An unusual foreign body in the urinary bladder.

A coiled electric cable was removed from the urinary bladder of a 35-year-old male, electrician by profession. Psychiatric evaluation revealed normal childhood and no psychiatric illness. Proper management by both a urologist and a psychiatrist is required in these cases.

Cell culture models and animal models of viral hepatitis. Part II: hepatitis C.

The lack of a preventive vaccine, coupled with common unresponsiveness to treatment and coinfection with HIV, has made HCV a major threat to public health. The authors review in vitro and in vivo models that are being used to study HCV and to develop new treatments and preventive measures.

Glucuronidation and the UDP-glucuronosyltransferases in health and disease.

This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.

Hepatocyte transplantation.

Numerous laboratory studies have shown that hepatocyte transplantation may serve as an alternative to organ transplantation for patients with life-threatening liver disease. Because of the successes of experimental hepatocyte transplantation, institutions have attempted to use this therapy in the clinic for the treatment of a variety of hepatic diseases. Unfortunately, unequivocal evidence of transplanted human hepatocyte function has been obtained in only one patient with Crigler-Najjar syndrome type I, and, even then, the amount of bilirubin-UGT enzyme activity derived from the transplanted cells was not sufficient to eliminate the patient's eventual need for organ transplantation. A roadmap for improving patient outcome following hepatocyte transplantation can be obtained by a re-examination of previous animal research. A better understanding of the factors that allow hepatocyte integration and survival in the liver and spleen is needed to help reduce the need for repeated cell infusions and multiple donors. Although clinical evidence of hepatocyte function can be used to indicate function of transplanted hepatocytes, definitive histologic evidence is difficult to obtain. In order to assess whether rejection is taking place in a timely fashion, a reliable way of detecting donor hepatocytes will be needed. The most important issue affecting transplantation, however, relates to donor availability. Alternatives to the transplantation of allogeneic human hepatocytes include transplantation of hepatocytes derived from fetal, adult or embryonic stem cells, engineered immortalized cells, or hepatocytes derived from other animal species.

Gene therapy as an alternative to liver transplantation.

Liver transplantation has become a well-recognized therapy for hepatic failure resulting from acute or chronic liver disease. It also plays a role in the treatment of certain inborn errors of metabolism that do not directly injure the liver. In fact, the liver maintains a central role in many inherited and acquired genetic disorders. There has been a considerable effort to develop new and more effective gene therapy approaches, in part, to overcome the need for transplantation as well as the shortage of donor livers. Traditional gene therapy involves the delivery of a piece of DNA to replace the faulty gene. More recently, there has been a growing interest in the use of gene repair to correct certain genetic defects. In fact, targeted gene repair has many advantages over conventional replacement strategies. In this review, we will describe a variety of viral and nonviral strategies that are now available to the liver. The ever-growing list includes viral vectors, antisense and ribozyme technology, and the Sleeping Beauty transposon system. In addition, targeted gene repair with RNA/DNA oligonucleotides, small-fragment homologous replacement, and triplex-forming and single-stranded oligonucleotides is a long-awaited and potentially exciting approach. Although each method uses different mechanisms for gene repair and therapy, they all share a basic requirement for the efficient delivery of DNA.

Therapeutic potential of hepatocyte transplantation.

Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future.

Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, 35 immortalized rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SV40Tag). The SV40Tag sequence and a suicide gene, herpes simplex virus thymidine kinase (HSV-tk), were flanked by loxP sequences so that they could be excised by Cre/lox recombination. When transplanted into the spleens of portacaval-shunted rats, 3 of the 35 immortalized hepatocyte clones prevented the development of hyperammonemia-induced hepatic encephalopathy. The protection was reversed by treatment with ganciclovir, which kills HSV-tk-expressing cells. Transplantation of alginate-encapsulated, immortalized hepatocytes into the spleens of cirrhotic rats resulted in significant improvement in prothrombin time, serum albumin and bilirubin levels, hepatic encephalopathy score, and duration of survival. The metabolic support provided by the immortalized cells equaled that observed after transplantation of primary rat hepatocytes. In conclusion, immortalized hepatocytes can function as well as primary hepatocytes following transplantation and can be engineered to contain safeguards that could make them clinically useful. Further investigation is warranted regarding the mechanisms of loss of mass or function of the transplanted hepatocytes over time and how the relatively few engrafted hepatocytes can ameliorate liver decompensation in cirrhosis.

A novel intronic mutation results in the use of a cryptic splice acceptor site within the coding region of UGT1A1, causing Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN-1) is characterized by severe unconjugated hyperbilirubinemia due to an inherited deficiency of hepatic bilirubin uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1), inherited as an autosomal recessive characteristic. CN-1 is potentially lethal because of the risk of bilirubin encephalopathy (kernicterus). Genetic lesions of the coding region of the UGT1A1 gene are known to cause CN-1. Here, we report a CN-1 patient who has a novel G > A mutation at the splice acceptor site in intron 4 (IVS4-1 G > A) on one allele, and a T > A substitution followed by a 13-nt deletion in exon 2 (877T > A 878-890del) of the other allele. As the UGT1A1 gene is expressed specifically in the liver, structural analysis of the expressed UGT1A1 mRNA requires liver biopsy. To use a noninvasive approach to determine the effect of the splice site mutation on splicing of the RNA transcript, we amplified the relevant region of the genomic DNA by long-range polymerase chain reaction (PCR). The amplicon was cloned in an expression plasmid and transfected into COS-7 cells. The expressed mRNA was amplified by reverse-transcription-primed PCR. Nucleotide sequence determination of the amplicon showed that the splice acceptor site mutation caused splicing of the 3'-end of exon 4 to a cryptic splice site within exon 5. This resulted in deletion of the first 7 nucleotides of exon 5, causing a frameshift and premature truncation of UGT1A1, with consequent inactivation of the enzyme.