Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease in industrialized countries worldwide, and has become a serious public health issue not only in Western countries but also in many Asian countries including Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease, which often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma. In turn, a large proportion of NAFLD/NASH is the liver manifestation of metabolic syndrome, suggesting that NAFLD/NASH plays a key role in the pathogenesis of systemic atherosclerotic diseases. Currently, a definite diagnosis of NASH requires liver biopsy, though various noninvasive measures are under development. The mainstays of prevention and treatment of NAFLD/NASH include dietary restriction and exercise; however, pharmacological approaches are often necessary. Currently, vitamin E and thiazolidinedione derivatives are the most evidence-based therapeutic options, although the clinical evidence for long-term efficacy and safety is limited. This practice guideline for NAFLD/NASH, established by the Japanese Society of Gastroenterology in cooperation with The Japan Society of Hepatology, covers lines of clinical evidence reported internationally in the period starting from 1983 to January 2012, and each clinical question was evaluated using the GRADE system. Based on the primary release of the full version in Japanese, this English summary provides the core essentials of this clinical practice guideline comprising the definition, diagnosis, and current therapeutic recommendations for NAFLD/NASH in Japan.(AU)

Smad9 is a new type of transcriptional regulator in bone morphogenetic protein signaling.

Smad1, Smad5 and Smad9 (also known as Smad8) are activated by phosphorylation by bone morphogenetic protein (BMP)-bound type I receptor kinases. We examined the role of Smad1, Smad5, and Smad9 by creating constitutively active forms (Smad(DVD)). Transcriptional activity of Smad9(DVD) was lower than that of Smad1(DVD) or Smad5(DVD), even though all three Smad(DVD)s associated with Smad4 and bound to the target DNA. The linker region of Smad9 was sufficient to reduce transcriptional activity. Smad9 expression was increased by the activation of BMP signaling, similar to that of inhibitory Smads (I-Smads), and Smad9 reduced BMP activity. In contrast to I-Smads, however, Smad9 did not inhibit the type I receptor kinase and suppressed the constitutively active Smad1(DVD). Smad9 formed complexes with Smad1 and bound to DNA but suppressed the transcription of the target gene. Taken together, our findings suggest that Smad9 is a new type of transcriptional regulator in BMP signaling.

An automated rapid detection system using the quenching probe method for detecting interleukin 28B and inosine triphosphatase single nucleotide polymorphisms in chronic hepatitis C.

Single nucleotide polymorphisms (SNPs) in the interleukin 28B gene (IL28B) are good pretreatment predictors of anti-hepatitis C virus (HCV) therapy with interferon. SNPs of the inosine triphosphatase (ITPA) gene are associated with reduced haemoglobin levels during treatment with ribavirin. The i-densy™ (Arkray, Inc.), which is based on the quenching probe (QP) method, automatically detects target genes in blood samples by fluorescence quenching within 100 min. Using a QP and primer set, a gene amplification response is generated that can quickly and easily detect a specific gene's arrangement by fluorometry. The present study was conducted to compare the utility of i-densy (QP method) with that of conventional direct sequencing (DS) for detecting SNPs in the IL28B and ITPA genes in chronic hepatitis C patients. Between June 2011 and January 2012, 73 consecutive patients underwent genotyping of IL28B, and 54 patients underwent genotyping of ITPA. All of the patients were seropositive for HCV-RNA. The IL28B and ITPA genotypes were tested for bi-allelic polymorphisms in rs8099917 (T/T, T/G and G/G; minor allele, G) and rs1127354 (C/C, C/A and A/A; minor allele, A), respectively. The results obtained with the QP method were identical to those obtained with the conventional DS method. The frequency of the IL28B genotypes TT, GT and GG were 74%, 24.7% and 1.4%, respectively, and those of the ITPA genotypes CC, AC and AA were 68.5%, 29.6% and 1.9%, respectively. These results indicate that the i-densy using the QP method can automatically, quickly and easily identify genotypes of IL28B and ITPA.

