Reduction of liver stiffness by antiviral therapy in chronic hepatitis B.

BACKGROUND: Liver stiffness (LS) has been reported to correlate with fibrosis stage (F). The correlation between LS and fibrosis stage and the reduction of LS by antiviral therapy were examined in patients with hepatitis B infection. METHODS: LS was measured by FibroScan in 212 patients infected with hepatitis B virus. Liver biopsies were done in 51 patients. Changes of LS were assessed in 29 patients treated with nucleotide or nucleoside analogs and 52 patients without antiviral therapy. RESULTS: LS was significantly correlated with fibrosis stage (ρ = 0.686, P < 0.0001). The optimal cut-off values of LS were 7.1 kPa for F ≥ 2, 10.7 kPa for F ≥ 3, and 16.0 kPa for F4. LS was significantly reduced by antiviral therapy, from 12.9 (range 6.2-17.9) kPa to 6.6 (4.4-10.3) kPa measured at an interval of 512 (range 366-728) days (P < 0.0001). Eleven of 19 (58%) patients with baseline fibrosis stages of F3-4 deduced from LS had 2-point or greater reductions of deduced stage at the last LS measurement. The change ratio of hyaluronic acid (P = 0.0390) was associated with a 2-point or greater reduction of deduced fibrosis stage. Without antiviral therapy, LS tended to increase, increasing from 6.1 (range 3.9-8.5) kPa to 6.3 (range 4.4-9.7) kPa at an interval of 422 (range 358-709) days (P = 0.0682). CONCLUSIONS: LS was significantly correlated with fibrosis stage in patients with chronic hepatitis B. The reduction of LS by antiviral therapy was significantly correlated with the reduction of hyaluronic acid. Thus, we conclude that LS can be useful to assess the progression and regression of liver fibrosis stage noninvasively.

Liver stiffness in extrahepatic cholestasis correlates positively with bilirubin and negatively with alanine aminotransferase.

AIM: Transient elastography is a non-invasive tool to measure liver stiffness (LS), which has been reported to correlate with stage of liver fibrosis. Extrahepatic cholestasis was reported to cause elevated LS, which is considered to be attributed to the increased hydrostatic pressure in the liver. In the present study, the correlation of LS with laboratory data was investigated in extrahepatic cholestasis. The change of LS after biliary drainage was also assessed. METHODS: LS was measured in 29 patients with extrahepatic cholestasis due to carcinomas in 12 and non-neoplastic diseases of biliary tract or pancreas in 17. RESULTS: In 15 patients, LS was 11.4 kPa or higher which suggested liver cirrhosis in chronic infection of hepatitis C virus. LS significantly correlated positively with serum bilirubin levels (r = 0.726, P < 0.0001) and negatively with serum aspartate aminotransferase (AST) levels (r = -0.481, P = 0.0082) and alanine aminotransferase (ALT) levels (r = -0.631, P = 0.0002). Biliary drainage led to a reduction of bilirubin by 13.5 to 0.9 mg/dL which was significantly correlated with a reduction of LS by 14.3 to 0.5 kPa (r = 0.524, P = 0.0257). CONCLUSION: In extrahepatic cholestasis, the elevation of LS which is probably attributed to the increased hydrostatic pressure in the liver, correlates positively with the accumulation of bilirubin but negatively with damage of hepatocytes indicated by ALT levels. Further studies on the mechanism underlying the elevation of LS should be helpful to elucidate the pathogenesis of extrahepatic cholestasis.

A new index for non-invasive assessment of liver fibrosis.

AIM: To construct and evaluate a new non-invasive fibrosis index for assessment of the stage of liver fibrosis. METHODS: A new fibrosis index (Fibro-Stiffness index) was developed in 165 of 285 patients with chronic hepatitis C, and was validated in the other 120 patients where liver biopsy was performed. Its usefulness was compared with liver stiffness (LS) measured by FibroScan, the aminotransferase-to-platelet ratio index, the Forns index and the FibroIndex. RESULTS: The Fibro-Stiffness index consists of LS, platelet count and prothrombin time. The values of the Fibro-Stiffness index differed significantly between neighboring fibrosis stages except F0-F1. The area under the receiver operating characteristics curves of the Fibro-Stiffness index for prediction of F ≥ 2 (0.90), F ≥ 3 (0.90) and F = 4 (0.92) in the estimation group and those for F ≥ 3 (0.93) and F = 4 (0.97) in the validation group were the highest among the 5 methods examined. The accuracy of the Fibro-Stiffness index had highest values for F ≥ 2, F ≥ 3 and F = 4 in both the estimation and validation groups. The diagnostic performance for F = 4 was improved by a combination of the Fibro-Stiffness index with serum hyaluronic acid level. CONCLUSION: The Fibro-Stiffness index was constructed and validated. It showed superior diagnostic performance to other indices for F ≥ 2, 3 and 4.

