Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.     

Radiofrequency ablation of pulmonary tumors and normal lung tissue in swine and rabbits

OBJECTIVE: The purpose of the present study was to examine the following during radiofrequency ablation (RFA): (1) the risk of hemorrhage from intrapulmonary large vessels; (2) the risk of incomplete ablation of pulmonary tumors; and (3) the late effect on lung tissue. MATERIALS AND METHODS: A 17-gauge, cool-tip-type radiofrequency electrode was used. The damage to the vessels and bronchi was examined by the injection of a colored silicone rubber, a liquid compound that hardens after injection. To examine the risk of hemorrhage from intrapulmonary large vessels, RFA was conducted at eight sites near the central pulmonary vessels in two swine. To examine the risk of an incomplete ablation for pulmonary tumors, 10 pulmonary nodules were made from a gelatin mixture in another two swine and were treated by RFA. To examine the late effect on lung tissue, RFA was conducted on the peripheral lung in 10 rabbits, and then the ablated regions were examined on days 1, 7, 14, 21, and 28 after RFA. RESULTS: The use of colored silicone rubber enabled us to examine the intrapulmonary vessels and bronchi for opening and leakage. RFA did not damage the large intrapulmonary vessels, even when they were located within the ablated regions. Lung tissue surrounding the gelatin nodules was hardly ablated over its entire circumference. Six of 10 gelatin nodules (60%) showed nonablated areas on the peripheral edges of the nodules. From 21 days after RFA, the ablated rabbit lung formed noninfectious cavities by communicating with the surrounding bronchi. CONCLUSION: It was improbable for hemorrhage to occur even when RFA was conducted near the large intrapulmonary large vessels. Because an incomplete ablation that left tumor cells at the site of ablation could occur during surgery due to the difficulty of ablating the entire tumor circumference, CT scan-guided RFA would be preferable to a surgical approach for making a safe margin. Cavity formation can occur beginning 21 days after RFA, which should be carefully followed up in a clinical setting to identify infection, especially in immunocompromised patients.     

Increased plasma levels of thioredoxin in patients with glucose intolerance

OBJECTIVE: The aim of the present study was to determine the effects of glucose intolerance on oxidative stress in patients with coronary artery disease (CAD). METHODS: The patients were divided into 3 groups, diabetes mellitus (DM), IGT or normal glucose tolerance (NGT) according to the criteria of the American Diabetes Association. PATIENTS: The present study consisted of 178 consecutive patients who underwent diagnostic coronary arteriography and a 75-g glucose tolerance test. RESULTS: The level of plasma thioredoxin, a marker of oxidative stress was measured in every patient during the fasting state. The levels of plasma thioredoxin were significantly higher in the DM and IGT groups than the NGT group. Furthermore, we found that there was a positive association between thioredoxin levels and glycosylated hemoglobin (sigma=0.225, p=0.018). In multivariate logistic regression analysis, glucose intolerance (DM or IGT) was only independently associated with the high levels of thioredoxin. The levels of plasma thioredoxin were significantly higher in the CAD group compared to the non-CAD group. In multivariate logistic regression analysis, high levels of thioredoxin, male, age and hypertension were independently associated with the presence of CAD. CONCLUSION: Glucose intolerance was associated with the high levels of thioredoxin. High levels of thioredoxin were related to the presence of CAD. The measurement of thioredoxin as the marker of oxidative stress may be useful for monitoring the development of the cardiovascular diseases.     

Presence of a translational stop codon in the precore region of HBVDNA in the HBeAg positive stage of chronic Hepatitis B

This work was Supported by a Grant-in-Aid for Scientific Research(B) from the Ministry of Education, Science and Culture, No 63480203.     

Hepatic Circulation and Hepatic Oxygen Consumption in Alcoholic and Nonalcoholic Fatty Liver

The purpose of this study was to determine the differences in hepatic circulation and oxygen consumption in two groups: those with nonalcoholic obesity-related fatty live and those with alcoholic fatty liver. Although the histological degree of fatty infiltration was equal in the two groups, the delta Er569-650, as an index of the regional liver blood flow estimated by spectrophotometric method, was significantly lower in alcoholic fatty liver than in nonalcoholic fatty liver, and the in vivo hepatic oxygen consumption (VO2), also determined by hepatic reflectance spectrophotometry during peritoneoscopy, tended to be lower in alcoholic fatty liver than in nonalcoholic fatty liver. The oxygen saturation of hemoglobin in local liver blood (SO2) was, however, significantly higher in alcoholic fatty liver than in nonalcoholic fatty liver. These results suggest that an increase in oxygen extraction to maintain oxygen consumption, which was indicated by the lowering of the SO2, was not found in alcoholic fatty liver, in spite of a reduction of oxygen supply to the liver. It is concluded that the impairment of hepatic circulation and hepatic oxygen consumption was more serious in alcoholic fatty liver than in nonalcoholic fatty liver, possibly contributing to a different prognosis for the two forms of fatty liver.     

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E₂ release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dosedependently stimulated prostaglandin E₂ release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A₂ activity. These results suggest that such an effect of EGF onde novo synthesis of cyclooxygenase protein and prostaglandin E₂ release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.     

Monocyte/Macrophage-Specific Marker CD163+ Histiocytic Sarcoma: Case Report with Clinical, Morphologic, Immunohistochemical, and Molecular Genetic Studies

We report a case of a very rare disorder, histiocytic sarcoma, from a review of our autopsy cases. The neoplastic cells that proliferated in organs throughout the body were large cells containing eosinophilic cytoplasm and pleomorphic nuclei with prominent nucleoli. In the bone marrow, erythrophagocytosis by neoplastic cells was observed. The neoplastic cells were positive not only for lysozymes and CD68 (KP-1, PG-M1, and Ki-M1P) but also for a monocyte/macrophage-specific marker, CD163. In contrast, the results of tests for markers of myeloid cells, lymphoid cells, and epithelial cells were all negative. In a polymerase chain reaction study of paraffin-embedded tissues, analyses for the rearrangement of immunoglobulin heavy chain and T-cell receptor-γ genes were negative. The current World Health Organization diagnostic criteria for histiocytic sarcoma regard immunohistochemical investigation as crucial. In this regard, the highly specific positivity for CD163 in this patient indicates that immunohistochemical staining of CD163 is very useful for the diagnosis of histiocytic sarcoma.     

Photoinducible phase-specific light induction of Cry1 gene in the pars tuberalis of Japanese quail

Prolactin (PRL) secretion is regulated by photoperiod in mammals and birds. In mammals, the pars tuberalis (PT) in the pituitary is involved in the regulation of photoperiodic regulation of PRL secretion. In birds, however, hypothalamic vasoactive intestinal peptide is implicated in PRL secretion, and physiological roles of the avian PT remain unknown. In the present study, we show that PRL secretion increases under long days and short days with a night interruptive schedule, both of which also cause gonadal growth in Japanese quail. We have also found Cry1 gene expression in the PT of Japanese quail. Cry1 expression was rhythmic under long and short photoperiods in the PT, and the peak was phase delayed under a lengthened photoperiod. Moreover, expression of Cry1 gene was induced by a light pulse but only when given during the photoinducible phase. In our previous study, we have shown rhythmic Per2 gene expression with a peak in the PT during the early day under various photoperiods. When taken together with the results from the present study, different phase relationships between Per2 and Cry1 in the Japanese quail PT under different photoperiods may decode photoperiodic information and regulate photoperiodic PRL secretion in a manner similar to that of mammals.