The RXR agonist LG100268 causes hepatomegaly, improves glycaemic control and decreases cardiovascular risk and cachexia in diabetic mice suffering from pancreatic beta-cell dysfunction

Aims/hypothesis. Although retinoid X receptor (RXR) and peroxisome proliferator activated receptor-γ (PPARγ) agonists have antidiabetic effects in hyperinsulinaemic animals, little information exists on their effects after pancreatic beta-cell failure. Thus, we examined if RXR and PPARγ agonists alter distinct metabolic pathways in animals suffering from impaired insulin secretion. Methods. Adverse side effects and antidiabetic responses were measured in db/db mice treated from 14–16 weeks of age with the RXR agonist, LG100268, and/or the PPARγ agonists, BRL49 653 or GW1929. Results. In animals treated with LG100 268 or BRL49653, serum glucose, glycohaemoglobin and the cardiovascular risk factor, fibrinogen, decreased to the same extent. Both of these agonists were equally effective at increasing insulin accumulation in beta cells, although neither agent had an effect on serum insulin concentrations. In contrast, the RXR agonist was less effective than the PPARγ agonists at lowering serum triglycerides and non-esterified fatty acids and increasing interscapular brown fat and body weight. Further, LG100 268 increased serum alkaline phosphatase and liver mass, hepatic fat accumulation, lauric acid hydroxylase activity, catalase-immunostaining and peroxisomal number more than the PPARγ agonists. Moreover, co-treatment with the RXR and PPARγ agonists reduced glucose, triglycerides, non-esterified fatty acids and cholesterol more than either agent alone. Conclusion/interpretation. These data suggest 1) RXR and PPARγ agonists decrease islet degeneration, cardiovascular risk and cachexia during later stages of diabetes, 2) RXR agonists are less effective than PPARγ agonists at decreasing serum lipids and causing weight gain and 3) RXR agonists have a more pronounced effect on liver metabolism (e. g. peroxisome accumulation and hepatomegaly) than PPARγ agonists. [Diabetologia (1999) 42: 545–554] Received: 13 July 1998 and in final revised form: 4 December 1998     

Significant Correlation Between Insulin Resistance and Serum Gamma-Glutamyl Transpeptidase (γ-GTP) Activity in Non-Drinkers

To understand the significance of elevated serum γ-GTP levels, factors relevant to the serum γ-GTP level were studied using data of health check-ups for the employees of a Japanese corporation. The γ-GTP level was positively correlated with levels of various liver function tests including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Except for them, the γ-GTP level was positively correlated with levels of insulin resistance, uric acid, total cholesterol, triglyceride, and body mass index. The correlation between the γ-GTP level and LDL cholesterol was also observed only when subjects who drank more than 5 times a week were selectively studied. When non-drinkers and opportunity drinkers were selectively studied, 63.6% of subjects whose γ-GTP level was more than 120 IU/liter showed elevated insulin resistance levels. Multiple factors including insulin resistance may affect serum γ-GTP activity in clinical subjects.     

Apoptosis and remodelling of beta cells by paracrine interferon-γ without insulitis in transgenic mice

Abstract Aims/hypothesis. To examine whether interferon-γ destroys islet beta cells directly or indirectly through lymphocyte activation, or whether direct action of interferon-γ on beta cells by itself induces diabetes without insulitis. Methods. To avoid possible nonspecific breakdown of beta cells by transgenic overexpression of interferon-γ by the insulin promoter, we generated transgenic mice expressing interferon-γ under the control of rat glucagon promoter (RGP-IFN-γ-Tg mice). Results. The absence of insulitis in RGP-IFN-γ-Tg mice enabled us to investigate the direct effects of paracrine interferon-γ. In RGP-IFN-γ-Tg mice, serum concentrations of interferon-γ and tumour necrosis factor-α (TNF-α) were 50 and 6 times higher than those in their littermates, respectively, and glucose-responsive insulin secretion decreased to one-half the level of that in the littermates. Transgenic interferon-γ induced remodelling of beta cells where apoptosis of many beta cells was compensated by their vigorous regeneration and diabetes did not occur in most of the RGP-IFN-γ-Tg mice. Conclusion/interpretation. Interferon-γ alone is insufficient for the complete destruction of beta cells in vivo, and factors other than interferon-γ including activated lymphocytes or other cytokines, are necessary in addition to interferon-γ for the development of Type I (insulin-dependent) diabetes mellitus. [Diabetologia (1999) 42: 566–573] Received: 16 November 1998 and in final revised form: 11 January 1999     

