Harumasa Yoshihara

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO2-. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO2- and HbCO. HbNO was, therefore, derived from NO, not from NO2-. These results show that free NO is produced in vivo by the stimulation of LPS.

Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C : relationship to interferon response

BACKGROUND/AIMS/METHODS The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be an important determinant of extracellular matrix deposition and breakdown. We measured serum MMP-1, MMP-2, TIMP-1 and TIMP-2 levels using the respective one-step sandwich enzyme immunoassays in 98 patients with chronic hepatitis C treated with interferon beta to examine their clinical significance for assessment of liver histology and to determine whether they can be useful as predictors of the interferon response. RESULTS Serum TIMP-1 levels showed a positive correlation with the degree of fibrosis (r(s)=0.30, p= 0.004). Serum MMP-2 levels revealed positive relationships with the degree of periportal necrosis (r(s)= 0.32, p=0.002), the degree of fibrosis (r(s)=0.26, p= 0.01) and total score of histological activity index (r(s)=0.24, p=0.02). Serum MMP-2 levels were significantly higher in patients with no response than in those with sustained and transient response (p<0.01 and p<0.05, respectively), while serum MMP-1 levels did not differ among the three groups. Compared with the levels in sustained responders, the total amounts of serum TIMP-1 were significantly lower in transient responders and non-responders (p<0.01 and p<0.001, respectively). As for serum TIMP-2 levels, a significant decrease was found in transient responders and non-responders (p<0.01). The ratios of serum MMP-2 to TIMP-1 levels were significantly higher in transient responders and non-responders than in sustained responders (p<0.001, respectively) even when HCV RNA levels were low in patients with HCV genome subtype 1b or when the HCV genome subtype was 2a or 2b. Sustained response was never found in type 1b patients with ratios of serum MMP-2 to TIMP-1 levels of over 6.0. In logistic multivariate regression analysis, the ratios of serum MMP-2 to TIMP-1 level (p=0.0001), HCV genome subtype (p=0.005) and serum TIMP-2 level (p=0.03) were the independent predictors for sustained response, while serum MMP-2 level (p=0.0006) was the only predictor for no response. CONCLUSIONS Serum MMP-2 and TIMP-1 levels might be useful for estimating the degree of liver fibrosis. The ratio of serum MMP-2 to TIMP-1 levels may serve as a new predictor of interferon response in patients with chronic hepatitis C.

Effect of interferon therapy on the incidence of hepatocellular carcinoma and mortality of patients with chronic hepatitis C: a retrospective cohort study of 738 patients.

The effect of interferon on the long‐term clinical outcome of patients with chronic hepatitis C remains unclear. This study included 594 patients with chronic hepatitis C who received interferon‐α therapy (Interferon group) and 144 patients with chronic hepatitis C who did not receive interferon (Control group). The patients in the Interferon group were classified into the following three groups based on the response of the serum aminotransaminase level of the patient during and after completion of the therapy protocol: sustained responders (n = 175), transient responders (n = 165), and non‐responders (n = 254). The age, sex, serum aminotransaminase level, platelet count, histological staging, hepatitis C virus (HCV) subtype, and HCV concentration at baseline were adjusted with the Cox proportional hazards model. The length of follow‐up for assessment of the risk for developing hepatocellular carcinoma (HCC) was 57.2 ± 13.9 months in the Interferon group and 67.7 ± 28.7 months in the Control group. Multivariate analysis showed that interferon therapy decreased the risk for developing HCC by 48% compared with that in the Control group (P = 0.064). The older the age, being male, having a low platelet count, and higher histological stage were independent factors associated with the development of HCC. The hazard rate ratio for development of HCC in the sustained responders, transient responders, and non‐responders was 0.16 (95% confidence interval [CI]: 0.04–0.62), 0.27 (95% CI: 0.09–0.79), and 0.74 (95% CI: 0.37–1.48), respectively. During follow‐up, 18 patients in the Interferon group died (10 from liver‐related diseases) and 17 patients in the Control group died (10 from liver‐related diseases). No sustained responder or transient responder in the Interferon group died of liver‐related disease. The cumulative survival rates of the Interferon and Control groups were nearly identical during the first 5 years following diagnosis. Thereafter, the cumulative survival rate of the Control group declined, resulting in an 8‐year survival rate in the Interferon and Control groups of 97% and 81%, respectively (P = 0.061). Similar trends were seen in the survival analysis of those who had died of liver disease: the 8‐year survival rates of the Interferon and Control groups were 98% and 88%, respectively (P = 0.32). Our study demonstrated that interferon therapy significantly lowered the incidence of HCC among patients with chronic hepatitis C who showed sustained normalization and among patients who showed transient normalization of the serum aminotransferase level after completion of interferon therapy. The survival analyses and determination of cause of death suggested that interferon therapy improves the long‐term survival of chronic hepatitis C patients who respond to this therapy, possibly by decreasing mortality from liver‐related diseases. Int. J. Cancer 87:741–749, 2000.

