Association of tyrosine with insulin resistance in hepatitis C virus-related chronic liver disease.

AIM: Insulin resistance (IR) increases during the early stages of hepatitis C virus (HCV)-related chronic liver disease and is a sign of poor prognosis as well as a risk factor for hepatic fibrosis and hepatocellular carcinoma. We aimed to determine the factors affecting IR in HCV-related chronic liver disease. METHODS: We retrospectively examined 71 patients with HCV-related chronic liver disease and analyzed various parameters, including amino acids, as possible predictors of IR. IR was assessed using the Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR). Amino acids were assayed by examining branched-chain amino acids (BCAA), tyrosine level, and the ratio of BCAA to tyrosine level (BTR). RESULTS: HOMA-IR was significantly correlated with body mass index, platelet count, prothrombin time, hemoglobin, total bilirubin, total protein, albumin, total cholesterol, fasting glucose, BTR (r = -0.46, P = 0.0001) and tyrosine (r = 0.55, P < 0.0001). However, BCAA were not significantly correlated with HOMA-IR (r = -0.21, P = 0.082). In multivariate analysis, only two factors were identified as independent parameters contributing to a HOMA-IR of 2.5 or more: total cholesterol (odds ratio [OR], 6.511; 95% confidence interval [95% CI], 1.554-27.284; P = 0.010) and tyrosine (OR, 4.839; 95% CI, 1.087-21.549; P = 0.039). CONCLUSION: Serum tyrosine levels may be associated with IR in patients with HCV-related chronic liver disease.

Systemic gemcitabine combined with hepatic arterial infusion chemotherapy with cisplatin, 5-fluorouracil, and isovorin for the treatment of advanced intrahepatic cholangiocarcinoma: a pilot study.

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) has a poor prognosis and usually presents as advanced disease. Hepatic arterial infusion chemotherapy (HAIC) is a promising option for advanced hepatocellular carcinoma; however, there have been few reports on the use of HAIC in patients with ICC. In the present study, we investigated the efficacy of treatment with systemic gemcitabine (GEM) combined with HAIC with cisplatin (CDDP), 5-fluorouracil (5-FU), and isovorin in patients with advanced ICC. METHODOLOGY: Seven patients with advanced ICC, who received systemic GEM combined with HAIC with CDDP, 5-FU, and isovorin were studied. RESULTS: The response rate after the first chemotherapy cycle was 57.1% (partial response, 4; stable disease, 2; progressive disease, 1). The cumulative survival rates at 1 and 2 years were 85.7% and 28.6%, respectively, and the median survival time was 22.3 months. With regard to grade 3 or 4 adverse reactions, the percentages of patients developing leukopenia, neutropenia, thrombocytopenia, anemia, and anorexia were 28.6%, 28.6%, 42.9%, 14.3%, and 14.3%, respectively. Na treatment-related deaths were encountered. CONCLUSIONS: Although this is a pilot study, we suggest that systemic GEM combined with HAIC with CDDP, 5-FU, and isovorin, may be a useful therapy for patients with advanced ICC.

Objective evaluation of tongue diagnosis ability using a tongue diagnosis e-learning/e-assessment system based on a standardized tongue image database.

