Keisuke Nakata

Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein.

BACKGROUND The sugar-chain structures of circulating alpha-fetoprotein in patients with hepatocellular carcinomas differ from those in patients with cirrhosis. We studied the reactivity of alpha-fetoprotein with two lectins, Lens culinaris agglutinin A and erythroagglutinating phytohemagglutinin, to monitor the evolution of hepatocellular carcinoma in patients with cirrhosis. METHODS Among 361 patients with cirrhosis caused mainly by chronic hepatitis B or hepatitis C virus infection, 33 with base-line serum alpha-fetoprotein concentrations > or = 30 ng per milliliter or more were found to have hepatocellular carcinomas during a mean follow-up of 35 months. The lectin-reactive profiles of the alpha-fetoprotein in the serum of these 33 patients were analyzed and compared with those in the serum of 32 patients with cirrhosis who had increased base-line serum alpha-fetoprotein concentrations and were followed for at least 24 months but in whom hepatocellular carcinoma did not develop. RESULTS At the time of tumor detection, 24 (73 percent) of the 33 patients with cirrhosis and hepatocellular carcinoma had higher percentages of L. culinaris agglutinin A-reactive alpha-fetoprotein (alpha-fetoprotein L3), erythroagglutinating phytohemagglutinin-reactive alpha-fetoprotein (alpha-fetoprotein P4+P5), or both than the 32 patients with cirrhosis but no hepatocellular carcinoma. Among the 24 patients, one or both of the markers were first elevated 3 to 18 months before the hepatocellular carcinoma was detected by imaging techniques. CONCLUSIONS Measurements of the alpha-fetoprotein L3 and alpha-fetoprotein P4+P5 fractions of serum alpha-fetoprotein allow the differentiation of hepatocellular carcinoma from cirrhosis in some cases and serve as predictive markers for the development of hepatocellular carcinoma during the follow-up of patients with cirrhosis.

Influence of interleukin-10 gene promoter polymorphisms on disease progression in patients chronically infected with hepatitis B virus

OBJECTIVES:The role of host genetic factors in chronic hepatitis B virus (HBV) infection is not fully understood. We studied the influence of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) gene promoter polymorphisms on disease progression in HBV carriers.METHODS:The sample population included 213 Japanese HBV carriers and 52 healthy volunteers. Of 213 HBV carriers, 66 were considered to be asymptomatic carriers based on the sustained normalization of serum ALT together with seropositivity for the antibody to hepatitis B e antigen (anti-HBe), and 147 were found to have chronic progressive liver disease including cirrhosis. Five biallelic polymorphisms in the TNF-α gene promoter and three biallelic polymorphisms in the IL-10 gene promoter were analyzed by polymerase chain reaction in combination with direct sequencing or restriction fragment length polymorphism assay.RESULTS:Allelic distributions of both gene promoters were not significantly different between HBV carriers and healthy volunteers. In HBV carriers, the TNF-α gene promoter polymorphisms were not linked to disease progression. In contrast, allelic frequencies of T and A at positions −819 and −592, respectively, in the IL-10 gene promoter, as well as the frequencies of ATA haplotype at positions −1082/− 819/− 592 (which is characterized with low capacity for IL-10 production), were significantly higher in asymptomatic carriers than in patients with chronic progressive liver disease. Even after adjusting for individuals positive for anti-HBe, such a relationship could be found between the two groups.CONCLUSION:In chronic HBV infection, inheritance of the IL-10 gene promoter polymorphisms is involved in a host genetic factor that is relevant to disease progression.

Interferon-|[alpha]| sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-|[kappa]|B inactivation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in a variety of cancer cells with little or no effect on normal cells. Human hepatoma cells, however, are resistant to TRAIL-induced apoptosis. Since interferon-α (IFN-α) is capable of enhancing TNF-α-induced apoptosis in certain cancer cells, we evaluated the effect of IFN-α on TRAIL-induced apoptosis of human hepatoma cells. IFN-α pretreatment enhanced TRAIL-induced apoptosis of HuH-7 and Hep3B cells, in which IFN-α upregulated the expression of DR5, a death receptor of TRAIL, and downregulated the expression of survivin, which has an antiapoptotic function. In contrast, IFN-α did not enhance TRAIL-induced apoptosis of HepG2 cells, in which expression of DR5 and survivin was not affected by IFN-α. On the other hand, TRAIL activated NF-κB composed of RelA-p50 heterodimer, a key transcription factor regulating cell survival, in HuH-7 and HepG2 cells. However, IFN-α pretreatment repressed the TRAIL-mediated activation of NF-κB and decreased its transcriptional activity in HuH-7 but not in HepG2 cells. Moreover, IFN-α pretreatment clearly augmented TRAIL-mediated caspase-8 activation in HuH-7 cells. Our results suggest that IFN-α could sensitize certain human hepatoma cells to TRAIL-induced apoptosis by stimulating its death signaling and by repressing the survival function in these cells.

