The escape of temperature-sensitive T antigen immortalized rat hepatocytes from conditional immortalization.

Conditionally immortalized hepatocytes (CIH) established with a gene for the temperature-sensitive mutant of the T antigen (tsT) have characteristics to stop proliferating and to differentiate at nonpermissive temperatures (37-39 degrees C) due to inactivation of the T antigen. Therefore, they may be a good alternative to primary hepatocytes for experimental investigations or clinical applications. Deinduction of the T antigen results in a transient increase of p53 in these cells, leading to reexpression of normal senescence because of the telomere attrition occurring during the early stages of immortalization. To determine this T antigen dependency for the maintenance of immortality, a type of rat CIH was cultured continuously at 39 degrees C. The frequency of occurrence of T-antigen-independent clones ranged from 0.053% to 0.093%. These clones maintained the temperature-sensitive property of the T antigen; nevertheless, they were able to progress to the S phase and proliferate without undergoing apoptosis at 39 degrees C as at 33 degrees C, a permissive temperature. The temperature-sensitive point mutation of tsT was not affected in these clones and the T antigen was functioning properly. The integrity of the p53 pathway was also maintained from the point of Western blot analysis of p21. Although the telomerase continued to be expressed and the telomere length was maintained, marked chromosomal damage could not be avoided in these cells. It is a plausible explanation that this escape phenomenon from conditional immortalization may be related to the change of other genes involved in cell cycles, which have yet to be elucidated. In conclusion, CIH could lose their temperature-sensitive characteristics without the change of tsT, itself, and the T antigen is not always necessary to maintain their immortality. Therefore, the results obtained from experimental investigations using these cells should be interpreted carefully, and unpredictable phenotypic changes should also be taken into consideration when using them in clinical applications.

[A case of non-traumatic hemobilia due to warfarin therapy].

Hemobilia is a hemorrhage into the biliary tract that may follow surgical trauma, liver biopsy, aneurysms, extra- or intra-hepatic tumors of the biliary tract, gallstones, and inflammatory lesion of liver, especially helminthic or pyogenic. Sometimes, it is associated with primary liver cancer. An 84 year-old woman was admitted because of continuous right upper quadrant pain 4 days before admission. Physical examination revealed decreased skin turgor, icteric sclerae and severe tenderness on right upper quadrant abdomen. She had no hepatosplenomegaly, and no rebound tenderness. She has been taking warfarin for 3 weeks before admission because of atrial fibrillation. On admission, serum bilirubin and transaminase were elevated. The level of hemoglobin and hematocrit were 11.3 g/dL and 37.4%, respectively. HBsAg was negative, but IgG anti-HBc and anti-HBs were positive and anti-HCV was negative. Parasite skin test and stool ova count demonstrated non-specific findings. Stool occult blood was strongly positive, and prothrombin time was markedly prolonged. According to endoscopic retrograde cholangiopancreatography, common bile duct was dilated, and filled with blood clot but there was no stone in bile tree. After two weeks, serum transaminase, bilirubin, hemoglobin, hematocrit, and CA19-9 were normalized. We report a case of hemobilia, occurring in a patient with continuous warfarin use.

Autoimmune cholangitis in a patient with thymoma.

Autoimmune cholangitis is characterized biochemically by chronic cholestasis and histopathologically by chronic non-suppurative destructive cholangitis. It is associated with positive antinuclear antibody test and negative antimitochondrial antibody test results. Recently, we experienced a case of a 35-year-old woman with autoimmune cholangitis associated with thymoma who presented with pruritus, jaundice, chronic fatigue and anterior chest discomfort. Her laboratory examinations revealed marked increases in levels of serum alkaline phosphatase and gamma-glutamyl transpeptidase. In serological tests, antinuclear antibody was found, but antimitochondrial antibody was not. Liver biopsy findings were compatible with chronic non-suppurative destructive cholangitis. On computed tomography (CT) of the chest, a large anterior mediastinal mass was found. The mass was totally resected and the patient was treated with ursodeoxy cholic acid. Thereafter, her clinical symptoms improved and liver functions completely returned to the normal range. We describe here an uncommon association of autoimmune cholangitis with thymoma, which has not been reported previously in the English-written literature.

