Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin.

The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg-IFN) and ribavirin for patients with chronic hepatitis C (CH-C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty-four patients with CH-C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg-IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c-EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given > or = 12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg-IFN could be reduced to 0.6 microg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose-dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (> or = 12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg-IFN alpha-2b plus ribavirin, especially in c-EVR patients.

Pegylated interferon alpha-2b (Peg-IFN alpha-2b) affects early virologic response dose-dependently in patients with chronic hepatitis C genotype 1 during treatment with Peg-IFN alpha-2b plus ribavirin.

Chronic hepatitis C (CH-C) genotype 1 patients who achieved early virologic response have a high probability of sustained virologic response (SVR) following pegylated interferon (Peg-IFN) plus ribavirin therapy. This study was conducted to evaluate how reducing drug doses affects complete early virologic response (c-EVR) defined as hepatitis C virus (HCV) RNA negativity at week 12. Nine hundred eighty-four patients with CH-C genotype 1 were enrolled. Drug doses were evaluated independently on a body weight base from doses actually taken. From multivariate analysis, the mean dose of Peg-IFN alpha-2b during the first 12 weeks was the independent factor for c-EVR (P = 0.02), not ribavirin. The c-EVR rate was 55% in patients receiving > or = 1.2 microg/kg/week of Peg-IFN, and declined to 38% at 0.9-1.2 microg/kg/week, and 22% in patients given <0.9 microg/kg/week (P < 0.0001). Even with stratified analysis according to ribavirin dose, the dose-dependent effect of Peg-IFN on c-EVR was observed, and similar c-EVR rates were obtained if the dose categories of Peg-IFN were the same. Furthermore, the mean dose of Peg-IFN during the first 12 weeks affected HCV RNA negativity at week 24 (P < 0.0001) and SVR (P < 0.0001) in a dose-dependent manner. Our results suggest that Peg-IFN was dose-dependently correlated with c-EVR, independently of ribavirin dose. Thus, maintaining the Peg-IFN dose as high as possible during the first 12 weeks can yield HCV RNA negativity and higher c-EVR rates, leading to better SVR rates in patients with CH-C genotype 1.

Roles of endothelin-1 and nitric oxide in the mechanism for ethanol-induced vasoconstriction in rat liver.

This study was designed to investigate the mechanism for ethanol-induced hepatic vasoconstriction in isolated perfused rat liver. Upon initiation of ethanol infusion into the portal vein at concentrations ranging from 25 to 100 mM, portal pressure began to increase in a concentration-dependent manner and reached maximal levels in 2-5 min (initial phase), followed by a gradual decrease over the period of ethanol infusion (escape phenomenon). Endothelin-1 antiserum significantly inhibited this ethanol-induced hepatic vasoconstriction by 45-80%. Cessation of infusion of endothelin-1 antiserum was followed by a subsequent increase in portal pressure. On the other hand, when a nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA), was infused into the portal vein simultaneously with ethanol, the initial phase of the response of portal pressure to ethanol was not altered and the peak values of portal pressure remained unchanged. However, after the peak increase in portal pressure, the rate of decrease was less than in the absence of L-NMMA. Thus, L-NMMA diminished the escape phenomenon and sustained the vasoconstriction. This study supports the hypothesis that two endothelium-derived vasoactive factors, endothelin-1 and nitric oxide, regulate hepatic vascular tone in the presence of ethanol.

Urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in dogs.

Clinicopathological, radiological, and histological studies were performed on urinary bladder neoplasia induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in five adult beagle dogs and in ten adult mongrel dogs. Tumors of the urinary bladder developed in dogs given various daily doses of BBN p.o. for different periods. The latent period of tumor induction was 4 years in dogs receiving a daily dose of 80 mg of BBN, 2 to 2.5 years in dogs receiving a daily dose of 160 mg of BBN, and 1.5 years in dogs receiving a daily dose of 240 mg of BBN. The total dose of BBN ingested by the dogs until the first tumors were observed by urological examinations was nearly the same in all groups, 100 to 140 g. These results suggest that there is a correlation between dose and induction time, but further dose-response studies are required. Histologically, tumors of the urinary bladder were transitional cell papillomas or transitional cell carcinomas resembling morphologically those found in human cases. It is possible to observe the process of development of urinary bladder tumors from initial lesions to invasive tumors using routine urological examinations. We believe that this experimental model is valuable for clinicopathological studies of urinary bladder tumors.

