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CAP biosynthesis in the pericarp of chili pepper fruits occurs with an ambiguous boundary in the placental septum and pericarp. Capsaicinoid (CAP) is a pungent ingredient of chili pepper fruits. Generally, CAP biosynthesis is limited to the placental septum of fruits, but it has been reported that its biosynthesis occurs even in the pericarp of some extremely pungent varieties, resulting in a substantial increase in total content. To examine the mechanism of CAP biosynthesis in the pericarp, comparative transcriptome analysis of a variety that produces CAP in the pericarp (MY) and a variety that does not (HB) was carried out. RNA-seq revealed that 2264 genes were differentially expressed in the MY pericarp compared with the HB pericarp. PCA analysis and GO enrichment analysis indicated that the MY pericarp has a gene expression profile more like placental septum than the HB pericarp. The gene expression of CAP biosynthesis-related genes in the MY pericarp changed coordinately with the placental septum during fruit development. In most Capsicum accessions including HB, the distribution of slender epidermal cells producing CAP was limited to the placental septum, and the morphological boundary between the placental septum and pericarp was clear. In some extremely pungent varieties such as MY, slender epidermal cells ranged from the placental septum to the pericarp region, and the pericarp was morphologically similar to the placental septum, such as the absence of large sub-epidermal cells and abundant spaces in the parenchymal tissue. Our data suggest that CAP biosynthesis in the pericarp occurred with an ambiguous boundary in the placental septum and pericarp. These findings contribute to further enhancement of CAP production in chili pepper fruits.

Grb2-associated binder 1 (Gab1) is an adaptor protein that is important for intracellular signal transduction by receptor tyrosine kinases that are receptors for various growth factors and plays an important role in rapid liver regeneration after partial hepatectomy and during acute hepatitis. On the other hand, mild liver regeneration is induced in livers of individuals with chronic hepatitis, where hepatocyte apoptosis is persistent; however, the impact of Gab1 on such livers remains unclear. We examined the role of Gab1 in chronic hepatitis. Gab1 knockdown enhanced the decrease in cell viability and apoptosis induced by ABT-737, a Bcl-2/-xL/-w inhibitor, in BNL.CL2 cells, while cell viability and caspase activity were unchanged in the absence of ABT-737. ABT-737 treatment induced Gab1 cleavage to form p35-Gab1. p35-Gab1 was also detected in the livers of mice with hepatocyte-specific Mcl-1 knockout (KO), which causes persistent hepatocyte apoptosis. Gab1 deficiency exacerbated hepatocyte apoptosis in Mcl-1 KO mice with posttranscriptional downregulation of Bcl-XL. In BNL.CL2 cells treated with ABT-737, Gab1 knockdown posttranscriptionally suppressed Bcl-xL expression, and p35-Gab1 overexpression enhanced Bcl-xL expression. Gab1 deficiency in Mcl-1 KO mice activated STAT3 signaling in hepatocytes, increased hepatocyte proliferation, and increased the incidence of liver cancer with the exacerbation of liver fibrosis. In conclusion, Gab1 is cleaved in the presence of apoptotic stimuli and forms p35-Gab1 in hepatocytes. In chronic liver injury, the role of Gab1 in suppressing apoptosis and reducing liver damage, fibrosis, and tumorigenesis is more important than its role in liver regeneration.

AIM In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis. METHODS Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin. RESULTS The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm2 /m2 , and the median body mass index was 23.4 kg/m2 . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men

Background & Aims Liver inflammation has been recognized as a hallmark of hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver inflammation has never been explored. Methods We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice by using the Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and time-dependent manner. Results After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice exhibited spontaneous development of hepatocyte death with elevated serum alanine aminotransferase levels and hepatic infiltration of macrophages. The removal of DOX in TG mice completely removed this effect, suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacologic inhibition of FoxM1 reduced liver inflammation in models of liver injury. Conclusions Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the treatment of liver injury but also for the prevention toward HCC., Graphical abstract