[The changes of ammonia and epidermal growth factor concentration in gastric juice before and after the Helicobacter pylori eradication].

BACKGROUND/AIMS: Helicobacter pylori (H. pylori) infection is the cause of peptic ulcer diseases, and gastric cancer. Hydrolysis of urea generating ammonia may cause cytotoxic effects on the gastric epithelium. The ammonia may induce the synthesis of epidermal growth factor (EGF) in gastric epithelium as an adaptive cytoprotective mechanism. The first aim was to examine the concentration of ammonia and EGF in gastric juice before and after H. pylori eradication in functional dyspepsia patients. The second aim was to examine the correlation among ammonia concentration, EGF concentration, and inflammatory score of gastritis. METHODS: The concentration of ammonia and EGF were measured by ELISA. The grade and severity of gastritis were measured according to the updated Sydney system. RESULTS: The concentration of ammonia in gastric juice was much higher in the H. pylori positive subjects (10,787 +/- 6,584 micro mol/L) than in the negative subjects (2,339 +/- 1,158 micro mol/L, p<0.0001). The concentrations of EGF in gastric juice was much higher in the positive subjects (1,462 +/- 393 pg/mL) than in the negative subjects (1,088 +/- 499 pg/mL, p<0.005). The concentration of ammonia and EGF in gastric juice showed significant correlation (r=0.63, p<0.0001). The concentrations of ammonia and histologic severities showed significant correlation (r=0.41, p<0.0001). Moreover, the level of EGF in gastric juice and histologic severities showed positive correlation (r=0.20, p<0.005). CONCLUSIONS: As the concentration of ammonia in gastric juices increased, the concentration of EGF was also increased in functional dyspepsia with H. pylori infection. The concentration of EGF in gastric juice may play a role in the adaptive cytoprotection in H. pylori- induced gastritis.

[Relationship between esophageal lesions observed by endoscopy and nasogastric intubation: a study of 185 cases of percutaneous endoscopic gastrostomy].

BACKGROUND/AIMS: Percutaneous endoscopic gastrostomy (PEG) is a method widely used for long-term enteral nutrition in dysphagia. Mostly, it is preceded by nasogastric intubation (NI) for short-term enteral nutrition; endoscopic findings associated with NI are encountered during PEG. The purpose of this study was to discuss such findings and to delineate a relationship between these findings, especially esophageal lesions and the duration of NI. METHODS: This study involved 185 individuals who had undergone PEG at Kyung Hee Medical Center from January 1999 to May 2002. The medical records were examined retrospectively. RESULTS: The dysfunction of the CNS comprised 98.4% of the causes of dysphagia. The duration of NI was 15.2 weeks on average, with median value of 8.7 weeks, indicating that PEG was performed relatively soon. Endoscopic findings revealed esophagitis in 63 cases, esophageal ulcers in 27 and active bleedings in another 10. The incidence of esophageal lesions was shown to be higher in subjects with duration of NI under 12 weeks than in those with duration over 12 weeks (p=0.032). CONCLUSIONS: PEG was carried out in many cases during the early stages of dysphagia, and NI-associated esophageal lesions appeared to be more prevalent within 12 weeks of NI duration. These results may be of help in deciding the timing of PEG.

Liver cirrhosis developed after ketoconazole-induced acute hepatic injury.

We describe a previously healthy woman who developed liver cirrhosis as a sequela of acute hepatic injury that was induced by ketoconazole administration to treat onychomycosis. The initial presentation of the disease was of a typical acute hepatitis, characterized by nausea, anorexia, fatigue, and jaundice that developed during the administration of ketoconazole. Many other causes of hepatitis were absent in the patient. Even though the hepatic injury was gradually resolved for several months after cessation of the drug, the liver function was not completely restored. Six months after the onset of illness, a follow-up abdominal computed tomography and peritoneoscopic liver biopsy were performed. They revealed a marked reduction in the liver volume and a definite cirrhotic change, which persisted for more than 5 years. The case suggests that the administration of ketoconazole can cause liver cirrhosis through acute hepatic injury within a short time under certain circumstances.

