Post-therapeutic needle biopsy in patients with hepatocellular carcinoma is a useful tool to evaluate response to proton irradiation.

AIM: Proton beam therapy is safe and more effective than conventional radiation therapy for the local control of nodular hepatocellular carcinoma (HCC). However, evaluating therapeutic response by imaging is not accurate during the early post-irradiation period. Therefore, we examined whether the histopathological study of biopsy specimens obtained at 3 weeks after irradiation can be used to more accurately assess therapeutic response. METHODS: Fifteen HCC lesions from 13 patients were treated with proton beam irradiation. Tissue biopsy samples were obtained using abdominal ultrasound-guided percutaneous fine-needle aspiration from the center of the tumor before, 3 weeks after and 1 year post-proton therapy. The specimens were examined after staining with hematoxylin-eosin (HE) and a MIB-1 antibody. RESULTS: MIB-1 labeling indices (LI) before treatment were 13.0 ± 8.5% (mean ± SD; range, 0.6-27.0), whereas those 3 weeks after proton therapy were significantly reduced to 3.2 ± 2.4% (range, 0.6-8.9) (P < 0.05). Although the tumor size was reduced, we did not observe a reduction in tumor blood flow by dynamic computed tomography or degenerative changes by HE. All lesions that displayed reduced MIB-1 LI at 3 weeks post-proton treatment were ultimately diagnosed as complete response at 1 year after treatment. In contrast, one case with increased MIB-1 LI at 3 weeks had significant tumor size progression at 1 year post-treatment. CONCLUSION: The percutaneous fine-needle aspiration biopsy of HCC is a safe and useful tool that can be used to evaluate the response to proton irradiation. In particular, MIB-1 LI may provide additional information to assess the therapeutic response of HCC during the early post-irradiated period.

Hepatitis C virus infection causes hypolipidemia regardless of hepatic damage or nutritional state: An epidemiological survey of a large Japanese cohort.

AIM: Infection with hepatitis C virus (HCV) is the leading cause of liver cirrhosis that develops into hepatocellular carcinoma. Previous studies have shown in vitro that lipids within hepatocytes are crucially important for a series of HCV infection-proliferation-release processes. On the other hand, in the patients with HCV, the serum total cholesterol (Total-C) and low-density lipoprotein cholesterol (LDL-C) levels have been reported to be lower. We conducted an epidemiological survey of a large cohort and investigated whether the lower serum lipid levels were caused by a direct or the secondary effects of HCV infection (i.e. hepatic damage or nutritional disorder). METHODS: Among 146 857 participants (male, 34%; female, 66%) undergoing public health examinations between 2002 and 2007 in Ibaraki Prefecture, Japan, the HCV positive rates determined by HCV antibody/antigen and/or RNA tests were 1.37% and 0.67% in males and females, respectively. RESULTS: In addition to Total-C and LDL-C, serum high-density lipoprotein cholesterol and triglyceride concentrations were also significantly lower in the HCV positive subjects compared with the negative subjects, regardless of sex, age or nutritional state evaluated by body mass index. Multivariate analysis showed that HCV infection was the strongest among the factors to be significantly associated with the lower level of these lipids. Particularly, the hypolipidemia was also confirmed in the HCV positive subjects with normal aminotransferase levels (alanine aminotransferase ≤30 and aspartate aminotransferase ≤30). CONCLUSION: This epidemiological survey in a large Japanese cohort suggests that the HCV infection itself might directly cause hypolipidemia, irrespective of host factors including age, hepatic damage and nutritional state.

The associated markers and their limitations for the primary screening of HCV carriers in public health examination.

AIM: Although the anti-hepatitis C virus (HCV) antibody test has been recommended to the whole Japanese population, most countries have not implemented it. The present study aims to re-evaluate the usefulness of markers examined in the general health examination for the initial screening of HCV carriers. METHODS: Of the overall population, 25 142 individuals (8876 males, 16 266 females) participated in health examinations with HCV tests in 2005, and the most commonly associated markers for HCV-positive subjects were explored by multivariate analysis, based on blood biochemical, physical, sphygmomanometric and hematological parameters. Thereafter, the efficiencies of the markers were estimated from a total population of 85 013 individuals (29 502 males, 55 511 females) in 2003-2005. RESULTS: The most significantly associated markers for HCV positivity were aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Optimal limits of ALT and AST by receiver-operator characteristic (ROC) analysis were 24 and 27 IU (male, 33 and 28 IU; female, 22 and 26 IU), respectively. However, one-quarter of HCV carriers were not found to be positive using the optimal limits of aminotransferases. CONCLUSION: The present study confirmed the limitation of serum aminotransferase levels as markers of HCV for primary screening. Therefore, at present, an anti-HCV antibody test is required for the efficient screening of HCV carriers in all health examinations.