In Japan, over 95% of the acreage is covered with plastic to force June-bearing (seasonal flowering, SF) strawberry cultivars to produce fruit from late fall to early summer. During the late 1960s, a forcing technique was developed that advanced flower bud initiation to late summer and prevented the transplants from becoming dormant during winter. This new forcing technique involved nitrogen starvation of nursery plants to induce floral initiation. Until about 1980, strawberry growers in Japan used runner plants produced in waiting beds, but most transplants are now produced in plastic pots under rain shelters to avoid soil-borne diseases. Recently, the use of tray plants produced from hanging runner cuttings has become popular. To induce early floral initiation, the following artificial low temperature (LT) treatments have been established: (1) “Yarei”, a combination of a short day with solar radiation and LT under darkness in cooling facilities (Yarei-ko); (2) “Kaburei”, continuous dark-LT with refrigeration facilities including industrial warehouses; and (3) “Kanketsu-reizo”, intermittent LT storage. An overview of the technologies applied to plant propagation and the control of floral initiation of Japanese SF cultivars is provided in this review.

The translocation of calcium (Ca) within the tomato plant and the causes of Ca deficiency, a factor associated with blossom-end rot (BER) in fruit, are still a matter of conjecture. The objective of this study was to determine the effect of defoliation on BER incidence and Ca transport into different size tomato fruit cultivars. Four experiments were conducted. The start and end dates for each experiment were; 14 March–2 May, 22 July–23 August, 30 August–7 October 2017, and 20 May–25 June 2018, for experiments 1, 2, 3, and 4, respectively. Five tomato cultivars including one large (‘Momotaro fight (MF)’, ≥ 200 g), three medium (‘Lui 60 (L60)’, ‘Tio cook (TC)’, and ‘Cindy sweet (CS)’, 30–80 g), and one small (‘Pepe (PP)’, ≤ 20 g) fruit cultivars, respectively, were grown under moderate water stress controlled by a combination of root zone restriction and solar mediated fertigation. Leaf area of plants was reduced by 20–30% by removing alternate leaflets on all leaves. Defoliation significantly reduced BER in all experiments. In experiment 4, no BER was observed in defoliated plants of L60 and PP, and in MF and TC, BER incidence decreased to a quarter of the control. Defoliation increased the fruit growth rate (FGR) in experiment 1, in which the temperature was the lowest, by a ratio of 1.42 and by 1.39 in experiment 4, in which the radiation was strongest and day length longest. Defoliation increased the rate of daily Ca transport into fruit (CTR) in MF, L60, TC, CS, and PP by average ratios of 1.64, 1.55, 1.35, 1.30, and 1.13, respectively. The increase in CTR in defoliated plants was highest in experiment 4 with a ratio of 1.68 followed by 1.37, 1.33, and 1.28 in experiments 1, 3, and 2, respectively. Defoliation increased both FGR and CTR and there were significant linear relationships between them. However, the degree of increase was larger in CTR than that in FGR, especially in the BER-sensitive large fruit cultivar MF, and defoliation increased the total Ca concentration in fruit accordingly. We conclude that under moderate water stress by root zone restriction and certain other BER inductive conditions, defoliation could be a promising approach to reduce BER incidence by improving Ca nutrition in susceptible large fruit cultivars.

Background & aims Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and hepatocellular carcinoma (HCC). However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. Approach & results Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce liver sinusoidal endothelial cells (LSECs) capillarization in mice and in vitro. LSECs capillarization was also confirmed in FALD patients. Mitogenic factor, sphingosine-1-phosphate (S1P) was increased in congestive liver and expression of sphingosine kinase 1 (SphK1), a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR)1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobials treatment lowered portal blood LPS concentration, LSECs capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. Conclusions In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for RHF patients with primary or metastatic liver cancer.

Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, which range from low pungency to extremely pungent. However, the molecular mechanisms underlying this quantitative variation have not been elucidated. Our previous study identified various loss-of-function alleles of the pAMT gene which led to low pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2 ) with different levels of putative aminotransferase (pAMT) activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1 , pamtL2 and a loss-of-function pamt allele reduced capsaicinoid levels to about 50%, 10% and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels than the functional type. RNA sequencing analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non-functional splice variants containing partial sequences of Tcc. The non-functional splice variants were more dominant in pamtL2 than in pamtL1 . This suggested that the difference in position of the intronic transposons could alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid content to different levels. Our results provide a striking example of allelic variations caused by intronic transposons; these variations contribute to quantitative differences in secondary metabolite contents.