[Clinical features of hepatocellular carcinoma in the 1990s].

BACKGROUND/AIMS: There has been a shift of the etiologies of chronic liver disease in the 1990s. Therefore, we studied clinical characteristics of hepatocellular carcinoma (HCC) in the 90s. METHODS: Medical records of 806 patients diagnosed as having primary HCC were reviewed. Etiology, clinical and laboratory characteristics were evaluated according to the time of diagnosis (the early period, 1992-1995; the late period, 1996-2000). RESULTS: The mean age was 55.7 years and male to female ratio was 4.6:1. The proportion of the symptomatic patients at the time of diagnosis was decreased from 67.4% of the early period to 41.3% of the late period. On the other hand, that of the patients detected by a periodic check-up was increased up to 58.7% in the late period from 32.6% in the early period (p<0.01). The majority of the patients accompanied cirrhosis (73.3%) and the main cause of HCC was HBV (78.6%) with no changes in the etiologic distribution according to the periods. The proportion of the candidates for surgical resection was significantly increased to 12.4% in the late period compared with 7.1% in the early period. CONCLUSIONS: Although the proportion of HCC which can be treated curatively has increased in the later half of the 1990s, its absolute number is still small. More meticulous periodic examination may be required in high risk patients.

[A case of chronic pouchitis resistant to medical treatment].

Pouchitis, a non-specific acute inflammation occurring in the ileal pouch, is one of the most common complications developed after the restorative proctocolectomy and ileal pouch-anal anastomosis (IPAA) performed for the treatment for the patients with ulcerative colitis and familial adenomatous polyposis. The prevalence of pouchitis is known to range from 20% to 50%. One to two percent of the cases are chronic and resistant to the drug therapy. The effective treatment for this chronic resistant pouchitis is to remove the ileal pouch and perform the permanent ileostomy. Hereby, we report one case of chronic pouchitis resistant to multiple drug therapy developed after IPAA performed for the treatment of ulcerative colitis in a patient.

Temporary amelioration of bilirubin conjugation defect in Gunn rats by transplanting conditionally immortalized hepatocytes.

BACKGROUND: Conditionally immortalized hepatocytes (CIH) have been used in hepatocyte transplantation as an alternative to primary hepatocytes to cope with the shortage of donor organs. However, CIH are known to undergo apoptosis at body temperature and survive in vivo for a short period. In the present study, we investigated whether CIH function or not and how long their function is maintained in vivo. METHODS: Various CIH cell lines that were established with temperature-sensitive Simian virus 40 large T antigen were transplanted into the spleen of Gunn rats, which are defective in bilirubin uridine diphosphate glucuronoside transferase (BUGT). Then, we measured biological changes over 3 months. RESULTS: Serum bilirubin of the syngeneic CIH recipients decreased by 30%, which was maintained for 8 weeks. Thereafter, it began to rise to basal levels. The recipients of allogeneic CIH showed a minor reduction of bilirubin, although this was not statistically significant. However, there was no significant change in the bilirubin level in recipients of BUGT-defective congeneic CIH throughout the study period. Bilirubin monoglucuronides in the bile were not detected in the recipients of BUGT-defective CIH. However, they appeared in recipients of non-defective CIH and made up approximately 41% of total bile pigments. CONCLUSIONS: Conditionally immortalized hepatocytes expressed hepatocyte function in vivo as well as in vitro, but the function lasted for a couple of months. According to our previous study, the limited functional duration may be related to the inevitable occurrence of apoptosis of these cells at body temperature. These data suggest that CIH can be used in hepatocyte transplantation only for temporary hepatic support.

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells.

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.