A scatter-compensated crystal interference factor in component-based normalization for high-resolution whole-body PET.

On a positron emission tomography (PET) scanner consisting of block detectors, coincidence responses to scattered radiation may differ from those to true depending on the crystal pair position within a coincidence block pair. Furthermore, these differences are considered to vary according to the radial position of the coincidence block pair. These conditions create ringing artifacts in the reconstructed image due to the lack of scatter compensation in detector normalization. In component-based normalization, a scatter-compensated crystal interference factor is therefore required in addition to the scatter-compensated block profile and intrinsic crystal efficiencies. In this study, we propose a scatter-compensated component-based normalization scheme using an annulus phantom, which provides true and scattered radiations over a large transaxial field of view, and evaluates the quality of three different-sized phantom images with whole-body PET. The results showed that the proposed normalization method significantly reduces the ringing artifacts in reconstructed images with different scattered/true fractions. The proposed algorithm, which introduced the scatter-compensated crystal interference factor, worked well under different scattered/true ratio conditions and was considered to be a robust, practical normalization method in high-resolution whole-body PET.

Involvement of JNK in the regulation of autophagic cell death.

Programmed cell death is a crucial process in the normal development and physiology of metazoans, and it can be divided into several categories that include type I death (apoptosis) and type II death (autophagic cell death). The Bcl-2 family proteins are well-characterized regulators of apoptosis, among which multidomain pro-apoptotic members (such as Bax and Bak) function as a mitochondrial gateway at which various apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we have previously reported that these cells still die by autophagy in response to various death stimuli. In this study, we found that jun N-terminal kinase (JNK) was activated in etoposide- and staurosporine-treated, but not serum-starved, Bax/Bak DKO cells, and that autophagic cell death was suppressed by the addition of a JNK inhibitor and by a dominant-negative mutant of JNK. Studies with sek1(-/-)mkk7(-/-) cells revealed that disruption of JNK prevented the induction of autophagic cell death. Co-activation of JNK and autophagy induced autophagic cell death. Activation of JNK occurred downstream of the induction of autophagy, and was dependent on the autophagic process. These results indicate that JNK activation is crucial for the autophagic death of Bax/Bak DKO cells.

Retrospective and prospective studies of hepatitis B virus reactivation in malignant lymphoma with occult HBV carrier.

BACKGROUND: In surface antigen of hepatitis B virus (HBsAg)-positive carrier for anticancer treatment of malignant lymphoma, it is well recognized that reactivation of hepatitis B virus (HBV) occasionally occurs. However, there have been only a few studies of HBV reactivation in serum HBsAg-negative and hepatitis B core antigen (HBcAb)-positive occult HBV carriers. We looked at both retrospective and prospective studies to determine the prevalence, clinical course and risk factor of HBV reactivation during chemotherapy in lymphoma patients. PATIENTS AND METHODS: Forty-eight of 127 (37.8%) lymphoma patients were HBsAg negative and HBcAb positive, and 24 of these patients were then given liver function tests and HBsAg tests monthly and serum HBV DNA every 3 months. RESULTS: HBV reactivation was observed in two patients (4.1%) who had received intensive chemotherapy including steroid and rituximab. Immediate administration of entecavir therapy after elevation of HBV DNA level was conducted, and this resulted in reduction of it and improvement of liver function test. CONCLUSIONS: Rituximab plus steroid-containing regimens may increase the risk of HBV reactivation in HBsAg-negative and HBcAb-positive lymphoma patients. More ambitious prospective studies are required to establish clinically useful or cost-effective follow-up methods for control of HBV reactivation in lymphoma patients with occult HBV infection.

Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma.

The programmed cell death 4 (PDCD4) gene was originally identified as a tumor-related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-beta1 (TGF-beta1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-beta1 signaling, protected cells from TGF-beta1-mediated apoptosis and suppressed TGF-beta1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-beta1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis.