Reduction of liver stiffness by interferon treatment in the patients with chronic hepatitis C.

AIM: To assess the regression of liver fibrosis after interferon (IFN) treatment in patients with chronic hepatitis C, liver stiffness (LS) was measured repeatedly and the factors associated with reduction of LS were assessed. METHODS: LS was measured by transient elastography before treatment, at end of treatment (EOT), and 1 year and 2 years after EOT in 145 patients with chronic hepatitis C treated by IFN with or without ribavirin. RESULTS: In the patients with sustained virological response (SVR) (n = 93) and relapsers (n = 28), LS significantly decreased at EOT (median, 5.4 [interquartile range, 4.0-8.6] kilopascals [kPa], P < 0.0001 and 6.8 [4.5-8.9] kPa, P = 0.0023) and 1 year after EOT (5.3 [4.2-7.0] kPa, P < 0.0001 and 6.8 [4.5-9.3] kPa, P = 0.0204) compared with baseline (8.0 [5.0-11.9] kPa and 10.6 [7.0-16.6] kPa). In SVR patients, LS significantly decreased 2 years after EOT (5.3 [4.1-6.3] kPa) compared with baseline (P < 0.0001) and LS at EOT (P = 0.0034). Two points or greater reduction of deduced stage at last LS measurement was observed in 78% of SVR patients, 59% of relapsers and 15% of patients with non-virological response whose pretreatment deduced stages were F3-F4. Fibrosis stage, hyaluronic acid levels, duration of treatment, response to treatment and alanine aminotransferase levels were associated with a 2-point or greater decrease of deduced fibrosis stage. CONCLUSION: IFN treatment reduced LS in SVR patients and relapsers. Significant reduction of LS is associated with milder fibrosis stage, lower hyaluronic acid levels, longer IFN treatment, virological response of SVR or relapse and higher alanine aminotransferase levels.

The loss of HBeAg without precore mutation results in lower HBV DNA levels and ALT levels in chronic hepatitis B virus infection.

BACKGROUND: The aim of this study was to investigate the correlation between precore (PC)/basal core promoter (BCP) mutations and the viral loads or activity of hepatitis in patients with chronic hepatitis B virus (HBV) infection. METHODS: HBV genotypes, PC mutations, BCP mutations, HBV DNA levels, and serological markers of HBV were analyzed in all the patients with chronic HBV infection seen in Fujita Health University Hospital from June 2004 to November 2008 (n=215). RESULTS: HBV genotype was C in 169 patients, B in 16, A in 3, F in 1, and unclassifiable in 5. Among the patients with genotype C, the prevalence of PC wild type was significantly lower in hepatitis B envelope antigen (HBeAg)(-) patients than in HBeAg(+) patients (9.5% versus 49.0%, P<0.0001). Among HBeAg(-) patients, the patients with PC wild type had significantly lower serum viral loads and alanine aminotransferase (ALT) levels compared with those with PC mutant (P<0.001). Among HBeAg(-) patients, the patients with genotype B had lower serum viral loads compared with those with genotype C (3.6+/-0.9 versus 4.6+/-1.6, P<0.05), and the prevalence of BCP wild type was significantly higher in those with genotype B than in those with genotype C (58.3% versus 10.8%, P<0.05). CONCLUSIONS: Among HBeAg(-) patients with genotype C, the patients with PC wild type had significantly lower viral loads and ALT levels than those with PC mutant. This suggests that the patients with PC wild type may have better prognosis than those with PC mutant among HBeAg(-) patients with genotype C.

Liver stiffness measured by transient elastography correlates with fibrosis area in liver biopsy in patients with chronic hepatitis C.