Declining Incidence of Hepatocellular Carcinoma in Osaka, Japan, from 1990 to 2003

Context Hepatitis C virus (HCV) infection in Japan began to spread during the 1920s, increased after World War II with an explosion in parenteral amphetamine use and paid blood donation, and decreased in the 1950s to 1960s with voluntary blood donation and penalties against amphetamine use. Evidence linking the trends in HCV infection to hepatocellular carcinoma rates in Japan is limited. Contribution Data from the Osaka Cancer Registry and 10 Osaka hospitals suggest that hepatocellular carcinoma rates began to decrease in 2000, mainly because of a decrease in HCV-associated cancer. Implication Control of HCV transmission within a population seems to be followed by a decrease in hepatocellular carcinoma. The Editors Primary liver cancer was the fifth most common cancer worldwide by 2000, with approximately 551000 new cases recorded (1). In most countries, hepatocellular carcinoma (HCC) comprises 85% to 90% of primary liver cancer cases. With some exceptions, developed countries, including the United States, have been experiencing an increase in the incidence of primary liver cancer, considered to be due at least in part to increased prevalence of chronic hepatitis C virus (HCV) infection (2). Japan has had one of the highest incidence rates of primary liver cancer among developed countries (age-standardized incidence rate in 1995, 25.5 per 100000 men and 7.7 per 100000 women) (3). Approximately 90% of liver cancer cases are HCC, which, in Japan, is mainly caused by chronic HCV infection rather than chronic hepatitis B virus infection (4). A recent report on the age-standardized incidence of primary liver cancer among Japanese men, which was calculated from 6 population-based cancer registries, showed a sharp increase that started in the mid-1970s but leveled off in the mid-1990s (5). These distinctive trends were thought to be due to the spread of HCV infection, which began in the 1920s and increased after World War II (68). Thus, HCV penetrated Japan earlier than Spain, Egypt, the United States, the former Soviet Union, South Africa, and Hong Kong, as evidenced by molecular clock analysis of the sequences of HCV isolates (8). However, recent temporal trends regarding incidence rates of HCC and the contribution of HCV infection have not been clearly documented in the Japanese population. We analyzed temporal trends for HCC incidence rates between 1981 and 2003 in Osaka Prefecture (population in 2005, 8.8 million) and interpreted these in the context of HCV infection rates. Methods Data Collection on Incident HCC Cases We obtained data on incident HCC cases from the Osaka Cancer Registry, which was established by the Osaka Prefectural Government in 1962. The registry collects reports on patients with newly diagnosed cancer, including demographic and cancer-related information, from all medical institutions in Osaka Prefecture (9). These have been routinely supplemented by death certificates gathered by the Osaka Prefectural Government (9). For patients with cancer who were enrolled in the registry on the basis of their death certificate, we contacted the issuing hospital to obtain information on diagnosis and treatment and to establish the date of HCC incidence, which we determined to be the time of diagnosis at that hospital. We site-coded the data according to the International Classification of Diseases for Oncology, Third Edition (10). We included patients with HCC (codes 8170 through 8180). The protocol was approved by the ethics committee of the Osaka Medical Center for Cancer and Cardiovascular Diseases. From 1981 to 2003, 48166 men and 15696 women with HCC were documented in the Osaka Cancer Registry. We calculated the annual age-standardized incidence rates of HCC (world population as a standard population) by sex between 1981 and 2003. To characterize temporal trends for HCC, we assessed 10-year, age-specific incidence rates of HCC between 1981 and 2003 in individuals age 50 to 79 years. We studied these particular age-specific rates because most HCV-related HCC cases in the Japanese population occur between the ages of 50 and 79 years (4). We used the annual population estimates from 1981 to 2003, which were based on the average population in each sex and age category for the Osaka Prefecture during the particular period, as denominators for calculating incidence rates. The annual population estimates were based on data from the 1980, 1985, 1990, 1995, 2000, and 2005 Japanese population censuses, with linear interpolation for the years in between. Statistical Analysis To identify years when a statistically significant change in the slope of the temporal trend in the incidence occurred, we applied the joinpoint regression model by using the Joinpoint Regression Program, version 3.0 (U.S. National Cancer Institute, Bethesda, Maryland). We assumed constant variance and uncorrelated errors (11) because we could not detect heteroskedasticity by the White test or autocorrelation by the Durbin-Watson test in men or women