The hepatitis B (HB) vaccine effectively prevents the incidence of hepatitis B virus (HBV) infection. However, vaccine failure occurs in 5–10% of the recipients. The precise mechanisms leading to responsiveness to the HB vaccine are poorly understood. High-throughput sequencing (HTS) may help clarify the immune response to the HB vaccine, so we applied this method to investigate whether the HB vaccine induced a specific change in the T-cell receptor (TCR) and B-cell receptor (BCR) repertoires. We conducted HTS of the TCR β chain and BCR IgG heavy (H) chain complementary determining region 3 (CDR3) repertoires in five volunteers before and after the second and third immunizations with the HB vaccine. The HB surface antibody (HBsAb) levels were >10 mIU/ml after the third vaccination in all five participants. The TCR β chain CDR3 repertoire diversity significantly increased, while the BCR IgG H chain CDR3 repertoire diversity significantly decreased after the second vaccination. Although there was no marked inter-individual variation in terms of the numbers of unique reads, it is possible that the TCR β chain and BCR IgG H chain CDR3 repertoires may have changed within the same numbers of unique reads. Our data failed to identify the specific dominant clones that responded to the HB vaccine. In summary, the TCR β chain CDR3 repertoire diversity significantly increased, while the BCR IgG H chain CDR3 repertoire diversity significantly decreased, after the second HB vaccination. These diversity changes might be associated with a better response to the HB vaccine.

Objective Since healthcare providers face an increased risk of hepatitis B virus (HBV) infection because of their work, vaccination plays a critical role in preventing HBV transmission. However, the duration for which acquired HBV surface antibodies (anti-HBs) persist remains unknown. To evaluate the primary immunologic response to HBV vaccination and its persistence in healthy Japanese adolescents. Methods In total, 690 young adults underwent HBV vaccination with a three-dose schedule. The primary response was determined by the anti-HBs titers at 1-2 months after the final dosage. Subjects with anti-HBs titers of 100 mIU/mL were classified as “non-responders,” “low-responders,” and “sufficient responders,” respectively. Anti-HB titers were re-measured at 1 or 2 years after vaccination. Results First, 95.8% and 72.8% of the subjects had anti-HBs titers of >10 and >100 mIU/mL, respectively, as a primary response. The anti-HBs titers measured at 1 and 2 years after vaccination were significantly correlated with those of the primary response (1 year: r=0.893, p100 mIU/mL at the primary response maintained an anti-HBs titer of >10 mIU/mL [1 year after vaccination, 208/209 (99.5%); 2 years after vaccination, 72/81 (90.1%)]. However, in subjects with a primary response of 10-100 mIU/mL the anti-HBs titer frequently declined; 17/38 (44.7%) and 9/10 (90.0%) subjects had a titer of

Background Rhododendrol (rhododenol), an inhibitor of tyrosinase activity, is used as a skin-whitening component. Many cases of leukoderma after the application have been reported, termed rhododenol-induced leukoderma (RIL). The aim of this study was to clarify the pathogenesis of RIL morphologically through comparison with vitiligo. Methods We examined 14 cases of RIL and 15 cases of vitiligo using routine histopathology and immunohistochemistry. Thirteen cases of RIL, six cases of vitiligo and specimens of the RIL mouse model were evaluated by electron microscopy. Results There were common findings in RIL and vitiligo at the light-microscopic level: (a) vacuolar changes in the dermo-epidermal junction, (b) melanophages in the papillary dermis, (c) perifollicular lymphocyte infiltration, (d) loss or decrease of basal melanin pigment and (e) decrease of melanocytes in the lesions. The ultrastructural observations showed specific findings of RIL: (a) remaining melanocytes in depigmented lesions, (b) inhomogeneous melanization in melanocytes and (c) degenerated melanosomes in melanocytes. Some of the findings were observed in a RIL mouse model. Furthermore, it is notable that cell organelles of melanocytes were intact in our RIL cases. Conclusion Morphological changes of RIL targeting melanosomes in melanocytes without degeneration of organelles reflect the reversible clinical course of most cases.