Regulatory T cells and liver pathology in a murine graft versus host response model.

AIM: We have previously reported in mice the hepatic inflammatory in graft versus host response (GVHR) model due to the disparity of major histocompatibility complex class-II. The regulatory T (Treg) cells have been reported to control excessive immune response and prevent immune-related diseases. This study aimed to investigate the pathogenesis profiles of chronic GVHR progression, focusing on the Treg cells. METHODS: GVHR mice induced by parental spleen CD4(+) T cell injection were sacrificed after 0, 2, 4, and 8 weeks (G0, G2, G4, G8). Further, one GVHR group received anti-IL-10 antibody in advance and were maintained for 2 weeks. Pathologic profiles of hepatic infiltrating inflammatory cells were evaluated by haematoxylin and eosin and immunohistochemistry staining with surface markers including Treg cell markers. RESULTS: Remarkable hepatic inflammatory in G2 significantly and gradually improved over time up to G8. In immunohistochemical staining, the increased IL-10 receptor beta(+) Tr1 cells in G2 were maintained through to G8; although other inflammatory cells decreased from G2 to G8. By contrast, in the anti-IL-10 antibody received-GVHR mice, the Tr1 cells were not detectable with significant inflammatory aggravation, while FoxP3(+) Treg cells significantly enhanced. CONCLUSIONS: These findings in the GVHR mice suggest that the expression and activity of Treg cells, especially the Tr1 cells, might be key factors for pathologic alteration in immune-related liver disease.

Serological survey of avian H5N2-subtype influenza virus infections in human populations.

To investigate the distribution of antibodies against H5N2 influenza virus in humans living in Ibaraki prefecture, Japan, 266 single serum samples were collected to perform serological tests. Results were compared to investigate the relationship between positive results and several factors. The number of positive serum neutralization antibody titers (> or = 40) against avian influenza virus A/H5N2 was significantly greater (P < 0.05) among poultry workers, in comparison to a Japanese healthy population. The geometric mean titers of serum neutralization antibody against A/H5N2 were significantly higher (P < 0.05) among Ibaraki inhabitants and poultry workers (P < 0.0001) when compared to a Japanese healthy population. Seropositivity against A/H5N2 virus was significantly (P < 0.05) associated with age (> or = 50 years old) in poultry workers. These results suggest that seropositivity against H5N2 virus in Ibaraki specimens is significantly higher than those of a Japanese healthy population and that the surveillance of avian influenza viruses is very important to evaluate the invasion or emergence of new pandemic influenza viruses from species other than humans.

Estimation of measles vaccine coverage needed to prevent transmission in schools.

BACKGROUND: The aim of the present study was to produce the first estimation in Japan of the basic reproduction number (R(0)) and the minimum level of vaccine coverage needed to prevent measles outbreaks (P(c)). METHODS: A questionnaire survey was conducted during two measles outbreaks among 12-15-year-old middle school students in one prefecture in spring, from the end of February to the beginning of May 2002, and a stochastic mathematical model was constructed to calculate vaccine effectiveness (VE) and the basic reproduction number (R(0)). P(c) was calculated from R(0) and VE. RESULTS: In outbreak 1 (school A), 62 (94%) of 66 patients responded to the questionnaire. Of a total of 601 students, 534 (88.9%) responded. Of these, 82.6% (441/534) had previously received measles vaccine. In outbreak 2 (school B), 20 (99%) of 21 patients responded. Of a total of 375 students, 373 (99.5%) responded. Of these, 317 (85.0%) received measles immunization. Mathematical analysis was as follows: in outbreak 1 R(0) was 7.40 (95% confidence interval [CI]: 7.36-7.44) and VE was 76.55% (95%CI: 53.24-87.54). In outbreak 2, R(0) was 18.89 (95%CI: 18.88-18.90) and VE was 98.54% (95%CI: 94.89-99.73). Consequently, P(c) was 112.97% (95%CI: 92.29-145.52) in outbreak 1 and 96.11% (95%CI: 93.81-98.53) in outbreak 2. CONCLUSION: Because of the lower VE in outbreak 1, measles virus transmission could not have been stopped even if all students received a single dose of vaccine. In outbreak 2, with higher VE, the outbreak could have been prevented by increasing the proportion of students who had been vaccinated.

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS.

We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio=3), approximately 833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77-2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47+/-0.07 nmol/mg protein, which coincided completely with the observed X0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS.

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol.