Microvessel density of hepatocellular carcinoma: its relationship with prognosis

Purpose: To elucidate the relationship between angiogenesis and prognosis after curative resection of hepatocellular carcinoma (HCC). Methods: An immunohistochemical study using anti-CD34 monoclonal antibody was carried out on surgical specimens from 78 HCC patients who had undergone curative resection; microvessel density (MVD) was counted and the overall survival and disease-free survival were analyzed retrospectively. Results: Blood vessels in the tumor were strongly stained by anti-CD34 antibody, but not those in the surrounding liver parenchyma. There were three types of tumor vessels: capillary-like (n = 59), sinusoid-like (n = 16) and mixed-type (n = 3). The median MVD count was 100 per field. The HCC were designated as hypovascular (n = 36) with an MVD count below 100, and hypervascular (n = 42) with an MVD count of 100 or more per field. The 5-year survival and disease-free survival rates were 49.7% and 42.8% respectively, and statistical analysis showed that the MVD level was not correlated with tumor size, capsule status, Edmondson's grade, α-fetoprotein level, associated cirrhosis, γ-glutamyltransferase, and serum HBsAg status. The sinusoid-like tumor vessels appeared more frequently in the more differentiated tumors (P < 0.05). No statistical difference in overall and disease-free survival between different MVD levels and microvessel types was found. Tumor size was the only predicting factor in the entire series. In patients with small HCC (≤ 5 cm, n = 40), 5-year survival and disease-free survival rates were 58.9% and 52.7% respectively, higher than the values in large HCC (39.8% and 32.0% respectively, P < 0.05). The MVD level was an independent predicting factor of disease-free survival, 5-year disease-free survival in the hypovascular group (74.6%) being better than that in the hypervascular group (34.7%, P < 0.05). Conclusions: The MVD level was not related to tumor size, capsule statuo, Edmondson's grade, α-fetoprotein level, associated cirrhosis, γ-glutamyltransferase and serum HBsAg status. In the entire series, tumor size was the only factor influencing survival after curative resection. However, in patients with small HCC, the MVD level was an independent factor of disease-free survival. The pathological and clinical implications of different types of tumor vessels in HCC remain to be studied. Received: 27 November 1998 / Accepted: 5 January 1999     

Peroxisome proliferator-activated receptor gamma ligand inhibits cell growth and invasion of human pancreatic cancer cells

Summary Background. Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in certain human cancers; ligand-induced PPARγ activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARγ ligands is not entirely known. Aim of Study. The purpose of this study was to examine the effect of PPARγ ligands on pancreatic cancer cell growth and invasiveness. Methods. The effect of two PPARγligands, 15 deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ₂ on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined. Results. Both 15d-PGJ₂ and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ₂ resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21^waf1 and p27^kip1. In addition, 15d-PGJ₂ treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ₂, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity. Conclusion. These results demonstrate that PPARγ ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.     

Visceral adiposity and leptin are independently associated with C-reactive protein in Korean type 2 diabetic patients

The inflammatory marker, C-reactive protein (CRP) is associated with long-term cardiovascular events. The aim of the study was to investigate the factors contributing to serum CRP, assess the relationship between CRP level and the parameters of visceral obesity, and examine the association between leptin and CRP level in type 2 diabetic patients. 150 patients with type 2 diabetes were enrolled. These patients were recently diagnosed (≤3 years) with type 2 diabetes and were drug naive or taking sulfonylureas only. BMI, WC, and serum concentration of CRP, glycosylated hemoglobin (HbA1c), glucose, lipids, plasminogen activator-1 (PAI-1) and leptin were measured. Insulin resistance was estimated by the insulin resistance index of homeostasis model assessment (HOMA-IR). We measured the carotid intima-media thickness (IMT). Fat mass assessed by dual-energy X-ray absorptionmetry and abdominal fat distribution was determined by CT scan. Serum concentration of CRP was significantly correlated with BMI (γ = 0.257, P < 0.01), WC (γ = 0.293, P < 0.01), fat mass (γ = 0.213, P < 0.01), total adipose tissue (γ = 0.263, P < 0.01), visceral adipose tissue (γ = 0.296, P < 0.01), insulin (γ = 0.189, P = 0.047), PAI-1 (γ = 0.206, P < 0.01), leptin (γ = 0.322, P < 0.01), mean IMT (γ = 0.132, P = 0.042), and HOMA-IR (γ = 0.172, P = 0.045). After adjustment for age and gender, multiple regression analysis showed that serum CRP was significantly associated with leptin (β = 0.326, P = 0.01) and visceral adipose tissue (β = 0.265, P = 0.035). In conclusion, serum CRP level is significantly associated with obesity, especially the visceral adipose tissue, and serum leptin is another important independent factor associated with CRP in Korean type 2 diabetic patients.     