in any age group. We computed the estimated slopes describing the average annual change of incidence rate per 100000 persons and the corresponding 95% CIs for each trend by fitting a piecewise regression line to the rates, using calendar year as a regression variable. We used the permutation test method to identify years when a statistically significant change had occurred (P< 0.05) and set the number of randomly permuted data sets at 4499. We set the number of joinpoints to a minimum of 0 and a maximum of 3 in the Joinpoint Regression Program. Data Collection on Prevalence of HCV Infection among Patients with HCC The Osaka Cancer Registry does not collect serologic data on HCV infection in the registered patients. Therefore, we used data on HCV seropositivity from patients with HCC that was diagnosed at 10 hospitals in Osaka Prefecture (1 university hospital, 2 cancer centers, and 7 general hospitals) to estimate the prevalence of HCV infection in patients with HCC. We considered the HCC diagnosis confirmed when the patient had positive histologic or positive radiologic results by enhanced computed tomography or hepatic angiography. We collected data on the patients sex, date of birth, date of diagnosis between 1990 and 2003, first Chinese letter of the family name, and presence of hepatitis B surface antigen and antibody to hepatitis C (anti-HCV) as assessed by any commercially available kit. We did not collect the full first and family name for reasons of confidentiality. Because anti-HCV testing first became available in Japan in 1990, we collected data on patients whose HCC diagnosis was between 1990 and 2003. One investigator checked for duplication of the data set, because some patients might have been registered multiple times among the participating hospitals as a result of referrals and recurrence of HCC. We defined HCV-related HCC as occurring in patients who were HCV-seropositive at the time of diagnosis. We calculated the sex-specific, age-specific (50 to 59, 60 to 69, or 70 to 79 years), and period-specific (1990 to 1992, 1993 to 1995, 1996 to 1998, 1999 to 2001, or 2002 to 2003) prevalences of HCV seropositivity for patients with HCC. We then multiplied prevalence rates by the corresponding strata of the HCC incidence rate obtained from the Osaka Cancer Registry data. Thus, we derived the denominators from the general population in Osaka through the denominators of the HCC incidence rate and obtained the numerators by multiplying the prevalence rates by the HCC incidence rate. We calculated the incidence rate of nonHCV-related HCC by subtracting HCV-related HCC from total HCC. Thus, we describe trends for the estimated incidence rates of HCV-related and nonHCV-related HCC between 1990 and 2003 in Osaka Prefecture. We calculated the CI of the estimated rates by multiplying the lower and upper limits of the CI of the prevalence based on SE by the corresponding HCC incidence rate. Role of the Funding Source This study was supported by the Osaka Prefectural Government between 1990 and 2000 and Grants-in-Aid for Hepatitis Research of the Japanese Ministry of Health, Labor, and Welfare. There is no conflict of interest in the study. The funding sources had no role in the collection, management, or analysis of data. Results The age-standardized incidence rate of HCC in men increased between 1981 and 1987 from 29.2 to 41.9 cases per 100000 persons, then fluctuated until 1995. After that, it steadily decreased to 24.0 cases per 100000 persons in 2003 (Figure 1). Among women, the age-standardized incidence rate of HCC increased between 1981 and 1996 from 6.6 to 10.8 cases per 100000 persons, then gradually decreased to 7.3 cases per 100000 persons in 2003 (Figure 1). Figure 1. Trends in age-standardized (world population) incidence of hepatocellular carcinoma in Osaka, Japan, 19812003. Figure 2 shows the trends in the incidence of HCC among men and women age 50 to 59 years, 60 to 69 years, and 70 to 79 years in Osaka between 1981 and 2003. The HCC incidence rate increased from 1981 to 1986 among men age 50 to 59 years, from 1981 to 1995 among men age 60 to 69 years, and from 1981 to 2000 among men age 70 to 79 years (average annual change of the incidence rate [per 100000 persons], 10.0, 10.7, and 6.2, respectively) (Table 1). A striking downward trend occurred after the year of peak incidence in the 3 age groups (7.9 until 1996, 22.3 until 2003, and 12.4 until 2003, respectively). Among men age 50 to 59 years, there was a second joinpoint (a change from rapid to moderate decrease) in 1996, resulting in a slope of 3.1 until 2003. Among women age 50 to 59 years, 60 to 69 years, and 70 to 79 years, the incidence rates of HCC peaked in 1991, 1997, and 2000, respectively (Table 1). The rates in women seemed to increase slightly from 1981 until the year of the joinpoint, with slopes of 0.43, 2.07, and 3.10, respectively. Thereafter, HCC incidence rates in women decreased through 2003 at a

Efficacy of interferon therapy in patients with chronic hepatitis C. Comparison between non-drinkers and drinkers.