Hypercholesterolemia is an important paraneoplastic syndrome in patients with hepatoma, but the nature of this defect has not yet been identified. We investigated the molecular mechanisms of hypercholesterolemia in a hepatoma-bearing rat model. Buffalo rats were implanted in both flanks with Morris hepatoma 7777 (McA-RH7777) cells. After 4 weeks, tumor weight was 5.5+/-1.7 g, and serum cholesterol level increased from 60+/-2 to 90+/-2 mg/dL. Protein and mRNA expression of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) was markedly higher in tumors than in livers. These increases were associated with activation of liver X receptor alpha (LXRalpha) as a result of the increased tissue oxysterol concentrations. The accumulation of oxysterols in the hepatomas appeared to be caused mainly by the upregulation of cholesterol biosynthesis, despite the increased tissue sterol concentrations. Overexpression of the sterol regulatory element-binding protein (SREBP) processing system relative to sterol concentration contributed to the resistance to sterols in this tumor. In addition, bile acid biosynthesis was inhibited despite the reduced expression of the small heterodimer partner (SHP) and activated LXRalpha, which also appeared to contribute to the accumulation of oxysterols followed by the acceleration of cholesterol efflux. In conclusion, hypercholesterolemia in McA-RH7777 hepatoma-bearing rats was caused by increased cholesterol efflux from tumors as a result of activation of LXRalpha. Overexpression of the SREBP processing system contributed to the activation of LXRalpha by maintaining high oxysterol levels in tissue.

Taurine inhibits oxidative damage and prevents fibrosis in carbon tetrachloride-induced hepatic fibrosis.

BACKGROUND/AIMS: The aim of the study was to examine the effects of taurine on hepatic fibrogenesis and in isolated hepatic stellate cells (HSC). METHODS: The rats of the hepatic damage (HD) group were administered carbon tetracholoride (CCl4) for 5 weeks and a subgroup received, in addition, a 2% taurine containing diet for 6 weeks (HDT). The HSC were isolated from normal rats and cultured for 4 days. RESULTS: The hepatic taurine concentration was decreased in the HD group. This loss and the hepatic histological damage and fibrosis (particularly in the pericentral region), were reduced following taurine treatment. Furthermore, the hepatic alpha-SMA, lipid hydroperoxide and 8-OHdG levels in serum and liver, as well as hepatic TGF-beta1 mRNA and hydroxyproline levels were significantly increased in the HD group, and most of these parameters were significantly reduced following taurine treatment. In contrast to the MAP-kinase and Akt expressions, which remained unchanged, the lipid hydroperoxide and hydroxyproline concentrations, as well as TGF-beta1 mRNA levels were significantly reduced by taurine in activated HSC. CONCLUSIONS: Oral taurine administration enhances hepatic taurine accumulation, reduces oxidative stress and prevents progression of hepatic fibrosis in CCl4-induced HD rats, as well as inhibits transformation of the HSC.

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.

The harmful effect of exercise on reducing taurine concentration in the tissues of rats treated with CCl4 administration.

BACKGROUND: We have previously reported that oral taurine administration reduced the frequency of painful muscle cramps in patients with liver cirrhosis, and that skeletal muscle taurine concentration was significantly decreased after exercise. The aim of this study was to examine taurine concentration in various tissues of a liver damaged with fibrosis (LD) in a rat model before and after exercise. METHODS: Rats were divided into normal (NML) and LD groups. The LD group received CCl(4) injection for 10 weeks. Thereafter, both groups were divided into control (NML/CTL, LD/CTL) and exercise (NML/EX, LD/EX) groups, respectively. The rats in the EX groups were subjected to treadmill running. Plasma, liver, brain, heart, and skeletal muscle taurine concentration, as well as plasma and liver lipid peroxidase (LPO) concentration, were measured. RESULTS: The liver, brain, and skeletal muscle taurine concentration in the LD groups was significantly decreased compared to that in the respective NML groups. Furthermore, the taurine concentration in the heart and skeletal muscles in the LD/CTL group was significantly decreased post exercise. The respective plasma and liver LPO concentration in the LD groups was significantly increased compared to that in the corresponding NML group. Moreover, plasma LPO concentration in the LD/EX group was significantly higher than in the LD/CTL group. CONCLUSIONS: Tissue taurine concentration, particularly in skeletal muscle, was significantly decreased in the LD model rats induced by CCl(4) administration, and furthermore, the significantly decreased concentration, except for liver, was aggravated by exercise, even though at lower intensity.

Significance of plasma 7alpha-hydroxy-4-cholesten-3-one and 27-hydroxycholesterol concentrations as markers for hepatic bile acid synthesis in cholesterol-fed rabbits.