A comparative study of the clinicopathological significance of E-cadherin and catenins (α, β, γ) expression in the surgical management of oral tongue carcinoma

Purpose: E-cadherin and catenins are important epithelial adhesion molecules in normal epithelium. Loss of E-cadherin-catenin adhesion is an important step in the progression of many epithelial cancers. E-cadherin and catenins expression in carcinoma of the tongue were evaluated in relation to their clinicopathological features and prognostic values. Method: Immunohistochemical staining was carried out with E-cadherin and (α, β, γ)-catenin monoclonal antibodies for 85 surgical specimens of oral tongue carcinoma, nine matched metastatic lymph nodes, and seven locally recurrent tumours. Results: There was under-expression in 85% of E-cadherin, 94% of α-catenin, 89% of β-catenin, and 83% of γ-catenin in the primary tumours. There was no correlation of E-cadherin/catenin expression with sex, age, cancer stage, and differentiation. Nodal metastasis was found in 68% of patients with weak expression of γ-catenin compared with 9% with strong expression in primary tumours (chi-square, P=0.02). E-cadherin was a significant prognostic factor for survival and recurrence; patients with weak E-cadherin expression had 53% 5-year survival compared with 85% with strong expression (Wilcoxon, P=0.0159). Conclusions: Both E-cadherin and catenins were highly under-expressed in oral tongue carcinoma, metastatic lymph node, and recurrent tumour. γ-catenin had predictive value for nodal metastasis. E-cadherin was, however, a more important prognostic factor for recurrence and survival. Received: 14 February 2000 / Accepted: 6 June 2000     

A male patient who developed late-onset primary biliary cirrhosis presenting with antinuclear envelope antibodies

 An 81-year-old man who had previously shown high levels of alkaline phosphatase (ALP), γ-glutamyltransferase (GTP), and total bilirubin presented with acute liver damage. He was positive for serum anti-gp210 and anti-p62 antibodies, but negative for serum antimitochondrial antibody. A liver biopsy revealed massive interstitial fibrosis and pseudolobulus, which were compatible with a diagnosis of primary biliary cirrhosis (PBC) at Scheuer's stage 4. He was given ursodeoxycolic acid at 600 mg/day. However, his condition deteriorated, and he eventually died of hepatic insufficiency in a state of malnutrition. We hypothesize that the presence of anti-gp210 and anti-p62 complex protein antibodies, rather than that of antimitochondrial antibodies, was correlated with the progression of PBC in this particular case. Received: December 18, 2000 / Accepted: December 26, 2001 Acknowledgments We thank Dr. Eng M. Tan of the W.M. Keck Foundation, Scripps Research Institute, LaJolla, California, USA, for the determination of antilamina-associated polypeptide (LAP)-2 antibodies in the SS patient included as a control in this study. We also thank Dr. P.A. Berg of Tubingen University for the determination of anti-M4 and anti-M8 antibodies. Correspondence to: K. Miyachi     

Expression of peroxisome proliferator-activated receptor γ (PPARγ) in normal human pancreatic islet cells