A novel strategy for cancer therapy by mutated mammalian degenerin gene transfer.

Mammalian degenerin (MDEG) is a member of the amiloride-sensitive sodium ion channel family, and its site-directed active mutant (MDEG-G430F) induces massive Na+ influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430F into cancer cells using tumor-specific promoters. In carcinoembryonic antigen (CEA)-producing gastric cancer cells, the level of cell death observed when MDEG-G430F was used with a CEA promoter was similar to that observed when using a potent nonspecific promoter such as the cytomegalovirus promoter. In an in vivo study, fusogenic liposome complexes containing MDEG-G430F driven by the CEA promoter were injected intraperitoneally into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead by 50 days postinoculation, 13 of the 15 mice treated with MDEG-G430F survived. These results indicate that transferring MDEG-G430F into cancer tissues using tumor-specific promoters can achieve striking and selective cancer cell death irrespective of the transcriptional efficiency of the promoters used in vivo, and suggest that this approach is a promising new strategy for cancer gene therapy.

Increase in survival of liver grafts after rinsing with warm Ringer's solution due to improvement of hepatic microcirculation.

Temperature increases membrane fluidity and decreases vascular resistance in isolated organs. Therefore, these studies were designed to determine if a rinse with warm buffer could increase survival time in the rat model of orthotopic liver transplantation by improving hepatic microcirculation. Brief periods of warm ischemia (3-8 min) did not damage the liver as indexed by minimal release of LDH. Survival of rats for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringer's solution; yet grafts stored for 8 hr in Euro-Collins solution and rinsed with 20 ml of cold (0-4 degrees C) Ringer's solution survived postoperatively only around 3 days. However, livers stored for 8 hr in Euro-Collins and rinsed with 20 ml of warm (37 degrees C) Ringer's survived longer than 30 days (i.e., permanently). Serum transaminase levels reached peak values around 6000 U/L one day postoperatively in the cold-rinsed group, and liver injury assessed histologically was substantial. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 23% of lung tissue examined and was associated with massive bleeding. Following a warm rinse, however, maximal SGOT levels and injury to both liver and lung were reduced significantly by 80-90% 24 hr postoperatively. Moreover, the warm rinse improved hepatic microcirculation. It accelerated blood flow into the liver approximately two-fold, as indexed by the half-time of changes in hemoglobin reflectance from the liver surface, improved the distribution of colloidal carbon in the organ observed macroscopically, and decreased vascular resistance by over 50%. These data support the hypothesis that a brief rinse of liver grafts with warm buffer markedly improves the hepatic microcirculation, leading to dramatic improvement in graft survival. This work demonstrates clearly that a brief warm rinse may be useful clinically in liver transplantation.

Evidence that adenosine is a key component in Carolina rinse responsible for reducing graft failure after orthotopic liver transplantation in the rat.

Recently, a rinse solution, Carolina rinse, designed to minimize reperfusion injury following liver transplantation in the rat has been developed. When used to rinse cold-stored grafts prior to completion of implantation surgery, Carolina rinse improved postoperative survival dramatically. Here we report the results of studies designed to determine the key components of Carolina rinse. Livers were explanted, stored for 12 hr in cold UW solution (0-4 degrees C), and rinsed with 15 ml of Ringer's solution immediately prior to completion of implantation surgery. In this group, 12/13 rats died within 2 days (nonsurvival conditions). In a second group, explants stored under identical conditions were rinsed with 15 ml of Carolina rinse. Carolina rinse increased average 30-day survival time significantly to over 75%. Furthermore, when grafts were rinsed with Carolina rinse lacking nicardipine or with the pH increased to 7.4, long-term survival of recipient rats was also about 75% (i.e., the modifications did not affect survival). However, Carolina rinse lost its efficacy (12% survival) when adenosine was omitted. In addition, when donor livers were rinsed with Ringer's solution containing adenosine (0.1 mM), average survival time was increased from 8% to 63%. Rinsing with Ringer's solution containing higher concentrations of adenosine (5 mM), however, did not improve survival significantly. Survival was also not improved by rinsing with Ringer's containing 0.1 mM ribose and 0.1 mM adenine, substrates for ATP synthesis that are not vasoactive. SGOT values were around 3000 U/L 1-3 days postoperatively in the nonsurviving group rinsed with Ringer's solution alone. Values were decreased over 6-fold by Carolina rinse but were not reduced significantly by Ringer's solution containing adenosine. Thus, adenosine improves survival following liver transplantation without preventing parenchymal cell injury, indicating that adenosine may work via nonhepatic mechanisms. Liver injury was also assessed by electron microscopy. After either adenosine or Ringer's rinse, sinusoidal thrombi and polymorphonuclear margination were observed together with a pattern of pericentral hepatocellular vacuolization and disruption of the sinusoidal lining characteristic of changes observed following hypoxia. With Ringer's rinse, Kupffer cells exhibited surface irregularity in pericentral regions indicative of activation. Following adenosine rinse, however, Kupffer cells appeared more flattened with less ruffling and reduced surface debris (i.e., they were less activated). Carbon uptake by Kupffer cells was also decreased significantly by Ringer's rinse when adenosine was present. Furthermore, adenosine lowered intracellular free calcium concentration in cultured Kupffer cells and improved hepatic microcirculation postoperatively. Adenosine rinse also affected extrahepatic systems: it reduced postoperative clotting time and diminished lung injury significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of effective portal venous flow in chronic liver diseases using echo-Doppler flowmetry combined with per jejunal portal scintigraphy.