Intensive use of a few elite sires has increased the risk of the manifestation of deleterious recessive traits in cattle. Substantial genotyping data gathered using single-nucleotide polymorphism (SNP) arrays have identified the haplotypes with homozygous deficiency, which may compromise survival. We developed Japanese Black cattle haplotypes (JBHs) using SNP array data (4843 individuals) and identified deleterious recessive haplotypes using exome sequencing of 517 sires. We identified seven JBHs with homozygous deficiency. JBH_10 and JBH_17 were associated with the resuming of estrus after artificial insemination, indicating that these haplotypes carried deleterious mutations affecting embryonic survival. The exome data of 517 Japanese Black sires revealed that AC_000165.1:g.85341291C>G of IARS in JBH_8_2, AC_000174.1:g.74743512G>T of CDC45 in JBH_17, and a copy variation region (CNVR_27) of CLDN16 in JBH_1_1 and JBH_1_2 were the candidate mutations. A novel variant AC_000174.1:g.74743512G>T of CDC45 in JBH_17 was located in a splicing donor site at a distance of 5 bp, affecting pre-mRNA splicing. Mating between heterozygotes of JBH_17 indicated that homozygotes carrying the risk allele died around the blastocyst stage. Analysis of frequency of the CDC45 risk allele revealed that its carriers were widespread throughout the tested Japanese Black cattle population. Our approach can effectively manage the inheritance of recessive risk alleles in a breeding population.

Background Calf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality. Results We identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1–180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease. Conclusions This study identified a deleted-type CNV associated with the mortality of 1–180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves.

Background Anisakiasis is a parasitic infection caused by Anisakis worms found in raw fish. Most cases of anisakiasis occur in the stomach and rarely occur in the intestine. It is extremely rare for live larvae to break through the intestine into the mesentery and cause severe intestinal ischemia. Anisakiasis can be treated conservatively, because the larvae will die in approximately 1 week, but, sometimes, a serious condition can arise, as in this case. We report the first case of extraluminal anisakiasis in which a live Anisakis worm caused severe intestinal ischemia. Case presentation The patient was a 26-year-old woman who ate squid a week prior. She had abdominal pain and was admitted to our emergency department. On physical examination, abdominal guarding and rebound tenderness were present in her lower abdomen. Contrast-enhanced computed tomography showed ascites, the whirl sign, localized submucosal edema of the intestinal wall, and a dilated small bowel segment with edema. We suspected the strangulated small bowel obstruction based on the CT-scan findings. To rule out the strangulated small bowel obstruction, laparoscopic exploration was performed. Bloody ascites in the pouch of Douglas and severe inflammation in 20 cm of the ileum were observed. An Anisakis larva had perforated the intestinal wall and was found alive in the mesentery. The ileum had developed a high degree of ischemia, so the affected section was resected. Histopathological examination revealed that the Anisakis worm body was in the inflamed mesentery and caused a high degree of ischemia in the intestinal tract. The patient was discharged 9 days after surgery. Conclusions A living Anisakis larva punctured the mesentery of the small intestine, resulting in severe intestinal ischemia. As seen in this case, intestinal anisakiasis may cause serious symptoms, and a low threshold for performing diagnostic laparoscopy for the early diagnosis of bowel ischemia secondary to anisakiasis can be useful in determining the definite diagnosis and indications for resection.

Background Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)–coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss. Methods This was a retrospective study of 58 HBV/HIV-1–coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1–coinfected patients with IRIS-HF or acute hepatitis B infection. Results During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1–coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF. Conclusions IRIS-HF was associated with HBsAg loss in HBV/HIV-1–coinfected patients.