Abstract Aims/hypothesis. Thiazolidinediones are reported to improve pancreatic islet morphology and beta-cell function in rodents, supporting the hypothesis of a direct action of thiazolidinediones on endocrine islet cells. In this study we examined the expression of the peroxisome proliferator-activated receptor γ, a nuclear receptor that is activated by naturally occurring fatty acids and synthetic thiazolidinediones, in normal human endocrine pancreatic cells. Methods. Human islets were isolated from pancreata harvested in ten brain-dead lean non-diabetic adult donors. We analysed the gene and protein expression of the human peroxisome proliferator-activated receptor γ and evaluated the effects of peroxisome proliferator-activated receptor γ agonist on insulin secretion in human islet preparations. Results. The RT-PCR carried out on total RNA from four distinct human islet preparations demonstrated the presence of peroxisome proliferator-activated receptor γ mRNA. Western blot analysis showed the consistent expression of peroxisome proliferator-activated receptor γ protein. Peroxisome proliferator-activated receptor γ was shown to be present in all three endocrine cell types studied (alpha, beta and delta cells) by immunohistochemistry. Conclusion/interpretation. We found that peroxisome proliferator-activated receptor γ is highly expressed in human islet endocrine cells, both at the mRNA and protein levels. These results support the hypothesis of a direct influence of peroxisome proliferator-activated receptor γ agonist on human pancreatic endocrine cells. [Diabetologia (2000) 43: 1165–1169] Received: 29 March 2000 and in revised form: 4 May 2000     

Role of AMP-activated protein kinase gamma 3 genetic variability in glucose and lipid metabolism in non-diabetic whites

Aims/hypothesis AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that acts as an intracellular fuel sensor, directing multiple metabolic pathways in a catabolic direction in times of nutrient shortage. In humans, three different γ-subunits (γ₁, γ₂, γ₃) have been identified as AMPK regulators. The AMPKγ3 (protein kinase, AMP-activated, gamma 3 non-catalytic subunit, PRKAG3) isoform plays a role in gene regulation in glucose/lipid metabolism and skeletal muscle glycogen content. We investigated whether PRKAG3, in addition to being expressed in skeletal muscle, is also expressed in human liver. We also investigated whether genetic variance in PRKAG3 is associated with glucose and/or lipid metabolism in non-diabetic whites. Materials and methods After sequencing a screening cohort (n = 50) in the PRKAG3 locus, we genotyped 1061 participants for frequently found single nucleotide polymorphisms (SNPs). Association analyses between genotypes/haplotypes and metabolic traits were carried out. Results We detected PRKAG3 expression in human liver and skeletal muscle. Two SNPs (rs692243, rs6436094) with minor allele frequencies of 0.16 and 0.26 respectively and in moderate linkage disequilibrium (D′ = 0.92; r ² = 0.47) were found. rs692243 (C/G) confers a Pro71Ala mutation, while rs6436094 (A/G) is located in the 3′ untranslated region. No associations with prediabetic traits such as body fat distribution, insulin resistance or insulin secretion were found (p > 0.15 for all). However, the minor alleles of both SNPs were significantly associated with higher serum LDL-cholesterol and apolipoprotein (Apo) B-100 levels (rs692243: CG:LDL 4.3%, ApoB-100 3.4%; GG:LDL 7.6%, ApoB-100 5.4%; p = 0.008 and p = 0.01 respectively; rs6436094: AG:LDL 3.3%, ApoB-100 1.7%; GG:LDL 11.3%, ApoB-100 11.1%; p = 0.009 and p = 0.05 respectively; dominant model). The GG/GG diplotype homozygous for both minor SNP alleles displayed the highest LDL-cholesterol among all frequent diplotypes (p = 0.059). Conclusions/interpretation While genetic variability in PRKAG3 does not seem to have a major effect on glucose metabolism, it may play an important role in lipoprotein metabolism in humans.     

Serum bile acid levels as a predictor for the severity of liver fibrosis in patients with chronic hepatitis C

Summary. Serum bile acids (SBAs) are commonly elevated in cholestatic liver diseases, but it is unclear if SBA levels are also elevated in noncholestatic chronic liver diseases and whether those levels correlate with disease severity. We analysed SBA levels of 135 consecutive patients with chronic hepatitis C virus infection and correlated these levels with the degree of liver fibrosis as determined by liver biopsy. In addition, we assessed the accuracy of SBA levels as a noninvasive predictor for liver fibrosis by its comparison to the patients’ FibroTest scores. Two‐thirds (90/135 patients, 67%) of the study patients had nonsevere liver fibrosis (Metavir F0–F2), and the others (45/135, 33%) had severe fibrosis or cirrhosis (Metavir F3–F4). The SBA levels were significantly higher in patients with severe fibrosis as compared to nonsevere fibrosis (11.46 ± 10.01 vs 6.37 ± 4.69, P < 0.0001). Furthermore, a receiver operator characteristics curve based on a model that included serum bile acids, age, body mass index, serum AST, glucose and cholesterol levels suggested that this combination reliably predicts the degree of liver fibrosis and is not inferior to the current noninvasive FibroTest score (areas under the curve of 0.837 vs 0.83, respectively, P = 0.87). We conclude that measurement of SBA levels may have a clinical role as a simple noninvasive tool to assess the severity of HCV‐induced liver disease. Combined with widely available laboratory parameters, SBA levels can predict disease severity with a high degree of accuracy.     

