Sergio Varotto

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 × 109 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 μmol/L before and 359 ± 46 μmol/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.

Hepatocyte transplantation in the treatment of acute liver failure: microencapsulated hepatocytes versus hepatocytes attached to an autologous biomatrix.

A liver transplant is considered today to be the only effective therapeutic solution for many otherwise intractable hepatic disorders. However, liver transplantation is beset by shortage of donors. Over the years, many liver support systems have been developed to supply the liver functions, mostly as a bridge to transplantation. Transplantation of isolated hepatocytes (HcTx) instead of whole liver has constituted one of the most appealing possibilities to treat several diseases. We compared two different models of HcTx in a surgical model of acute liver failure in pigs, using microencapsulated hepatocytes (MHcTx) and hepatocytes attached to a porcine biomatrix (PBMHcTx), both transplanted into peritoneum. The collected data were survival, laboratory findings, hemodynamic parameters, light microscopy, histology, MTT, and glycogen content. The group with PBMHcTx has a better outcome than the group with MHcTx (p < 0.05). Histology showed normal morphology of the hepatocytes, high glycogen content, 75% viability, positive MTT, and 95% adhesion of the hepatocytes to the biomatrix. Our biomatrix (PBM) provides cell-to-cell contact and interaction with extracellular matrix, which have been shown to play major roles in hepatocyte survival and physiologic regulation of gene expression, and guarantee a prompt engraftment and an adequate neovascularization. PBMHcTx is a useful method to treat acute liver failure and it indicates a possible liver-direct gene therapy in the treatment of inherited and acquired disorders.

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.

Parathyroid localization in primary hyperparathyroidism: double-tracer scintigraphy and venous sampling techniques combined. A first evaluation

The authors report their first experience with two techniques for preoperative localization of hyperfunctioning parathyroid glands: double-tracer (99mTc-201TI) scintigraphy and venous sampling from thyroid veins. Nineteen patients affected by primary hyperparathyroidism were examined by both methods and subsequently submitted to surgery. Scintigraphy showed a sensitivity, of 77.3% while the positivity of sampling was 63.6%; with the two techniques combined a correct localization was obtained in 90.9% of all cases. The authors emphasize the usefulness of both methods combined for locating enlarged hyperfunctioning parathyroids.

Oligonucleotide probe array for p53 gene alteration analysis in DNA from formalin-fixed paraffin-embedded breast cancer tissues.

Mutations in the TP53 tumor‐suppressor (p53) gene represent the most common molecular changes in various malignancies, including breast cancer (BC). We sequenced the p53 gene in DNA extracted from archival paraffin‐embedded BC tissues and compared the results obtained from direct sequencing with those obtained by oligonucleotide probe array (OPA). DNA was extracted from 34 samples. OPA correctly detected 13 genetic alterations in 14 cases, with a mutation frequency of 41.2%. No changes were detected in exons 3, 4, 9, 10, and 11 and no polymorphisms were found. Direct manual sequencing in which DNA was amplified by PCR showed 21 genetic mutations in 19 (55.9%) cases. Eight mutations were identified by both OPA and PCR methods. Although OPA detected fewer gene alterations than direct sequencing, the difference was not significant (P= 0.11). In conclusion, the OPA may be safely used to identify individual genetic variations of human p53 gene in BC specimens.