BACKGROUND Alcohol has been reported to be an important factor that modulates the development and prognosis of chronic viral hepatitis; however, little is known about interaction of alcohol intake and chronic hepatitis C. The aim of this study was to examine whether alcohol drinking affects the effectiveness of interferon (IFN) therapy for chronic hepatitis C. METHODS Thirty-nine patients with chronic hepatitis C were divided into three groups on the basis of the amount of alcohol intake before IFN therapy: group I (n = 15), non-drinkers; group II (n = 14), less than 70 g/day; and group III (n = 10), more than 70 g/day of ethanol intake for at least 10 years. The IFN (total dose, 330 +/- 206 MU) was administered daily for 2 weeks and then intermittently. Drinkers stayed abstinent for at least 1 month before, during, and after IFN therapy. The sustained responder was defined as the patient who showed normal alanine aminotransferase (ALAT) levels continuously for more than 6 months after the therapy. The liver histology (HAI score) and serum hepatitis C virus (HCV) RNA were also examined before and after the therapy. RESULTS There was no significant difference among the three groups in the level of ALAT before IFN therapy, age, total dose of IFN, and liver histology. The rates of sustained responders in groups I, II, and III were 53.3%, 42.9%, and 0%, respectively, resulting in a significantly lower rate in group III than in groups I (p < 0.01) and II (p < 0.01). The serum HCV-RNA turned negative after the therapy in 58.3%, 20.0%, and 12.5% of groups I, II, and III, respectively, leading to a significantly lower rate of disappearance of HCV-RNA in group III than in group I (p < 0.05). CONCLUSION The IFN therapy for chronic hepatitis C was less effective in heavy drinkers than in non-drinkers.

Risk of non-Hodgkin's lymphoma in patients with hepatitis C virus infection

Hepatitis C virus (HCV) has been suggested to play an etiological role in the development of B‐cell non‐Hodgkins lymphoma (NHL) in Italy. However, another study in Scotland questioned increased risk of development of NHL in patients with chronic HCV infection. A total of 2,162 patients admitted to 3 hospitals in Osaka, where the incidence of HCV‐related hepatitis is highest in Japan, during the period from 1957 to 1997 were followed up from the date of diagnosis of chronic HCV‐related hepatitis until 30 October 1997. Overall, 12,404.5 person‐years of observation were accrued with a follow‐up period ranging from 0.25 to 40.4 (average 5.74) years. NHL of the B‐cell type developed in 4 patients. The interval between onset of chronic HCV and NHL ranged from 6 to 36 (median 13) years. Expected number of cases of NHL in the sex‐, age‐ and calender year‐matched general population was 1.90, which gave a relative risk (RR) of 2.10 (95% confidence interval 0.57–5.38; p = 0.247). Taking the much higher RR for hepatocellular carcinoma among patients with HCV infection into account, chronic HCV infection was considered to be moderately associated with increased risk of NHL. Int. J. Cancer 80:237–239, 1999.

Effect of acute and chronic ethanol consumption on hepatic tissue oxygen tension in rats

In vivo hepatic tissue oxygenation was investigated in chronically ethanol-treated rats using micro oxygen electrode and reflectance spectrophotometry. Effect of acute ethanol administration was also studied. Hepatic oxygen tension of rats treated with ethanol chronically (daily ethanol intake, 9-12 g/kg for 9 months) was very low as compared with that of normal rats, the decrease being comparable to that of rats treated with carbon tetrachloride. The hepatic oxygen tension in normal controls shortly after ethanol ingestion increased from basal level (median, 23 mmHg) to 40-70 mmHg, while in chronically ethanol-treated rats, the hepatic oxygen tension decreased transiently, followed by a gradual increase, but it still remained low. In CCl4-treated rats, the hepatic oxygen tension decreased further after the ethanol ingestion. It is concluded that chronic ethanol consumption in rats resulted in hepatic hypoxia with decreased liver blood flow and volume. Also acute ethanol administration does not induce hepatic hypoxia in normal rats, while in rats with injured liver it induces hypoxia.

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

Summary.  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 ≥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 μg/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 (≥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.

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.

IgG Oligosaccharide Alterations Are a Novel Diagnostic Marker for Disease Activity and the Clinical Course of Inflammatory Bowel Disease

BACKGROUND AND AIMS:Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.METHODS:Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohns disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.RESULTS:The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P < 0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.CONCLUSIONS:G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.