Possible mechanisms of elevation of serum transaminase levels during interferon-β therapy in chronic hepatitis C patients

Background. Serum transaminase levels are frequently elevated in patients with chronic hepatitis C who are receiving interferon (IFN)-β therapy, despite hepatitis C virus (HCV)-RNA being eradicated from the serum. We examined liver histology to determine the reason for this elevation. Methods. Patients with chronic hepatitis C, diagnosed by liver histology and positive serum HCV-RNA, were given intravenous injections of IFN-β, at a daily dose of 6 MU, every day for periods of 6 to 12 weeks. When serum alanine transaminase (ALT) levels during the therapy were higher than three times the levels before the therapy, liver biopsy was performed. Histological findings on light microscopy were compared in liver biopsy specimens obtained before and during the therapy. Results. An increase in serum ALT levels was found in 19 of the 102 patients who received the IFN-β therapy. Autoimmune hepatitis was not contributory in any of these 19 patients, because serum antinuclear antigen was negative and IgG levels were not increased. Liver histology was examined in 10 of these 19 patients. The period between the start of IFN-β therapy and the biopsy during the therapy ranged from 14 to 46 days. In 2 patients, the extent of mononuclear cell infiltration in the liver and hepatocyte necrosis was less than the extent before the therapy. In the remaining 8 patients, the grade of chronic hepatitis was unchanged during the therapy, but vacuole formation and apoptotic nuclei in hepatocytes were found in 2 patients, and centrilobular necrotic areas in 1 patient. Conclusions. The elevation of serum ALT levels during IFN-β therapy in chronic hepatitis C patients was not a result of increased hepatitis activity. Degenerative, apoptotic, and necrotic changes in hepatocytes, probably a result of the cytotoxic effects of IFN-β, may have contributed to this elevation of ALT levels. However, such changes were absent in most of the patients, suggesting that decisions on the discontinuation of IFN-β therapy must be made in accordance with liver histology findings. Received: March 7, 2001 / Accepted: June 22, 2001     

Intrahepatic distant recurrence after radiofrequency ablation for a single small hepatocellular carcinoma: Risk factors and patterns

Background. The pathogenesis of frequent intrahepatic recurrence of hepatocellular carcinoma (HCC) after surgical resection or local ablation therapy remains uncertain. Risks and patterns of intrahepatic distant recurrence (IDR) of a single, primary HCC lesion after radiofrequency (RF) ablation were examined. Methods. Ninety patients with a single primary HCC lesion of less than 3 cm who had complete RF ablation were enrolled in the study. Risk factors for IDR and the patterns of IDR after RF ablation were analyzed. Results. The median follow-up was 37.4 months. IDR was observed in 44 (48.9%) patients. The cumulative rate of IDR was 10.4%, 52.5%, and 77.0% at 1, 3, and 5 years, respectively. Univariate analysis revealed that a pretreatment serum α-fetoprotein (AFP) level of ≥50 ng/ml (P = 0.0324), a des-γ-carboxy prothrombin (DCP) level of ≥40 mAu/ml (P = 0.006), an ablative margin of <5 mm of the ablation zone (P = 0.0306), and a prothrombin time of <70% (P = 0.0188) were related to IDR. A multivariate stepwise Cox proportional hazards regression model revealed that pretreatment serum AFP and DCP level and the ablative margin were independent risk factors for IDR pretreatment. Serum DCP level ≥ 40 mAu/ml (P = 0.025), local tumor progression (P = 0.011), and ablative margin < 5 mm (P = 0.024) were related to multiple IDR. Conclusions. HCC patients with high serum AFP or DCP before RF ablation should be carefully followed up to monitor any IDR. A suffi cient ablative margin in RF ablation for HCC is required to prevent IDR.     