Portal circulation changes due to the progression of chronic liver disease and portal venous flow are also affected by pharmacotherapy. Thus, noninvasive measurement of effective portal venous flow (EPVF) is highly desirable. We evaluated EPVF under steady-state conditions using echo-Doppler flowmetry combined with per jejunal portal scintigraphy in 32 patients with chronic liver disease. After introduodenal administration of 37 MBq (1 mCi) of 123I-iodoamphetamine, scintigraphy of the pulmonary and hepatic regions was performed and a portosystemic shunt index (SI) calculated. EPVF was calculated as follows: EPVF = PVFx (1-SI/100). EPVF in chronic hepatitis, compensated cirrhosis and decompensated cirrhosis was 12.0 +/- 1.8 ml/min/kg, 10.3 +/- 1.6 ml/min/kg and 8.0 +/- 2.5 ml/min/kg, respectively. There were significant differences in EPVF between all groups, although PVF was similar in each group. EPVF correlated with liver function tests and was a better indicator of liver function than PVF. Measurement of EPVF may provide useful information in the management of patients with chronic liver disease.

Ethanol-induced disturbance of hepatic microcirculation and hepatic hypoxia.

The hypothesis was tested whether ingestion of ethanol might disturb the hepatic microcirculation with resulting hepatic hypoxia. Infusion of ethanol increased the portal pressure concentration-dependently in rat livers perfused with Krebs-Henseleit buffer at a constant flow rate (Emax = 11.5 cm H2O, EC50 = 90 mM). This increase in portal pressure was due to hepatic vasoconstriction, since it diminished in the presence of sodium nitroprusside, a direct acting vasodilator. The regional hepatic tissue hemoglobin concentration after perfusion with added erythrocyte suspension (hematocrit 1%), measured by tissue-reflectance spectrophotometry, was significantly diminished by the infusion of ethanol, indicating the impairment of the microcirculation of the superficial layer of the liver. When the absorption spectrum of the liver was examined by reflectance spectrophotometry, infusion of ethanol caused a parallel reduction of all the mitochondrial respiratory cytochromes in a concentration-dependent fashion, concomitant with the increase of portal pressure, indicating a marked reduction of oxygen concentration in superficial liver tissue. The reduction of the respiratory cytochromes was also associated with the decrease in oxygen consumption of the liver, indicating that the hepatic hypoxia was due to the reduction of oxygen delivery to hepatocytes rather than the increased oxygen consumption of the liver. The reduction of the respiratory cytochromes was correlated with the increase in portal pressure and was inhibited by sodium nitroprusside. These data indicate that the ethanol-induced hepatic vasoconstriction disturbs hepatic microcirculation, resulting in hepatic hypoxia and reduction of mitochondrial respiratory cytochromes.

Wy-14,643 but not 2-ethylhexanol increases intracellular free calcium in cultured Kupffer cells.

