factor therapies may provide a previously unrecognized pharmaceutical approach to soft tissue tumors. The data also suggest that although synovial sarcoma of the head and neck and synovial sarcoma of the extremities have similar morphologic features, they may be clinically and mechanistically distinct entities.     

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.     

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines against Neisseria meningitidis groups Y and W135

The glycoconjugate vaccines against Neisseria meningitidis groups Y and W135 consist of pools of selected oligosaccharides conjugated to the protein carrier (CRM197). Consistent production of these vaccines requires control and thus determination of the average degree of polymerisation of the oligosaccharides used for conjugation. Acid hydrolysis generates group Y and W135 oligosaccharides with N-acetylneuraminic acid at the reducing end. A method, involving NaBH4 reduction and quantification of this terminal N-acetylneuraminic acid by use of high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) following acid hydrolysis (2M TFA), was developed. The average degree of polymerisation is calculated from the ratio of reduced N-acetylneuraminic acid to total N-acetylneuraminic acid. The assay was qualified by application to group C, Y and W135 oligosaccharide standards characterised by liquid chromatography, mass and NMR spectroscopy.     

Blunt liver trauma: efficacy of non-operative management

All haemodynamically stable patients with blunt abdominal trauma can be managed conservatively by non-operative management which is the treatment of choice in 80% of liver trauma. Non-operative management, when feasible, yields very good results in terms of survival, need for blood transfusions, morbidity and reduction of hospital stay. The Authors retrospectively analyse their recent experience with liver trauma. Of 53 patients with blunt liver trauma, 36 underwent surgical treatment whereas 17 haemodynamically stable patients received non-operative management. After CT scans, all patients on non-operative management underwent haemodynamic monitoring, US and blood examinations. A percentage of 47.1 % had a > or = grade III trauma. The mortality and morbidity rates were 0% and no blood transfusions were needed. The efficacy of non-operative management was 100% and none of the patients needed subsequent surgical treatment. Our clinical experience demonstrates that non-operative management is feasible and effective even for higher grade traumas, but always requires strict clinical, haemodynamic and US monitoring and careful patient selection. Current progress in the field and recent evidence reported in the literature indicate the likelihood of further developments and more widespread utilisation of non-operative management in liver trauma.     

Onset of inflammatory bowel diseases during combined alpha-interferon and ribavirin therapy for chronic hepatitis C: report of two cases

We report two patients who developed an inflammatory bowel disease (IBD) shortly after beginning combined alpha-interferon and ribavirin treatment for HCV-related chronic hepatitis. The previous clinical history was negative for IBD in both patients, who developed diarrhoea and rectal bleeding 10 days and 6 months, respectively, after the initiation of therapy. The history, therapeutic management and the possible causal relationships of these cases are discussed.