Background: Hepatitis A virus (HAV) infection, a sporadically occurring disease in general populations, is a major cause of acute hepatitis, but rarely causes death. It is important to know the current situation regarding this infection rate among the general population so that an effective strategy and action plan for HAV vaccination or treatment can be established. Here, we investigated the seroprevalence of IgG antibodies against HAV (anti-HAV) in Iwate Prefecture, Japan, in 1998 and 2019, and compared the seroprevalence patterns over these past two decades. Methods: Altogether, 777 randomly selected serum samples were obtained from health check-up residents in Iwate prefecture in 1998 and 2019. Serum anti-HAV IgG levels were measured by radioimmunoassay (in 1998) and chemiluminescent immunoassay (in 2019). Results: Altogether, 586 serum samples (278 from males, 308 from females; mean age 53 years; range 30–89 years) and 191 serum samples (96 from males, 95 from females; mean age 54 years; range 30–89 years) from 1998 and 2019 were tested, respectively. The overall anti-HAV IgG prevalence in 2019 [16.8% (95% confidence interval, CI; 1.8–22.8%)] was significantly lower than that in 1998 [47.8% (95% CI 43.7–51.9%)] (p

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Neuropathological examinations of the brain in cases of brain death are usually insufficient because of autolysis. We examined a case of sporadic-type cerebral amyloid angiopathy-related hemorrhage (sCAA-H) in a 74-year-old Japanese woman who had been clinically established as brain dead 7 days before cardiac arrest. The brain was macerated, and a huge hematoma was evident in the right parieto-occipital region. Ordinary neuropathological examination was unable to clarify where the hematoma was located in the brain parenchyma or the subarachnoid space (SAS). Immunohistochemistry for amyloid-β (Aβ) and synaptophysin revealed that: (i) the hematoma affected the cerebral sulcus, cerebral cortex (CC) and subcortical white matter; (ii) the CC was destroyed at the depth of the cerebral sulcus; (iii) in three 6-μm-thick sections, ruptured Aβ-positive vessels were seen only in the intrasulcal hematoma and not in the CC or intracerebral hematoma; and (iv) in the CC adjacent to the intrasulcal hematoma, a few macrophages were observed, indicating a fresh infarct of the CC. These findings indicate that sCAA-H occurred first in the cerebral sulcus due to rupture of multiple meningeal vessels, as had been documented in our previous reports. The present study shows that even in an autolytic dead brain, immunohistochemistry is more useful than ordinary staining methods. Other than double-barreled vessels, several vascular changes such as fibrinoid degeneration, segmental dilatation (so-called micro-aneurysmal dilatation), and hyalinous onion-like change of the intima were seen in the intrasulcal hematoma, SAS and CC. Interestingly these vascular changes were not observed in the ruptured Aβ-positive vessels. More detailed studies will be needed to examine the correlation between these vascular changes and vessel rupture.

Objective There are few reports on the outcomes of 12-week paritaprevir, ombitasvir, and ritonavir (PTV/OBV/r) treatment in real-world clinical settings. We aimed to evaluate the efficacy and safety of 12-week treatment with ritonavir-boosted paritaprevir and ombitasvir in patients with hepatitis C virus (HCV) genotype 1 infection in a real-world setting. Methods Fifty-eight patients with chronic hepatitis or compensated hepatic cirrhosis and genotype-1 HCV infection were treated with PTV/OBV/r and followed for 24 weeks after the completion of treatment in 10 centers in northern Tohoku. The efficacy and safety of this 12-week treatment regimen was analyzed. Results Among the 58 treated patients, 18 (31%) had compensated liver cirrhosis, while 11 (19%) patients had experienced treatment failure with another treatment regimen. NS5A resistance-associated variants (RAVs) were detected at baseline in 3 patients (5.2%), including Y93H in two patients and L31M in two patients. One patient had NS5A RAVs at both positions 93 and 31. The overall sustained virological response (SVR) 24 rate was 96.6%. Three patients with NS5A RAVs also achieved an SVR24. The SVR24 rate was not significantly affected by age, sex, prior treatment, prior history of HCC, or liver stiffness. The mean alanine aminotransferase (ALT) levels decreased significantly during this treatment. Adverse events occurred in 15 patients (26%), 26% of which were grade 1 or 2. No severe adverse events occurred. Conclusion In this real-world study, 12-week PTV/OBV/r treatment was effective and safe for treating patients with HCV-1 infection who had chronic hepatitis or compensated hepatic cirrhosis.