Hypolipidemic drugs and phthalic ester plasticizers induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. Recent evidence from this laboratory suggests that many agents in this class of chemicals are uncouplers of mitochondrial oxidative phosphorylation both in vitro and in vivo. Uncoupling of oxidative phosphorylation decreases ATP required for ion pumps and could thereby indirectly increase intracellular free calcium. The goal of these experiments, therefore, was to compare the effect of the potent uncoupler and non-genotoxic carcinogen Wy-14,643 with the weaker agent 2-ethylhexanol on intracellular free calcium in cultured Kupffer cells. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic growth factors that may modulate cell proliferation. In this study, the cytosolic free calcium concentration in Fura-2-loaded cultured Kupffer cells was increased significantly from 78 +/- 11 to 838 +/- 112 nM following incubation with Wy-14,643 (1.25 mM). The increase in intracellular calcium due to Wy-14,643 was both time- and dose-dependent. At equimolar concentrations, ethylhexanol had no effect on intracellular calcium (65 +/- 20 nM). However, at higher concentrations (3 mM), ethylhexanol also increased intracellular calcium. These data suggest that elevation of intracellular calcium in Kupffer cells may be involved in the mechanism of action of this interesting class of non-genotoxic carcinogens.

Effect of hepatic blood oxygenation on bile secretion in rats.

The effect of hepatic hemodynamics and hepatic tissue blood oxygenation on bile flow was studied in anesthetized rats by reflectance spectrophotometry. The hepatic hemodynamics and blood oxygenation were assessed by reflectance spectrophotometry. The hepatic ischemia was induced by partial ligation of portal vein and hepatic hypoxia was induced by inhalation of low concentration of oxygen. 1. The ischemia decreased hepatic blood volume index and hepatic blood oxygenation, and diminished bile flow. 2. Respiratory hypoxia suppressed hepatic oxygenation with minimal change of hepatic blood volume, and it also reduced the bile flow. 3. Bile flow was related hyperbolically with hepatic oxygenation and its dependency in hepatic ischemia and respiratory hepatic blood hypoxia identical. It is concluded that the hepatic tissue blood oxygenation affects hepatic energy metabolism, thus affecting the bile secretion.

Effect of ethanol on hepatic oxygenation: evidence of hepatic hypoxia.

The effects of low (1 g/kg body wt) and high doses (4 g/kg body wt) of ethanol on hepatic oxygenation in rats was investigated employing an in vivo microscopic and spectrophotometric system and also a micro oxygen electrode. The low dose of ethanol increased sinusoidal blood hemoglobin oxygenation (ISO2) at periportal regions in the liver lobule. The high dose of ethanol increased ISO2 at periportal regions, but decreased ISO2 at pericentral regions. The low dose of ethanol increased hepatic surface tissue PO2, but the high dose decreased this PO2. From these data it is concluded that the increase of hepatic blood flow, after administration of a low dose of ethanol, compensated for the increase of oxygen demand in hepatocytes but that administration of a high dose of ethanol reduced hepatic tissue oxygenation via an imbalance between delivery and demand of oxygen in pericentral regions of the liver lobule, resulting in local hypoxia in surface hepatic tissue.

Binding cells of 125I-iodoamphetamine in rat liver.

We recently reported that transrectal or intestinal portal scintigraphy with 123I-iodoamphetamine (IMP) could be a useful method for the non-invasive and quantitative evaluation of the portosystemic shunt in portal hypertension, but what cells in the liver trap IMP has not been clarified. This study was aimed at elucidating whether IMP was extracted by parenchymal cells, sinusoidal endothelial cells, Kupffer cells or fat storing cells. Each type of liver cell was isolated from rats and cultured. The cells were incubated with 125I-IMP and the radioactivity of the lysate was determined. Nonspecific binding was assessed in the presence of an excess of unlabeled IMP, and specific binding was determined by subtracting the nonspecific from total binding. Specific binding observed in parenchymal cells, endothelial cells and Kupffer cells was 70.2 +/- 0.4, 4.2 +/- 1.4 and 2.3 +/- 0.8 pmol/well, respectively, but no specific binding was observed in fat storing cells. The binding in parenchymal cells was much higher than that in endothelial cells or Kupffer cells (p < 0.005). In addition, the binding to parenchymal cells reached equilibrium within 20 min and was not saturable over the concentration range tested (0.5-10 microM). These findings indicate that IMP is mostly extracted by parenchymal cells in the liver.

[Experience with microsurgical two-layer vasovasostomy].