Association between nonalcoholic fatty liver, markers of obesity, and serum leptin level in young adults.

OBJECTIVES:The aim of the present study was to clarify the risk factors for nonalcoholic fatty liver in young adults.METHODS:One thousand two hundred two students, aged 18–21 yr, received matriculation health examinations, including measurements of body mass index and percent body fat and determination of serum levels of ALT at Nagasaki University in 1998. One hundred twenty-nine were found to have borderline or elevated levels of serum ALT, and 105 of the 129 students (75 men and 30 women) were subjected to further analysis for the presence of fatty liver using ultrasonography, by which both the degree of steatosis and the abdominal wall fat index (AFI) corresponding to the ratio of visceral to s.c. adipose tissue (V/S ratio) were evaluated, in addition to determination of the serum level of leptin.RESULTS:Of 105 students, 74 (70%) had fatty liver. The incidence of moderately to severely fatty liver was significantly higher in men than in women. In parameters related to obesity, the close correlation between body mass index and percent body fat was observed in both sexes. The serum level of leptin correlated well with percent body fat and AFI (V/S ratio) in women, whereas it did not correlate with AFI (V/S ratio) in men. Multiple logistic regression analysis indicated that AFI (V/S ratio) was the only independent risk factor for fatty liver in both sexes.CONCLUSIONS:These results suggest that visceral fat distribution is a key risk factor for nonalcoholic fatty liver in young adults.

Treatment of hepatocellular carcinoma with transcatheter arterial embolization. Analysis of prognostic factors

Background. Transcatheter arterial embolization (TAE) is a useful treatment modality for hepatocellular carcinoma (HCC). To study the prognostic factors, survival time of patients with HCC after TAE was analyzed retrospectively using clinical manifestations of coexisting liver cirrhosis and clinical features of HCC.

Exacerbation of Thyroid Autoimmunity by Interferon α Treatment in Patients with Chronic Viral Hepatitis

In the present studies, the long term effects of IFN alpha on thyroid function and thyroid autoantibodies were evaluated in 42 patients with chronic viral hepatitis type C treated with IFN alpha for at least 4 months. Before IFN treatment, 41 patients tested were all euthyroid. Five (12%) out of 24 patients tested had positive tests for thyroid autoantibodies. MCHA/TPOAb was detected in all 5 and TGHA/TGAb in 3 out of these 5 patients. Six to 10 x 10(6) units (U) of recombinant or natural IFN alpha were given intramuscularly daily for the first 2 to 4 weeks, followed by 3 to 10 x 10(6) U thrice weekly for the subsequent 14 to 22 weeks. Thyroid dysfunction and/or rises in titers of thyroid autoantibodies were observed in 6 patients during IFN alpha treatment; clinically overt thyroid dysfunctions, destructive thyroiditis and thyrotoxicosis of unidentified etiology, developed in 2 patients 4 to 5 months after start of IFN treatment, subclinical hypothyroidism with a slight increase in serum TSH concentrations but no serum thyroid hormone alternations was observed in 2 patients, and increases in titers of thyroid autoantibodies without thyroid dysfunction were found in 2 patients. Thus, IFN alpha exacerbated thyroid autoimmunity exclusively in all patients with positive tests for thyroid autoantibodies prior to treatment, but did not induce thyroid autoimmunity in thyroid autoantibody-negative patients. These data suggest that the prolonged IFN alpha therapy can lead to exacerbation of thyroid autoimmunity in susceptible (thyroid autoantibody-positive) patients.

Evaluation of Nontumorous Tissue Damage by Transcatheter Arterial Embolization for Hepatocellular Carcinoma

The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6-diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE.