α-Chemokine growth factors for adenocarcinomas; a synthetic peptide inhibitor for α-chemokines inhibits the growth of adenocarcinoma cell lines

Purpose: The experiments aimed to determine if α-chemokine inhibitors are effective suppressors of the growth of adenocarcinomas, a neoplasm with a high mortality rate. Methods: Expression of growth-related oncogene (GROα) and interleukin-8 (IL-8) was determined by enzyme-linked immunosorbent assay. Inhibition of α-chemokine binding to tumor cells was assessed in the presence and absence of the hexapeptide, antileukinate. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assays were performed to determine the effect of α-chemokines, monoclonal antibodies (mAb), and antileukinate on cell proliferation. Finally, antileukinate inhibition of human, lung adenocarcinoma tumor growth, was determined in BALB/c nude mice. Results: All of the adenocarcinomas tested produced either GROα or IL-8 or both. Proliferation of lung, stomach and colon adenocarcinoma cells was inhibited by anti-GROα mAb and/or anti-IL-8 mAb while recombinant human GROα stimulated the proliferation of lung and stomach adenocarcinomas. Antileukinate inhibited GROα binding to specific receptors on adenocarcinoma cells and inhibited the proliferation of all adenocarcinomas tested. Colon-derived adenocarcinomas specifically bound IL-8 and this binding was also inhibited by antileukinate. Administration of antileukinate in vivo inhibited the tumor growth of adenocarcinoma A549. Conclusions: GROα and IL-8 are necessary for the growth of lung, stomach and colon adenocarcinomas, and can be inhibited by the hexapeptide, antileukinate. The findings suggest the possibility of using α-chemokine receptor inhibitors in the treatment of adenocarcinoma. Received: 24 March 1999 / Accepted: 26 July 1999     

Expression level of glutamine synthetase is increased in hepatocellular carcinoma and liver tissue with cirrhosis and chronic hepatitis B

Studies have suggested that glutamine synthetase (GS) is a potential marker of hepatocellular carcinoma (HCC). We aimed to evaluate the expression of GS in non-malignant liver tissue and serum GS levels in HCC, liver cirrhosis (LC), chronic hepatitis B (CHB), five kinds of extrahepatic diseases patients and healthy subjects. Immunohistochemistry (IHC) was used to assess GS expression in 260 liver tissue samples (from 120 HCC, 90 CHB stage 4, and 50 CHB stage 1–3 patients). Enzyme-linked immunosorbent assays of 325 samples (from 100 healthy donors, 33 CHB stage 1–3, 43 CHB stage 4, 111 HCC, and 45 extrahepatic diseases patients) were used to further analyze GS levels in serum. IHC studies showed the expression of GS in 70% of HCC patients, 46.7% of CHB stage 4 patients and 38% of CHB stage 1–3 patients. The χ² tests showed significant difference between HCC samples and non-tumor tissues (P = 0.001 for HCC vs. CHB stage 4, P = 0.000 for HCC vs. CHB). Consistent with this, serum GS levels are increased in HCC and CHB stage 1–4 patients. There are significant differences among all samples (P = 0.000 for all), except CHB stage 1–3 versus CHB stage 4 (P = 0.552). Based on multiple linear regressions, HCC, CHB stage 1–4 and AFP were significantly associated with serum GS levels. In addition, in HCC group, TNM and Child-Pugh were significantly associated with GS levels. Expression of GS is increased in HCC, LC, and CHB. It may be a new serum marker for liver disease.     

HFE Gene Mutations, Serum Ferritin Level, Transferrin Saturation, and Their Clinical Correlates in a Korean Population

The aim of this study was to investigate HFE gene mutations, blood iron indices, and their clinical correlates in a Korean population. In 484 prospectively enrolled health-check examinees, HFE gene mutations and iron indices with clinical and laboratory variables were analyzed. Although neither the C282Y nor S65C gene mutation were found, the H63D heterozygote was detected in 41 subjects (8.5%). The mean serum ferritin and transferrin saturation (TS) were 136.2 ± 129.8 μg/dl and 39.2 ± 15.7%, respectively. The H63D genotype was not significantly associated with iron indices. High serum ferritin was associated with old age, the male gender, high body mass index (BMI), and the presence of nonalcoholic fatty liver disease (NAFLD). High TS was associated with the male gender and alcohol drinking. HFE gene mutation is rare; however, TS seems to be higher in Koreans compared to Caucasians or other ethnic groups. Serum ferritin reflects iron store as well as the presence of NAFLD.     