Excellent results of vasectomy reversal have been reported by Silber and Owen in 1977 using microsurgical technique without splints. Herein, we report two cases of male sterility due to previous voluntary vasectomy in which fertility was restored by microsurgical vasovasostomy. The reason for vasectomy reversal in these cases was a subsequent marriage. Case 1: A 36-year-old male who had had vasectomy 3 years earlier, underwent vasovasostomy on September 20th 1976. The sperm granuloma was not found, but sperm was observed in the fluid from the proximal cut-end of the vas deferens at the time of operation. He fathered a baby on October 2, 1977. Case 2: A 43-year-old male who had had vasectomy 10 years ago underwent vasovasostomy on February 5th 1980. Sperm granuloma was observed at the time of operation. He fathered a baby on February 25th 1981. The important factors that determine the success of vasectomy reversal are the method and time of reversal. 1) Microsurgical two-layer vasovasostomy is the most reliable method among the various operations for vasectomy reversal. 2) A shorter duration between vasectomy and it's reversal, and the existence of sperm granuloma after previous vasectomy increase the possibility to restore fertility.

[Clinical study of urothelial tumors of the upper urinary tract. 1: Primary renal pelvic tumors].

Forty primary renal pelvic tumors treated at our University between 1963 and 1981, were reviewed retrospectively. The conclusions of this study are as follows. Sex and age distribution of the patients were 30 males and 10 females (3: 1), and average age was 60.5 years old. The major symptoms were hematuria and flank pain; however, palpable mass was rare. The majority of patients were admitted to our clinic within 6 months from manifestation of symptoms. The major findings of IVP were non-functioning kidney and filling defect. The positive rate of urinary cytology was 46.7%. Total nephroureterectomy with bladder cuff was performed in 20 out of 32 cases. Histologically, 29 cases were transitional cell carcinoma and 4 cases were squamous cell carcinoma with renal calculi. Simultaneous urothelial tumors were seen in 10 cases, 3 in the ureter and 7 in the bladder. A subsequent ureteral tumor was found in one out of 12 cases in which ureters were resected incompletely, and 7 subsequent bladder tumors were found out of 32 cases receiving surgical treatment in the follow-up period. The 5-year survival rate by the actuarial method was 75.9%. Among several factors, grade and stage of the tumor were the most influencing factors for prognosis. An effective method of post-operative treatment could not be established.

[Clinical study of urothelial tumors of the upper urinary tract. 2: Primary ureteral tumors].

Twenty-five primary ureteral tumors treated at our University between 1963 and 1981, were reviewed retrospectively. The conclusions of this study are as follows. Sex and age distribution of the patients were 18 males and 7 females (2.6: 1), and average age was 63.04 years old. The major symptom was hematuria. The majority of the patients were admitted to our clinic within 6 months from manifestation of symptoms. The major finding of IVP was non-functioning kidney. The positive rate of urinary cytology was 63.2%. Total nephroureterectomy with bladder cuff was performed in 21 out of 23 cases. Histologically, 22 cases were transitional cell carcinoma and one case was squamous cell carcinoma. Simultaneous urothelial tumor was found in 13 cases in the bladder. Most of the ureteral tumors (63.6%) were found in the lower third segment of the ureter. Subsequent urothelial tumors were seen in 3 bladders and one urethra out of 22 cases receiving surgical treatment in the follow-up period. The 5-year survival rate by actuarial method was 39.4%. Among several factors, grade and stage of tumor were the most influencing factors for prognosis. An effective method of post-operative treatment could not be established.

[Fundamental studies on intravesical instillation of carboquone for treatment of urinary bladder tumors--on the effects of intravesical instillation of carboquone in normal beagle dogs].

The effects of intravesical instillation of Carboquone at the clinically used doses of 5 and 10 mg on the normal mucosa of female beagle dogs was compared with that of 10 mg Mitomycin C used as the control drug. Intravesical instillation for 48 hours of 10 mg carboquone/20 ml phosphate buffer solution (PBS) after bilateral cutaneous uretrostomies produced severe inflammatory changes in all layers of the bladder wall. However, no secondary effects were observed in blood laboratory examinations or histological examinations of the whole organ after autopsy. Phosphate buffer solution produced no remarkable secondary effects in animals. Five milligrams carboquone per 20 ml PBS was instilled intravesically once a week for 3 weeks in normal animals. Cystoscopically , the bladder mucosa recovered normally. Blood laboratory examinations showed no abnormal results, but the bladder epithelium had regenerative epithelial hyperplasia and slightly inflammatory changes in the submucosal layers. Two of the three control animals given instillation of 10 mg of Mitomycin C/20 ml PBS had slight leucopenia at 7 days after the last intravesical instillation, but leucocyte count was normal at the end of the experiment. Cystoscopic and histological examination of the epithelium of the urinary bladder revealed severe inflammatory changes in 2 of the 3 animals.