Reciprocal regulation of α-fetoprotein and albumin gene expression by butyrate in human hepatoma cells

Abstract Background/Aims: Butyrate, a product of colonic bacterial flora, functions as an antiproliferative agent and induces cell differentiation in a variety of cell types. In the present study, the effects of butyrate on cell growth and expression of α-fetoprotein (AFP) and albumin genes in HuH-7 human hepatoma cells were investigated. Methods: The HuH-7 cells were treated with sodium butyrate (0-1 mmol/L), and numbers of viable cells were counted at 24, 48, and 72 hours after treatment. To elucidate the effects of sodium butyrate on AFP and albumin gene expression, Northern blotting and transient chloramphenicol acetyltransferase plasmid transfection experiments were performed. Results: Cell growth was dose dependently inhibited by sodium butyrate. By Northern blot analysis, the level of AFP messenger RNA was reduced by treatment with sodium butyrate, whereas the level of albumin messenger RNA was elevated by this treatment. In transient chloramphenicol acetyltransferase plasmid transfection experiments, sodium butyrate repressed the AFP promoter activity but did not change the AFP enhancer or silencer activities. In contrast, the albumin promoter activity was stimulated by sodium butyrate. Conclusions: These results suggest that butyrate leads to the reciprocal differentiating regulation of AFP and albumin gene expression at the transcriptional level in human hepatoma cells.

Relationship between sustained elevation of serum alanine aminotransferase and progression from cirrhosis to hepatocellular carcinoma: comparison in patients with hepatitis B virus- and hepatitis C virus-associated cirrhosis.

Abstract  Most patients with hepatocellular carcinoma (HCC) in Japan have hepatitis B virus (HBV)‐or hepatitis C virus (HCV)‐associated cirrhosis. In the present study, the risk of HCC in patients with cirrhosis was analysed by the levels of serum alanine aminotransferase (ALT). One hundred and one (78%) of 129 patients with cirrhosis registered from April 1979 were followed at monthly intervals with the measurement of serum ALT. Of 101 patients, 38 tested positive for hepatitis B surface antigen (HBsAg) but negative for antibody to HCV (anti‐HCV; HBV group), 47 tested negative for HBsAg but positive for anti‐HCV (HCV group) and nine tested positive and seven tested negative for both. Mean serum ALT during follow‐up was calculated on the basis of monthly values during the observation period that started at enrolment and ended with the detection of HCC or at the end of March 1994. By the end of March 1994, 37 (37%) patients developed HCC; 12 were in the HBV group, 21 in the HCV group and four were in the group positive for both. Mean serum ALT during the observation period was similar in patients who developed HCC and those who did not develop HCC in the HBV group. In contrast, the value was significantly higher in patients who developed HCC than in patients who did not develop HCC in the HCV group (P < 0.05).

Spontaneous loss of hepatitis B surface antigen in chronic carriers, based on a long-term follow-up study in Goto Islands, Japan

Abstract: Annual mass examination was performed between 1972 and 1997 in Tomie-town, Goto Islands, Japan, where hepatitis B virus (HBV) infection is very prevalent. In the present study, the incidence of spontaneous loss of hepatitis B surface antigen (HBsAg) in HBsAg carriers was determined in this area. Three thousand and nineteen inhabitants were tested for HBsAg two or more times in our annual surveys. Among them, 131 (4.3%) were defined as chronic HBsAg carriers based on the persistence of HBsAg for 1 or more years. These 131 subjects were followed for 12.2 ± 7.6 years. During the follow-up period, spontaneous loss of HBsAg occurred in 38 (29%) of the 131 carriers, with a yearly incidence of 2.5%. This loss was seen more frequently in carriers aged 40 years or more on enrollment than in those aged less than 40 years during the same observation periods (P = 0.0141), irrespective of sex or the results of liver function tests. The values for liver function test results were similar before and after loss of HBsAg in these carriers. Stored serum samples were available for later analysis of HBV-DNA by polymerase chain reaction in 32 carriers with loss of HBsAg. The HBV-DNA sequence was detected in 26 (81%) and 2 of the 32 carriers (6%) before and after loss of HBsAg, respectively. These results indicate that spontaneous loss of HBsAg, largely attributable to clearance of viremia, occurs age-dependently in chronic carriers.