Background: In Kampo medicine, tongue examination is used to diagnose the pathological condition "Sho," but an objective evaluation method for its diagnostic ability has not been established. We constructed a tongue diagnosis electronic learning and evaluation system based on a standardized tongue image database. Purpose: This study aims to verify the practicality of this assessment system by evaluating the tongue diagnosis ability of Kampo specialists (KSs), medical professionals, and students. Methods: In the first study, we analyzed the answer data of 15 KSs in an 80-question tongue diagnosis test that assesses eight aspects of tongue findings and evaluated the (i) test score, (ii) test difficulty and discrimination index, (iii) diagnostic consistency, and (iv) diagnostic match rate between KSs. In the second study, we administered a 20-question common Kampo test and analyzed the answer data of 107 medical professionals and 56 students that assessed the tongue color discrimination ability and evaluated the (v) correct answer rate, (vi) test difficulty, and (vii) factors related to the correct answer rate. Result: In the first study, the average test score was 62.2 ± 10.7 points. Twenty-eight questions were difficult (correct answer rate, <50%), 34 were moderate (50%-85%), and 18 were easy (≥85%). Regarding intrarater reliability, the average diagnostic match rate of five KSs involved in database construction was 0.66 ± 0.08, and as for interrater reliability, the diagnostic match rate between the 15 KSs was 0.52 (95% confidence interval, 0.38-0.65) for Gwet's agreement coefficient 1, and the degree of the match rate was moderate. In the second study, the difficulty level of questions was moderate, with a correct rate of 81.3% for medical professionals and 82.1% for students. The discrimination index was good for medical professionals (0.35) and poor for students (0.06). Among medical professionals, the correct answer group of this question had a significantly higher total score on the Kampo common test than the incorrect answer group (85.3 ± 8.4 points vs. 75.8 ± 11.8 points, p < 0.01). Conclusion: This system can objectively evaluate tongue diagnosis ability and has high practicality. Utilizing this system can be expected to contribute to improving learners' tongue diagnosis ability and standardization of tongue diagnosis.

Construction of a Standardized Tongue Image Database for Diagnostic Education: Development of a Tongue Diagnosis e-Learning System.

Tongue examination is an important diagnostic method for judging pathological conditions in Kampo (traditional Japanese medicine), but it is not easy for beginners to learn the diagnostic technique. One reason is that there are few objective diagnostic criteria for tongue examination findings, and the educational method for tongue examination is not standardized in Japan, warranting the need for a tongue image database for e-learning systems that could dramatically improve the efficiency of education. Therefore, we constructed a database comprising tongue images whose findings were determined on the basis of votes given by five Kampo medicine specialists (KMSs) and confirmed the educational usefulness of the database for tongue diagnosis e-learning systems. The study was conducted in the following five steps: development of a tongue imaging collection system, collection of tongue images, evaluation and annotation of tongue images, development of a tongue diagnosis e-learning system, and verification of the educational usefulness of this system. Five KMSs evaluated the tongue images obtained from 125 participants in the following eight aspects: (i) tongue body size, (ii) tongue body color, (iii) tongue body dryness and wetness, (iv) tooth marks on the edge of the tongue, (v) cracks on the surface of the tongue, (vi) thickness of tongue coating, (vii) color of tongue coating, and (viii) dryness and wetness of tongue coating. Medical students (MSs) were given a tongue diagnosis test using an e-learning system after a lecture on tongue diagnosis. The cumulative and individual match rates (%) (individual match rates of 100% (5/5), 80% (4/5), and 60% (3/5) are shown in parentheses, respectively) were as follows: (i) tongue body size: 92.8 (26.4/26.4/40.0); (ii) tongue body color: 83.2 (10.4/20.8/52.0); (iii) tongue body dryness and wetness: 88.8 (13.6/34.4/40.8); (iv) tooth marks on the edge of the tongue: 88.8 (6.4/35.2/47.2); (v) cracks on the surface of the tongue: 96.8 (24.0/35.2/37.6); (vi) thickness of tongue coating: 84.8 (7.2/21.6/56.0); (vii) color of tongue coating: 88.0 (15.2/37.6/35.2); and (viii) dryness and wetness of tongue coating: 74.4 (4.8/19.2/50.4). The test showed that the tongue diagnosis ability of MSs who attended a lecture on tongue diagnosis was almost the same as that of KMSs. We successfully constructed a tongue image database standardized for training specialists on tongue diagnosis and confirmed the educational usefulness of the e-learning system using a database. This database will contribute to the standardization and popularization of Kampo education.

Diagnosis and treatment of portal hypertension.

Major progress has been made in the area of diagnosis and treatment of portal hypertension. Multi-detector row computed tomography (MD-CT) enabled the revelation of the precise overview of portal hemodynamics and was applied into the decision of therapeutic strategies. A noninvasive liver stiffness measurement device, transient elastography, is expected to be applied in the prediction of existence of esophageal varices. Balloon-occluded retrograde transvenous obliteration (B-RTO) is performed mainly in Japan to eradiate the fundic varices and high eradiation rate and low recurrence rate are reported. Partial splenic embolization (PSE) has been performed to improve hypersplenism without serious side-effect and excellent result is reported. Angiotensin receptor blockers (ARBs) are promising drugs for its portal hypotensive effect and antifibrotic effect and more investigation is necessary. Anti-fibrotic therapy, including autologous bone marrow therapy, has the possibility to improve not only liver fibrosis but also portal hypertension.