Aim We prospectively screened patients treated with direct-acting antivirals (DAA) in order to detect and analyze serum markers that are present prior to the development of drug-induced liver injury (DILI). Methods The levels of various serum markers among DILI, non-DILI and control groups were compared. The DILI group consisted of eight patients whose alanine aminotransferase (ALT) levels exceeded 32 IU/L during the DAA treatment. Eight patients without DILI were selected for the non-DILI group via a matched-group design based on age, sex and disease severity. Additionally, eight healthy volunteers were employed as the controls. Serum measurements of cytokines/chemokines, cytokeratin-18 fragment (CK-18F) and super oxidase dismutase-2 (SOD2) were evaluated on the date at which hepatitis C virus RNA was absent (baseline). For patients with DILI, serum measurements taken before treatment, 1 week before pronounced transaminase elevation (prominence-1 W) and on the date at which pronounced elevation of transaminase occurred (prominence) were also evaluated. Results All patients treated with DAA had normalized transaminase levels at baseline. In patients with DILI, interferon-inducible protein-10 (IP-10) levels were higher at prominence-1 W than at baseline. Those patients also had significantly higher levels of SOD2 and CK-18F at prominence-1 W than at baseline. Conclusion Elevated IP-10 may be a preconditioning chemokine for DAA-induced liver injury, and damage markers associated with cell death and mitochondrial dysfunction are potential predictive serum markers for DILI.

In Japan, honey bees, bumble bees, and flies are used in strawberry production to produce normally shaped fruits. These insects may determine the inbreeding or outbreeding of strawberry flowers. Whole-genome DNA of the strawberry cultivar ‘Shizutakara’ showed no amplification of DNA fragments when PCR was performed with a F3H CAPS DNA marker. However, in other ordinary strawberry cultivars, amplification of DNA fragments was observed on PCR with the F3H. Thus, growing one plant of ‘Shizutakara’ with other cultivars and a single insect species, harvesting ‘Shizutakara’ fruit, and performing DNA analysis of the seeds could show whether the seed resulted from inbreeding or outbreeding, as the DNA fragments would be amplified from outbred but not from inbred seed. On this basis, differences in pollination mode among these three pollinators were analyzed. DNA analysis showed that DNA fragments could not be detected in seeds obtained by fly pollination in the greenhouse. Flies appeared to promote inbreeding. For honey bees and bumble bee, amplified DNA fragments could be detected. Some seeds pollinated by honey bees and bumble bee appeared to be of outbred origin. From these results, differences in pollination mode among insect pollinators were identified.

Seedlessness is a desirable trait to improve eases of processing and fresh consumption of sweet pepper (Capsicum annuum L.). Genetic resources for seedless pepper breeding are limited, compared with other Solanaceae crops. A new seedless mutant named tn-1, was identified by screening an ethylmethane sulfonate mutagenized population derived from ornamental chili pepper. Wild-type fruit contained about 40 seed; tn-1 contained no, or a few seed. Ninety-nine individuals in the F2 population derived from a cross between tn-1 and other cultivars were classified into the groups: Normal with more than 3 seed in each fruit, and Seedless with less than 3 seed in each fruit. The Seedless group had smaller fruit compared with the Normal group. The average values of fruit weight, length, and width of Normal group were 2.94 g, 3.64 cm and 1.40 cm, respectively. On the other hand, mean values for the Seedless group were 0.64 g, 2.11 cm, and 0.90 cm, respectively. Genetic analysis indicated that seedlessness was determined by a single recessive gene. Bulk segregant analyses combined with genotyping-by-sequencing were conducted to identify the genomic region responsible for seedlessness in tn-1. It appears the gene responsible for seedlessness may be located near the end of chromosome 12. The tn-1 mutant will be a useful breeding material to develop seedless sweet pepper.