Envenomation by the madagascan colubrid snake, Ithycyphus miniatus

We report two cases of envenomation by a Madagascan opisthoglyphous snake, Ithycyphus miniatus. In both cases, the snake bit the finger of a human who was preparing an experiment by tying a string around the snake body. Symptoms of the first case included temporal severe local pain and extensive bleeding. In the second case, severe pain accompanying obvious local swelling was caused and lasted for several hours. The present observations indicate that bite by I. miniatus potentially causes serious physiological effects in humans although the snake is basically calm and reluctant to bite.

Revisiting [3 + 3] route to 1,3-cyclohexanedione frameworks: hidden aspect of thermodynamically controlled enolates.

We have revisited the traditional consecutive Michael-Claisen [3 + 3] process (MC-[3 + 3]) promising the synthesis of a cyclohexane-1,3-dione derivatives from nonactivated simple ketones and enoates and evaluated its potential in modern organic synthesis. Twenty to thirty examples were demonstrated to be effective. The reactions exhibited remarkable regioselectivity with the Michael addition proceeding through nucleophilic attack by the more hindered site of the ketones without exception. The subsequent Claisen condensation resulted in the formation of carbon-carbon bonds between less hindered site of the ketones and acyl carbon of the enoates. The MC-[3 + 3] process described is useful for the synthesis of Taxol A-ring synthons in multigram quantities and for the synthesis of other six-membered carbocyclic compounds. A number of control experiments have been conducted to provide strong support for the mechanism of this MC-[3 + 3].

Gln27Glu beta2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is associated with the metabolism of lipid, glucose and energy. Beta-adrenergic receptors play an important role in the regulation of energy expenditure, in part, by stimulating lipid mobilization through lipolysis. METHODS: To assess whether it is common for the beta2-adrenergic receptor (B2AR) gene polymorphisms in codons 16 and 27 to play a role in the development of fatty liver, we investigated 251 unrelated healthy Japanese males who were drug-free and showed no signs of heavy drinking. RESULTS: The allelic frequency of B2AR gene mutation in codons 16 and 27 did not differ between obese subjects (BMI>25.0 kg/m(2), n=151) and non-obese subjects (BMI

Association of the Pro90Ser CD36 mutation with elevated free fatty acid concentrations but not with insulin resistance syndrome in Japanese.

BACKGROUND: CD36 deficiency is reportedly an underlying factor about insulin resistance, defective fatty acid metabolism and hypertriglyceridemia in spontaneously hypertensive rat (SHR), and may be involved in the pathogenesis of insulin resistance and hyperlipidemia in humans. METHODS: We examined 831 adults undergoing health screening. The majority (780) was Pro90 homozygous for the CD36 gene product, but 51 displayed a CD36 mutation (2 homozygous and 49 heterozygous for Ser90). This is the major mutation site involved in CD36 deficiency in Japanese. RESULTS: Among parameters related to insulin resistance, there were no differences in body mass index (BMI), HDL cholesterol, total cholesterol, triglycerides, insulin and insulin resistance index (HOMA IR), or blood pressure between 91 normal subjects (45 male and 46 female) randomly selected from the 780 Pro90 homozygotes and the 51 (29 male and 22 females) CD36-deficient subjects (Ser90 homozygote and Pro90Ser heterozygote). Free fatty acid concentrations, however, were higher in Ser90 CD36 subjects than in Pro90 control subjects. CONCLUSIONS: The CD36Pro90Ser mutation is not necessarily related to the insulin resistance syndrome, but is associated with high free fatty acid concentrations in Japanese.

Congenital muscular dystrophy with primary partial laminin alpha2 chain deficiency: molecular study.

The authors report a case of congenital muscular dystrophy with mild nonprogressive muscle weakness, white matter hypodensity, and absence of the laminin alpha2 chain in muscle fibers with two antibodies, but not with four others. They identified mutations in LAMA2, which explain the partial laminin alpha2 deficiency. Analysis of this case and two others allows us to refine the epitopes of two of the commercial antibodies, and illustrate the importance of using antibodies directed against different domains of the protein.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Inhibitory effects of an antisense oligonucleotide in an experimentally infected mouse model of influenza A virus.