A new hepatic encephalopathy model to monitor the change of neural amino acids and astrocytes with behaviour disorder.

BACKGROUND/AIMS: To elucidate the pathogenesis of hepatic encephalopathy (HE), we developed a new HE model with behaviour disorder. METHODS: Male Wistar rats were divided into four treatment groups: a HE model: acetaminophen (APAP)+3-methylcholanthrene (3-MC) group (APAP+MC group); control group: acetaminophen group; 3-methylcholanthrene group; and a no-treatment group. We monitored the changes of neural amino acids in the synaptic cleft and astrocytes in the brain during behaviour disorder. RESULTS: In the APAP+MC group, alanine amino transferase, blood ammonia and glucose increased from 3 h and total bilirubin increased at 6 h. Prothrombin time was prolonged from 3 h in the APAP+MC group. The APAP+MC group exhibited centrilobular necrosis in the liver after 8 h. In the APAP+MC group, rats jumped vertically and this vertical activity increased significantly from 4 to 7 h. During the behaviour disorder, we found that glutamate and aspartate increased in the synaptic cleft from 4 h after treatment with APAP+3-MC, glutamate increased 23.9-fold at 7 h and aspartate increased 16.1-fold at 4 h, whereas glutamine did not change. At that time, we observed morphological changes of the astrocytes by immunostaining for the glial fibrillary acidic protein. CONCLUSIONS: Our new HE model demonstrated that increased excitatory neural amino acids and morphological change in astrocytes were involved in the behaviour disorder that occurs with HE.

Web-Based Evaluation System to Measure Learning Effectiveness in Kampo Medicine.

Measuring the learning effectiveness of Kampo Medicine (KM) education is challenging. The aim of this study was to develop a web-based test to measure the learning effectiveness of KM education among medical students (MSs). We used an open-source Moodle platform to test 30 multiple-choice questions classified into 8-type fields (eight basic concepts of KM) including "qi-blood-fluid" and "five-element" theories, on 117 fourth-year MSs. The mean (±standard deviation [SD]) score on the web-based test was 30.2 ± 11.9 (/100). The correct answer rate ranged from 17% to 36%. A pattern-based portfolio enabled these rates to be individualized in terms of KM proficiency. MSs with scores higher (n = 19) or lower (n = 14) than mean ± 1SD were defined as high or low achievers, respectively. Cluster analysis using the correct answer rates for the 8-type field questions revealed clear divisions between high and low achievers. Interestingly, each high achiever had a different proficiency pattern. In contrast, three major clusters were evident among low achievers, all of whom responded with a low percentage of or no correct answers. In addition, a combination of three questions accurately classified high and low achievers. These findings suggest that our web-based test allows individual quantitative assessment of the learning effectiveness of KM education among MSs.

Evidence-based clinical practice guidelines for liver cirrhosis 2015.

The Japanese Society of Gastroenterology revised the evidence-based clinical practice guidelines for liver cirrhosis in 2015. Eighty-three clinical questions were selected, and a literature search was performed for the clinical questions with use of the MEDLINE, Cochrane, and Igaku Chuo Zasshi databases for the period between 1983 and June 2012. Manual searching of the latest important literature was added until August 2015. The guidelines were developed with use of the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system. This digest version in English introduces selected clinical questions and statements related to the management of liver cirrhosis and its complications. Branched-chain amino acids relieve hypoalbuminemia and hepatic encephalopathy and improve quality of life. Nucleoside analogues and peginterferon plus ribavirin combination therapy improve the prognosis of patients with hepatitis B virus related liver cirrhosis and hepatitis C related compensated liver cirrhosis, respectively, although the latter therapy may be replaced by direct-acting antivirals. For liver cirrhosis caused by primary biliary cirrhosis and active autoimmune hepatitis, urosodeoxycholic acid and steroid are recommended, respectively. The most adequate modalities for the management of variceal bleeding are the endoscopic injection sclerotherapy for esophageal varices and the balloon-occluded retrograde transvenous obliteration following endoscopic obturation with cyanoacrylate for gastric varices. Beta-blockers are useful for primary prophylaxis of esophageal variceal bleeding. The V2 receptor antagonist tolvaptan is a useful add-on therapy in careful diuretic therapy for ascites. Albumin infusion is useful for the prevention of paracentesis-induced circulatory disturbance and renal failure. In addition to disaccharides, the nonabsorbable antibiotic rifaximin is useful for the management of encephalopathy. Anticoagulation therapy is proposed for patients with acute-onset or progressive portal vein thrombosis.