Repair of potentially lethal damage by total and quiescent cells in solid tumors following a neutron capture reaction

Purpose: We analyzed the time-course of changes in the sensitivity of total (proliferating + quiescent and quiescent (Q) cell populations within solid tumors in situ following a neutron capture reaction and compared it with that after γ-ray irradiation. Methods: After continuous labeling of proliferating cells with BrdU for 5 days, mice bearing SCC VII tumors received thermal neutron irradiation with or without a ¹⁰B-labeled compound (sodium [¹⁰B]borocaptate, BSH, or dl-p-[¹⁰B]boronophenylalanine, BPA), or γ-ray irradiation. From 5 min to 72 h after treatment, tumors were excised, minced, and trypsinized. Cell suspensions were incubated for 48 h with the cytokinesis blocker cytochalasin-B. The micronucleus frequency for BrdU-unlabeled cells, Q cells at treatment, was then determined by immunofluorescence staining for BrdU. The micronucleus frequency for total cells was obtained from tumors that had not been pretreated with BrdU labeling. The sensitivity was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (micronucleus frequency). Results: Overall, Q cells showed greater repair capacities than total cells. γ-Ray irradiation and neutron irradiation with BPA induced larger repair capacities in each cell population. In contrast, thermal neutron irradiation without a ¹⁰B-labeled compound induced the smallest repair capacity in both cell populations. The use of a ¹⁰B-labeled compound, especially BPA, widened the difference in sensitivity between total and Q cells, resulted in an increase in repair capacity in both cell populations, and made the repair patterns of the two cell populations look like those induced by γ-ray irradiation. Conclusion: Differences in sensitivity and repair patterns following the neutron capture reaction were thought to depend on differences in the distribution of the ¹⁰B-labeled compound between the proliferating and Q cell populations. Received: 18 February 1999 / Accepted: 4 June 1999     

Increased intercellular adhesion molecule-1 serum concentration in cholestasis

Background/Aims: Increase of serum levels of the soluble intercellular adhesion molecules in patients with the cholestatic liver diseases primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are known and have been thought to indicate activation of the immune system and the grade of the inflammatory process. In hepatitis and cholestatic diseases, expression of adhesion molecules was found on the surface of bile duct epithelia and hepatocytes.Materials and Methods: Serum levels of sICAM-1 in patients with intrahepatic cholestasis in PBC (n=42) and extrahepatic cholestasis (n=18) due to choledocholithiasis were investigated. sICAM-1 levels and “classical” cholestasis parameters as alkaline phosphatase (ALP), γ-glutamyl-transpeptidase (γ-GTP) and bilirubin levels were compared. Furthermore, sICAM-1 concentrations and “classical” cholestasis parameters were analysed before and after therapy with ursodeoxycholic acid (UDCA). In addition, sICAM-1 was detected in serum and bile fluid of four patients with cholestasis due to choledocholithiasis. Soluble ICAM-1 levels in sera and, if accessible, in bile fluids were determined using a commercially available ELISA system. Statistics were done by Wilcoxon's signed rank exact test and Spearman's rank correlation test. Sensitivity and specificity of cholestasis parameters and sICAM-1 concentrations was analysed by receiver operating characteristic (ROC) curves.Results: Increased sICAM-1 serum concentrations in a similar range were found in patients with PBC (range 251–2620 μg/l; median 966 μg/l) as well as in patients with extrahepatic cholestasis (257–2961 μg/l; median 760 μg/l) compared to healthy controls (n=12; 220–500 gmg/l; median 318 μg/l). sICAM-1 levels correlated significantly to histological stage I to IV (p<0.001), ALP (range 107–1877 U/l; median 545 U/l; r=0.496, p=0.0008), bilirubin (range 0.3–26 mg/dl; median 0.8 mg/dl; r=0.52; p<0.0004) and γ-GTP levels (range 43–705 U/l; median 221 U/l; r=0.36; p=0.02) in PBC patients. In PBC patients a histological stage III or IV (n=21) could be predicted with high sensitivity (95%) and specificity (85%) if sICAM-1 levels were above 840 μg/l. After treatment of PBC patients with UDCA, sICAM-1 levels decreased significantly with decline of other “classical” cholestasis parameters. Increased sICAM-1 levels (range 257–2961, median 745 μg/l) in extrahepatic cholestasis correlated also significantly with serum concentrations of bilirubin (r=0.8; p<0.01; range 0.3–19.7, median 1.6 mg/dl), γ-GTP (r=0.55; p=0.03; range 33–1401, median 179 U/l) and ALP (r=0.61; p=0.1; range 110–1378, median 562 U/l). sICAM-1 2as detectable in bile fluid (264–919 μg/l) of four patients with extrahepatic cholestasis and nose-biliary catheterisation.Conclusions: sICAM-1 concentrations were found to discriminate between histological stage I/II and stage III/IV of PBC with higher sensitivity and specificity than “classical” cholestasis parameters. Increased serum concentrations for sICAM-1 in intra- and in extrahepatic cholestasis and detection of sICAM-1 in the bile may indicate that sICAM-1 is eliminated through the bile. In other words, not only increased synthesis but also decreased elimination may be responsible for increased sICAM-1 serum levels in patients with cholestatic liver diseases.     