AIM: Liver stiffness (LS) measured by transient elastography (TE) has been reported to correlate with liver fibrosis, which is usually semiquantitatively assessed. In the present study, the fibrosis area was measured by image analysis software in liver biopsy specimens and its correlation with LS was assessed. METHODS: LS was measured by TE in all 165 patients with chronic hepatitis C virus (HCV) infection who underwent liver biopsy consecutively in Fujita Health University Hospital from July 2004 to September 2007. RESULTS: Fibrosis area was significantly correlated with fibrosis stage as assessed by the Metavir score (rho = 0.733, P < 0.0001). The optimal cut-off value of fibrosis area was 1.6% for F > or = 2, 3.1% for F > or = 3, and 3.8-6.4% for F4. LS was significantly correlated with fibrosis stage (rho = 0.734, P < 0.0001). The optimal cut-off value of LS was 7.1 kPa for F > or = 2, 9.6 kPa for F > or = 3 and 11.6-16.9 kPa for F4. Multiple linear regression analysis selected fibrosis area (P = 0.0002), alanine aminotransferase (ALT) (P = 0.0237), gamma-glutamyltransferase (gamma-GTP) (P = 0.0114), prothrombin time (P = 0.0114) and hyaluronic acid (P < 0.0001) as factors correlating with LS. CONCLUSION: The correlation between LS and liver fibrosis was confirmed by the objective measurement of fibrosis area. ALT was significantly correlated with LS, suggesting that inflammatory activity also affects LS values. Despite some limitation, LS measurement is a useful method for the diagnosis of liver fibrosis.

Genetic polymorphisms of cyclooxygenase-1 (COX-1) are associated with functional dyspepsia in Japanese women.

BACKGROUND: Prostaglandins (PGs), catalyzed from arachidonic acid by cyclooxygenase (COX), are involved in a variety of physiological processes in the stomach. COX-1 has been regarded as a constitutively expressed enzyme that generates prostaglandins for gastrointestinal integrity. We investigated the association between the potentially functional polymorphism T-1676C in the COX-1 gene promoter and functional dyspepsia (FD) in the Japanese population. METHODS: The study was performed in 272 subjects (185 with no upper abdominal symptoms and 87 with FD). We employed the PCR-SSCP method to detect the gene polymorphism. RESULTS: Overall, female sex had a 2-2.5-fold risk for the development of FD compared with male sex, whereas the COX-1 gene polymorphism and Helicobacter pylori infection were not associated with susceptibility to FD. However, in female subjects, -1676T allele carriers had a significantly increased risk for the development of FD (OR 2.70, 95%CI 1.04-6.99, p = 0.041), especially the epigastric pain syndrome (EPS) subgroup (OR 4.07, 95%CI 1.15-14.4, p = 0.029). CONCLUSIONS: Our results provide the first evidence that the COX-1 gene polymorphism is significantly associated with the development of the EPS subgroup of FD in female subjects.

The influence of promoter polymorphism of nuclear factor-erythroid 2-related factor 2 gene on the aberrant DNA methylation in gastric epithelium.

Aberrant promoter methylation is an important mechanism for gene silencing. Inflammation-related reactive oxygens contribute to this CpG island methylation. The nuclear factor-erythroid 2-related factor 2 gene (Nrf2) is known to regulate the expression of detoxifying and antioxidant genes. We investigated the relationship between promoter polymorphisms of Nrf2 gene and the CpG island methylation in non-cancerous gastric mucosa. The study was performed in 85 subjects (46 without gastric malignancies, non-GC group, and 39 with gastric cancer, GC group). The promoter methylation status of p14(ARF), p16(INK4a) and p21(Waf1) genes was determined by methylation-specific-polymerase chain reaction. The Nrf2 gene genotypes were determined by the PCR-SSCP method. In the 85 subjects, CpG island methylation was found in 25.9% for p14, 15.3% for p16, none for p21. The frequency of the methylated genes was significantly higher in GC group than non-GC group (OR, 2.67; 95% CI, 1.10-6.49; p=0.029). In particular, the frequency of p16 gene methylation was much higher in GC group (p=0.0023). The Nrf2 -686/-684 G/G haplotype was positively associated and A/G haplotype was inversely associated with the development of CpG island methylation, especially p14 gene methylation (OR, 3.28; 95% CI, 1.26-8.59; p=0.015, and OR, 0.38; 95% CI, 0.15-0.96; p=0.040, respectively). In Helicobacter pylori (H. pylori) infected subjects, the number of -686/-684 G/G allele was positively correlated and that of A/G allele was inversely correlated to the methylation status, especially p14 methylation, by the adjusted analysis (OR, 2.90; 95% CI, 1.14-7.36; p=0.026, and OR, 0.33; 95% CI, 0.13-0.88; p=0.027, respectively). Our results suggested that the promoter polymorphisms of Nrf2 gene may affect the methylation status of tumor-related genes, especially the p14 gene, under the influence of H. pylori-induced gastric inflammation.

Functional promoter polymorphisms of the macrophage migration inhibitory factor gene in gastric carcinogenesis.

The macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. Two functional polymorphisms have been identified in the promoter region of the MIF gene. We attempted to clarify the associations of these polymorphisms with the development of gastric cancer. The study was performed in 229 patients with gastric cancer and 428 subjects with no evidence of gastric malignancies on the upper gastro-duodenal endoscopy. The severity of histological chronic gastritis was classified according to the updated Sydney system. Overall, the 5-CATT carriers had a reduced risk of developing gastric cancer (OR, 0.67; 96% CI, 0.48-0.93; p=0.015), especially the diffuse type cancer. In subjects >60 years, the adjusted risk for gastric cancer among individuals who were -173C carriers was 1.71 (range, 1.03-2.84; p=0.038) compared to the G/G homozygous genotype. The number of 7-CATT alleles was also positively correlated with the development of intestinal type gastric cancer (adjusted OR, 1.70; 95% CI, 1.02-2.58; p=0.043). In subjects <60 years, the 7/7-CATT homozygous genotype was linked with a risk for the progression of atrophic gastritis (adjusted OR, 8.74; 95% CI, 1.31-58.6; p=0.026). In addition, the number of 7-CATT alleles was significantly correlated with the activity and inflammation scores (p=0.010 and 0.030, respectively). Our results suggested that functional promoter polymorphisms of the MIF gene are associated with the progression of gastric mucosal inflammation and the development of mucosal atrophy at an early stage in life and these genotypes may increase the risk for the subsequent development of gastric cancer, especially the intestinal type, in older subjects.

The influence of polymorphisms of interleukin-17A and interleukin-17F genes on the susceptibility to ulcerative colitis.

We investigated the association between ulcerative colitis (UC) and polymorphisms of IL-17A (rs2275913, G-197A) and IL-17F (rs763780, 7488T/C) genes. We employed the multiplex PCR-SSCP method to detect gene polymorphisms. Both the numbers of -197A (IL-17A) and 7488T (IL-17F) alleles were significantly correlated to the development of UC. The frequencies of -197A/A and 7488T/T genotypes in the UC group were significantly higher than those in the non-UC group. An adjusted analysis revealed that -197A and 7488T alleles were independent risk factors for the developing UC. In addition, both polymorphisms were significantly associated with the pancolitis phenotype. Furthermore, -197A allele was significantly correlated to the chronic relapsing phenotype and -197A/A homozygote was more frequent in steroid-dependent cases, whereas 7488T allele was correlated with the chronic continuous phenotype. Our results provided the first evidence that -197A (IL-17A) and 7488T (IL-17F) alleles may influence the susceptibility to and pathophysiological features of UC independently.

Association between promoter polymorphisms of nuclear factor-erythroid 2-related factor 2 gene and peptic ulcer diseases.

Transcription factor Nrf2 regulates the expression of detoxifying and antioxidant genes. Three promoter polymorphisms of this gene have been identified. We attempted to clarify the association of these polymorphisms with the development of peptic ulcer diseases. The study was performed with 384 stocked DNAs obtained from subjects with no evidence of gastric malignancy. In all 384 DNAs, 77 and 48 were obtained from gastric and duodenal ulcer patients, respectively. By an unadjusted analysis, infection with Helicobacter pylori (H. pylori), male gender and the -686/-684 G/G carrier (OR, 2.52; 95% CI, 1.19-5.45; p=0.016) were associated with a significantly increased risk for developing gastric ulcer, whereas the -686/-684 A/G homozygote was linked to a significantly reduced risk for developing gastric ulcer (OR, 0.26; 95% CI, 0.099-0.67; p=0.0055). On the other hand, infection with H. pylori and male gender were significantly associated with the development of duodenal ulcer, whereas Nrf2 promoter polymorphisms were not. By the analysis, after adjustment for age, gender, non-steroidal anti-inflammatory drug/aspirin use and H. pylori infection status, the -686/-684 A/G homozygote was associated with a significantly reduced risk for gastric ulcer (OR, 0.25; 95% CI, 0.088-0.73; p=0.011). Our results suggest that promoter polymorphisms of the Nrf2 gene are associated with the susceptibility to gastric ulcer.

Genetic polymorphisms of molecules associated with inflammation and immune response in Japanese subjects with functional dyspepsia.