Prolyl 4-hydroxylase inhibitor is more effective for the inhibition of proliferation than for inhibition of collagen synthesis of rat hepatic stellate cells.

The aim of this study was to investigate whether a prolyl 4-hydroxylase inhibitor (HOE 077) prevents the proliferation and collagen synthesis of rat hepatic stellate cells (HSCs). Rat HSCs were isolated and cultured with 100, 500, 1000 or 2000 &mgr;g/ml of HOE 077 with or without hepatocytes. After 4 day culture, the cell cycle of HSCs was examined by flow cytometry along with messenger RNA expression of procollagen type I. After 4 days of culture, HSCs had DNA synthesis and a high concentration (2000 &mgr;g/ml) of HOE 077 significantly reduced this DNA synthesis. However, HOE 077 incubated with hepatocytes could significantly reduce mitosis at a lower concentration of HOE077 (500 &mgr;g/ml) without significant reduction of alpha(2)(I) procollagen mRNA expression. These results indicate that the prolyl 4-hydroxylase inhibitor is more effective for the inhibition of proliferation than for inhibition of collagen synthesis of HSCs.

Th1 down-regulation at the single-lymphocyte level in HCV-related liver cirrhosis and the effect of TGF-beta on Th1 response: possible implications for the development of hepatoma.

Patients with hepatitis C virus (HCV) related-liver cirrhosis (LC) often develop hepatoma. The type 1 helper T cell (Th1) response presents an antitumor effect. We evaluated the Th1 response in patients with HCV-related LC at the single-cell level and examined the influence of transforming growth factor (TGF)-beta, an immunosuppressive cytokine, on the Th1 response. We determined the ratios of Th1-type cytokine (IFN-gamma, IL-2)-producing cells to CD3-positive cells in 14 patients (LC group) and in 16 normal controls using flow cytometry and measured serum TGF-beta(1) and TGF-beta(2) levels by ELISA. We then incubated, peripheral blood mononuclear cells from seven healthy volunteers with recombinant TGF-beta(1) or TGF-beta(2) for 48 h, and determined the ratio of IFN-gamma producing cells to CD3-positive cells. The IFN-gamma ratio was significantly lower in the LC group (29.7+/-0.3 vs. 44.2+/-15.0%, P<0.01). The serum TGF-beta(2) level was significantly increased in the LC group (601+/-232 vs. 329+/-118 pg/ml, P<0.001). TGF-beta(2) significantly suppressed IFN-gamma production at the single-cell level (10.0+/-4.3 vs. 7.3+/-2.0%, P<0.05). These findings indicated that the enhanced down-regulation of Th1 by TGF-beta(2) in patients with HCV-related LC might be effective against hepatoma.

Antioxidant, N-acetyl-L-cysteine inhibits the expression of the collagen alpha2 (I) promoter in the activated human hepatic stellate cell line in the absence as well as the presence of transforming growth factor-beta.