Th1/Th2 cytokine profiles and their relationship to clinical features in patients with chronic hepatitis C virus infection

Purpose. An imbalance in helper T-cell type 1 (Th1) and type 2 (Th2) cytokines is suggested to play an important role in the pathogenesis of chronic viral infections, but this issue is not resolved in patients with hepatitis C virus (HCV) infection. The aim of this study was to clarify the relationship between the balance of Th1 and Th2 cytokines and liver damage. Methods. We investigated cytokine levels in the peripheral blood and liver tissue of patients with chronic HCV infection (n = 59) by three different methods; we used flow cytometry to detect intracellular cytokines, and we measured cytokine titers in sera and in the supernatants of lymphocyte cultures with enzyme-linked immunosorbent assays (ELISAs). Results. In both CD4+ and CD8+ cells, interferon (IFN) γ-producing cell populations increased, while there was no difference in interleukin (IL)-10 production, indicating a shift to a Th1 cytokine profile with the progression of liver disease. With respect to the ratio of IFN-γ to IL-10, a correlation was found in CD4+ cells between peripheral blood and liver tissue (r = 0.98; P = 0.0011). Th1 cytokine was predominant in intrahepatic CD4+ cells, while it was predominant in peripheral blood CD8+ cells. Conclusions. These findings indicate a correlation between dominant Th1 response and disease activity and progression. In addition, we suggest that intrahepatic CD4+ T cells play a pathogenetic role in the hepatic injury of HCV infection. Received: August 10, 2000 / Accepted: February 2, 2001     

The relationship between HBcrAg and HBV reinfection in HBV related post-liver transplantation patients

Background

Post-transplant hepatitis B virus (HBV) reinfection is one of the major problems facing patients who undergo HBV-related liver transplantation (LT). We analyzed the clinical impact of serum hepatitis B core-related antigen (HBcrAg) on HBV reinfection in post-LT patients with HBV-related liver diseases.

Methods

Serum hepatitis B surface antigen (HBsAg), HBV DNA, and HBcrAg were measured over time in 32 post-LT patients. Twenty-one out of 32 patients had HCC at LT. The effects of HBcrAg, hepatocellular carcinoma (HCC) recurrence, and HBs gene mutation on HBV reinfection and withdrawal from hepatitis B immune globulin (HBIG) were analyzed.

Results

Sixteen out of 32 patients (50 %) were positive for HBcrAg even though only six patients were thought to have experienced HBV reinfection based on reappearance of either HBV DNA or HBsAg during a median follow-up time of 75 months. Three of these six patients who became re-infected with HBV experienced HCC recurrence after LT. The HBV DNA reappearance rate was significantly higher in patients with HCC recurrence after LT (p < 0.001). Two HBV re-infected patients without HCC recurrence had HBs gene mutations G145R and G145A, respectively. Anti-HBs antibody development rate by HB vaccination was similar between HBcrAg-positive and negative patients (p = 0.325).

Conclusions

HBV reinfection is more common than is usually considered based on conventional measurement of HBsAg and HBV DNA. HCC recurrence and mutations in the HBV S gene were associated with HBV reinfection after LT.