Inflammatory changes in the gastric mucosa are commonly observed in Japanese patients with functional dyspepsia (FD). However, detailed data regarding the relationship between the genetic regulatory factors of inflammation and FD are not available. We investigated the associations between FD and genetic polymorphisms of molecules associated with inflammation or immune response (IL-17A, -17F and MIF). The study was performed with 278 subjects (188 with no upper abdominal symptoms and 90 with FD according to the Roma III criteria). We employed the PCR-SSCP (multiplex PCR for IL-17A and -17F) method to detect the gene polymorphisms. Overall, the polymorphisms of the IL-17A, -17F and MIF genes were not correlated with the susceptibility to FD. However, the MIF -173C allele carrier had a significantly increased risk for the development of epigastric pain syndrome (EPS) of FD (OR, 2.12; 95% CI, 1.00-4.49; p=0.0497). In Helicobacter pylori (H. pylori)-infected cases, the number of IL-17F 7488T alleles was positively correlated with the development of EPS (OR, 11.3; 95% CI, 1.23-103.2; p=0.032), while the IL-17F T/T homozygote and the MIF -173C carrier had an increased risk for EPS (OR, 10.4; 95% CI, 1.17-92.3; p=0.036 and OR, 3.66; 95% CI, 1.19-11.3; p=0.024, respectively). In addition, a significant interaction between the IL-17F 7488 polymorphism and H. pylori infection was shown to increase the activity and inflammation scores (p=0.043 and 0.042, respectively). There were no significant associations between the IL-17A polymorphism and FD. Our results provide the first evidence that the IL-17F and MIF gene polymorphisms are significantly associated with the development of FD, particularly EPS, a subgroup of FD, in H. pylori-infected subjects. The genetic polymorphisms of inflammation or immune response-related molecules are involved in the development of one of the FD subgroups via H. pylori-induced gastric inflammation.

Functional polymorphisms in the promoter region of macrophage migration inhibitory factor and chronic gastritis.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator, which plays a pivotal role in inflammatory and immune diseases. We attempted to clarify associations of the functional polymorphisms of the MIF gene promoter with the development of chronic gastritis. The study was performed with 290 stocked DNAs from subjects with no evidence of gastric malignancy. We employed the PCR-SSCP method to detect gene polymorphisms. The severity of histological chronic gastritis in antral biopsy specimens was classified according to the updated Sydney system. Both the 7/7-CATT repeat at position -794 and the -173 C/C genotypes were significantly associated with a risk of developing severe gastric mucosal atrophy (OR, 9.69; 95% CI, 1.29-72.5; and OR, 4.60; 95% CI, 1.05-20.2, respectively). In subjects younger than 60 years old, the number of 7-CATT alleles was significantly correlated with both the activity and inflammation scores (p=0.0079 and 0.0080, respectively). Our results suggested that functional promoter polymorphisms of the MIF gene might be associated with the severity of gastric mucosal inflammation in younger subjects and with the subsequent development of mucosal atrophy.

Association between genetic polymorphisms in the cyclooxygenase-1 gene promoter and peptic ulcers in Japan.

Cyclooxygenase-1 (COX-1) has been regarded as a constitutively expressed enzyme that generates prostaglandins for gastrointestinal integrity. We attempted to clarify the association between potentially functional polymorphisms (T-1676C and A-842G/C50T) in the COX-1 gene promoter and gastroduodenal disorders in a Japanese population. The study was performed with 480 stocked DNAs from subjects (gastric ulcers in 93 subjects and duodenal ulcers in 44) with no evidence of gastric malignancy. We employed the PCR-SSCP method to detect gene polymorphisms. The severity of histological chronic gastritis in antral biopsy specimens was classified according to the updated Sydney system. The T-1676C polymorphism was not associated with either gastric mucosal atrophy or infiltration of inflammatory cells into gastric mucosa. In non-NSAID (non-steroidal anti-inflammatory drug) users, male gender and Helicobacter pylori (HP) infection were significantly associated with both gastric and duodenal ulcers, whereas the -1676T allele carrier was significantly associated with only gastric ulcers (OR, 2.86; 95% CI, 1.29-6.34). In NSAID users, the number of -1676T alleles was significantly associated with developing gastroduodenal ulcers (OR, 5.80; 95% CI, 1.59-21.1), whereas male gender and HP infection were not. The -842T/ C50T polymorphism was not detected in any of the 480 Japanese subjects. In conclusion, a carrier of the -1676T allele in the COX-1 gene promoter, as well as HP infection and male gender, seem to be significant risk factors for developing gastric ulcers, and the number of -1676T alleles was also a significant risk factor for the NSAID-induced ulcer, whereas the frequency of the A-842G polymorphism was thought to be very rare in the Japanese population.