Although recent studies suggest the inhibitory property of N-acetyl-L-cysteine (NAC) on activation of hepatic stellate cell (HSC), the effects on once-activated HSCs are not well clarified. We investigated the influences of NAC on human-derived once-activated HSC line, LI90 with a focus on the collagen alpha2 (I) (COL1A2) promoter expression. Plasmid containing whole length of COL1A2 promoter linked to firefly luciferase gene and its various 5'-deletions were transiently transfected to LI90. The luciferase activity was determined with or without 10 mM of NAC in the absence or presence of 1 ng/ml of transforming growth factor (TGF)-beta. The effects of NAC on generation of intracellular reactive oxygen species (ROS) in LI90 were also analyzed. As a result, NAC significantly (P<0.05) suppressed the COL1A2 promoter expression in the absence or presence of TGF-beta. The expression was much more inhibited when used the deletion containing only AP-1/NF-kappaB binding sites than that including only three SP-1 binding sites. The ROS production was also comparably inhibited by NAC in both condition. These results indicated NAC suppressed, through its anti-oxidative action, the COL1A2 promoter expression in once-activated HSCs in the absence or presence of TGF-beta at least partly by affecting the signal transduction cascade encompassing AP-1/NF-kappaB activation.

Effects on liver functions of gastric variceal therapy by endoscopic ligation using a detachable snare and sclerotherapy with O-ring ligation.

Although various effects of therapy for gastric varices, a complication of portal hypertension, on portal hemodynamics have been suggested, the effects of the therapy on liver functions have not yet been clarified. The aim of this study was to examine the effects of endoscopic gastric variceal therapy on liver functions in patients with hepatic cirrhosis. The subjects were 18 hepatic cirrhosis patients with the complication of gastric varices, who could be monitored for a long term (at least 1-year after therapy). They consisted of patients receiving prophylactic treatments after being confirmed to have a risk of rupture of gastric varices (16 prophylactic cases) and those with a history of bleeding and receiving palliative treatments to prevent rebleeding (two palliative cases). To evaluate liver functions, blood tests before the therapy, just after the therapy and at 1, 3, 6 and 12 months after the therapy were retrospectively examined and the levels of albumin, total bilirubin (T.Bil), ALT, choline esterase (ChE), platelets and the Child-Pugh classification were assessed. The albumin levels tended to increase after the therapy, and the level at 6 months after the therapy was significantly higher than the pre-treatment value (P=0.0311). At 1 year after the therapy, the level was higher than the pre-treatment value, but there was no significant difference. The ChE level tended to decrease just after the therapy, but it tended to be increased at 3 and 6 months after the therapy. Although significant improvement was not seen in T.Bil, ALT, platelet levels or the Child-Pugh classification after the therapy, none of these worsened. Endoscopic gastric variceal ligation therapy significantly improved liver functions, or at least albumin synthesis.

Timeline for development of autologous bone marrow infusion (ABMi) therapy and perspective for future stem cell therapy.

Liver cirrhosis patients generally progress to liver failure. To cure this progressive disease, we developed a novel cell therapy using bone marrow cells; autologous bone marrow cell infusion (ABMi) therapy. We previously described the possible action mechanism of ABMi therapy in the cirrhotic liver, and showed the timeline and results of clinical studies of ABMi therapy. We have also carried out other clinical studies using bone marrow cells and granulocyte colony-stimulating factor. Here, we report a new randomized clinical trial to evaluate the effects of ABMi therapy. However, ABMi therapy may not be possible in patients who are unable to undergo general anesthesia; therefore, we have started to develop a next-generation stem cell therapy using cultured mesenchymal stem cells.

Reprogramming efficiency and quality of induced Pluripotent Stem Cells (iPSCs) generated from muscle-derived fibroblasts of mdx mice at different ages.

Induced pluripotent stem cells (iPSCs) hold promise as a potential treatment for Duchenne muscular dystrophy (DMD). To determine the impact of the donor's age on reprogramming, we generated iPSCs from muscle-derived fibroblasts (MuFs) of mdx mice aged 6 weeks, 6 months, and 14 months. MuFs from 14-month-old mdx mice showed lower proliferative activity and lower reprogramming efficiency, compared with those from younger mdx mice. Furthermore, iPSCs derived from 14-month-old mdx mice (14m-MuF-iPSCs) gradually lost Nanog expression, and regressed in conventional ES medium during passages. Interestingly, inhibition of TGF-ß signaling and BMP signaling stabilized Nanog expression and promoted self-renewal of 14m-MuF-iPSCs. Finally, rescued mdx-derived iPSCs efficiently differentiated into the skeletal muscle lineage.