The antiviral effects of a 20-mer antisense phosphorothioate oligonucleotide, PB2-as, on influenza A virus infection in mice were examined and compared to those of PB2-as encapsulated with several cationic liposomes. Intravenous injection of PB2-as, as a complex with DMRIE-C, a cationic liposome, was most effective for prolonging the mean survival time in days (MSDs) and increasing the survival rates of mice infected with the influenza A virus. In addition, the liposomal PB2-as significantly inhibited viral growth in lung tissues. These results suggest that PB2-as encapsulated with DMRIE-C may be active against the influenza A virus infection through the inhibition of virus replication in the mouse lung.

Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma.

Although matrix metalloproteinases (MMPs) are thought to be involved in the invasion and metastasis of a variety of malignant tumors, including human hepatocellular carcinoma (HCC), the mechanisms for the expression of MMPs in HCC are not known. To understand the mechanism(s) of MMP expression, the expression of matrilysin (MMP-7) and several genes of the Ets transcription factor family was investigated in human HCC and hepatoma-derived cell lines. The role of Ets-1 gene expression in HCC was also studied. Analysis by semiquantitative reverse transcription-PCR revealed that MMP-7 and Ets-1 are overexpressed and closely associated in HCC. To clarify the role of Ets-1, hepatoma cells were transduced with human Ets-1 or targeted with the Ets-1-specific antisense oligonucleotides. Cells stably transduced with the Ets-1 gene showed increased MMP-7 expression compared to parental and mock-transfected cells. Cells targeted with Ets-1-specific antisense oligonucleotides showed reduced expression of MMP-7. Cotransfection of cells with a MMP-7 promoter-reporter gene plasmid and an Ets-1 expression vector yielded an increase in MMP-7 promoter activity in an Ets-1-responsive element-dependent manner. Taken together, these data suggested that the Ets-1 oncogene is up-regulated and involved in the overexpression of MMP-7 in human HCC and may contribute to the progression of HCC.

Necrotizing myopathy in a patient with chronic hepatitis C virus infection: a case report and a review of the literature.

We describe a 61-year-old man presenting with necrotizing myopathy associated with chronic active hepatitis due to hepatitis C virus (HCV) infection. Thirteen patients with HCV-associated myopathy have been reported previously. In most of these cases, varying degrees of inflammatory changes were observed in the muscle tissue. In 2 patients, myopathy developed after initiation of interferon therapy for chronic HCV hepatitis. Our case was unusual due to long-standing elevation of creatine kinase values which improved following interferon therapy and the non-inflammatory features of the muscle tissue where the HCV RNA minus strand, a marker for replicative intermediates of the virus, was undetectable. The association of myopathy with HCV infection might represent a unique clinical entity, although the underlying pathological mechanisms remain unknown.

Effect of age on rat aortic responses to acetylcholine and nitric oxide donor (NOC-18).

The purpose of the present study was to examine the effects of age on vascular responses to acetylcholine (ACh) and NO-releasing compound (NOC-18). The studies were performed in young (4 months old, n = 8) and old (22 months old, n = 6) male rats. Responses to ACh and NOC-18 were examined in vitro by using isolated abdominal aortic rings. The maximum relaxation response to ACh, an endothelium-dependent vasodilation, was lower in aortas from old rats. Sensitivity (mean effective concentration; EC50) of ACh in old rats was significantly less than in young rats. There were no differences in maximum NOC-18-induced relaxation, an endothelium-independent vasodilation, in aortas from young and old rats. On the other hand, the concentration-response curve for NOC-18 was shifted to the right and the sensitivity (EC50 to NOC-18) was lower in old rats. These results indicated that both endothelium-dependent vasodilation induced by ACh and endothelium-independent vasodilation induced by NOC-18 are impaired in aorta from old rats.