Plasma 7alpha-hydroxy-4-cholesten-3-one has been used as an index of hepatic bile acid synthesis. The aim of the current study was to ascertain whether the level of this oxysterol reflects hepatic cholesterol 7alpha-hydroxylase activity when plasma cholesterol concentrations are markedly changed. In addition, the relationship of hepatic sterol 27-hydroxylase activity with plasma concentrations of 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid was studied. We used New Zealand white rabbits fed 2% cholesterol for 5 or 10 days and/or constructed bile fistula. Feeding cholesterol markedly increased and bile drainage reduced plasma cholesterol concentrations. Initially, in these models there was no correlation between plasma 7alpha-hydroxy-4-cholesten-3-one concentrations and hepatic cholesterol 7alpha-hydroxylase activities (r = -0.24, n = 10). Cholesterol feeding was associated with downregulated 7alpha-hydroxylase activities, while plasma 7alpha-hydroxy-4-cholesten-3-one concentrations were elevated in the presence of increased plasma cholesterol levels. However, this discrepancy was overcome and significant correlation was observed (r = 0.73, P <.05, n = 10) by expressing 7alpha-hydroxy-4-cholesten-3-one levels relative to cholesterol. In contrast, hepatic sterol 27-hydroxylase activities were not significantly correlated with plasma absolute (r = 0.23, difference not significant [NS], n = 10) nor cholesterol-related levels of 27-hydroxycholesterol (r = -0.13, NS, n = 10), or 3beta-hydroxy-5-cholestenoic acid concentrations (r = 0.30, NS, n = 10). In conclusion, plasma 7alpha-hydroxy-4-cholesten-3-one concentrations reflected hepatic cholesterol 7alpha-hydroxylase activities when the sterol levels were adjusted to plasma cholesterol concentrations in rabbits with hypercholesterolemia. The results suggest that plasma 7alpha-hydroxy-4-cholesten-3-one relative to cholesterol is a better marker for hepatic cholesterol 7alpha-hydroxylase activity than the absolute concentration when hypercholesterolemia is present. In contrast, 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid levels in plasma did not reflect hepatic sterol 27-hydroxylase activities even if the levels were adjusted to plasma cholesterol concentrations.

Progression of autoimmune-mediated hepatic lesions in a murine graft-versus-host reaction by neutralizing IL-10.

C57BL/6 (B6) spleen T cells which were injected into major histocompatibility complex (MHC) class II-disparate (B6.C-H-2(bm/2)xB6) F1 hybrid mice induced autoimmune graft-versus-host reaction (GVHR). Early production of interferon (IFN)-gamma and delayed production of interleukin (IL)-10 might play an important role in the formation of GVHR hepatic lesions. To clarify whether blocking of IL-10 deteriorate autoimmune-mediated hepatic lesions induced by GVHR, and to elucidate the change of the T helper (Th)1/Th2 cytokines in the liver, anti-IL-10 monoclonal antibodies (mAbs, 500 µg) were given 4 h before the induction of GVHR. We evaluated the change of splenomegaly and GVHR-induced hepatic lesions. The changes of the expressions of IFN-gamma and IL-4 mRNA isolated from liver-infiltrating lymphocytes were measured by real-time polymerase chain reaction (PCR). In GVHR with anti-IL-10 mAbs mice splenomegaly and periportal cellular infiltration was significantly increased compared with those of GVHR mice. In these mice, both IFN-gamma and IL-4 mRNA expression levels were significantly elevated by the neutralization of IL-10. These findings suggest an important role of IL-10 in murine GVHR due to MHC class II disparity. IL-10 may play a crucial role in down-regulating autoimmune-related hepatic lesions.

Progression of autoimmune-mediated hepatic lesions in a murine graft-versus-host reaction by neutralizing IL-10.

C57BL/6 (B6) spleen T cells which were injected into major histocompatibility complex (MHC) class II-disparate (B6.C-H-2(bm/2)xB6) F1 hybrid mice induced autoimmune graft-versus-host reaction (GVHR). Early production of interferon (IFN)-gamma and delayed production of interleukin (IL)-10 might play an important role in the formation of GVHR hepatic lesions. To clarify whether blocking of IL-10 deteriorate autoimmune-mediated hepatic lesions induced by GVHR, and to elucidate the change of the T helper (Th)1/Th2 cytokines in the liver, anti-IL-10 monoclonal antibodies (mAbs, 500 &mgr;g) were given 4 h before the induction of GVHR. We evaluated the change of splenomegaly and GVHR-induced hepatic lesions. The changes of the expressions of IFN-gamma and IL-4 mRNA isolated from liver-infiltrating lymphocytes were measured by real-time polymerase chain reaction (PCR). In GVHR with anti-IL-10 mAbs mice splenomegaly and periportal cellular infiltration was significantly increased compared with those of GVHR mice. In these mice, both IFN-gamma and IL-4 mRNA expression levels were significantly elevated by the neutralization of IL-10. These findings suggest an important role of IL-10 in murine GVHR due to MHC class II disparity. IL-10 may play a crucial role in down-regulating autoimmune-related hepatic lesions.

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver.

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.