This research reveals that the up-regulated expression of multiple capsaicinoid biosynthetic genes in pericarp tissue leads to the elevation of total capsaicinoid content in chili pepper fruit. Capsaicinoids are health-functional compounds that are produced uniquely in chili pepper fruits. A high capsaicinoid level is one of the major parameters determining the commercial quality and health-promoting properties of chili peppers. To investigate the mechanisms responsible for its high contents, we compared an extremely pungent cultivar ‘Trinidad Moruga Scorpion Yellow’ (MY) with other cultivars of different pungency levels (Fushimi-amanaga, Takanotsume, Red Habanero). Capsaicinoid concentrations were markedly higher in MY fruit (23.9 mg/g DW) than in other pungent cultivars including ‘Red Habanero’ (HB) fruit (14.3 mg/g DW). Comparative analysis of MY and HB reveals that both cultivars accumulated similar capsaicinoid concentrations in the placental septum, with that in the HB pericarp (1.8 mg/g DW) being markedly lower than that in the placental septum (69.1 mg/g DW). The capsaicinoid concentration in HB fruit is dependent on the placental septum, as reported in other accessions. Therefore, even though placental septum tissue contains high capsaicinoid concentrations, those in the pericarp and seeds attenuated its total content. In contrast, the MY pericarp exhibited a markedly higher concentration (23.2 mg/g DW). A qRT-PCR analysis revealed that multiple capsaicinoid biosynthetic pathway genes (Pun1, pAMT, KAS, and BCAT) were strongly up-regulated in placental septum of pungent cultivars. The genes were expressed exclusively in the MY pericarp, but were barely detected in the pericarps of other pungent cultivars. Collectively, the present study indicates that the up-regulated expression of these genes not only in placental septum but also in pericarp plays an important role in driving capsaicinoid accumulation in the whole fruit.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1–6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI ( ApoAI) and ATP-binding cassette subfamily A member 1 ( ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.

OVOL1 and OVOL2, ubiquitously conserved genes encoding C2H2 zinc finger transcription factors in mammals, control epithelial cell proliferation, and differentiation, including those in skin. OVOL1 and OVOL2 expression is coordinately mediated via the Wnt signaling pathway, and OVOL1 negatively regulates OVOL2 expression in a transcriptional manner. Our previous study of OVOL1 expression in human skin revealed that OVOL1 is preferentially expressed in the inner root sheath of the hair follicle. Therefore, we hypothesized that the OVOL1-OVOL2 axis is involved in normal and neoplastic follicular differentiation. Immunohistochemical analysis showed that OVOL1 and OVOL2 were strongly expressed in a mutually exclusive manner in the cytoplasm of inner root sheath cells and matrix cells, respectively, in normal follicles. OVOL2 was also expressed in pilomatricoma, with only partial expression of OVOL1. Cultured human keratinocytes expressed OVOL1 and OVOL2 on both the mRNA and protein levels. The expression of OVOL2 was higher in keratinocytes transfected with siRNA of OVOL1. Ketoconazole, a hair growth stimulant, up-regulated the expression of OVOL1 but did not affect OVOL2 expression. These results indicated that the OVOL1-OVOL2 axis may actively contribute to cell differentiation and proliferation in the hair bulb, suggesting that the OVOL1 and OVOL2 may be therapeutic targets of hair disorders, including alopecia, and play important roles in the tumorigenesis of pilomatricoma.

Rationale Hepatitis B virus (HBV) reactivation caused by immunosuppressive therapy or chemotherapy is well known. The administration of direct-acting antiviral agents (DAAs) to treat hepatitis C virus (HCV) infection has also been reported to cause HBV reactivation. We report a rare case of HBV reactivation in a patient with HCV infection after DAA therapy. Patient concerns In 1996, a 53-year-old female was identified as infected with HCV at a medical check-up, following which she visited our hospital. She was infected with HCV genotype 2b, and at follow up in 1997, was found to be hepatitis B surface antigen (HBsAg) and antibody against HBsAg negative, antibody against HBV core positive. She then experienced malignant lymphoma in 2001 at 58 years of age. Complete remission was achieved following chemotherapy and autologous peripheral blood stem cell transplantation. In 2014, she remained negative for HBsAg and antibody against HBsAg but positive for antibody against HBV core. In 2015, 12 weeks of sofosbuvir and ribavirin treatment for HCV was started. Serum HCV RNA levels rapidly decreased, and HCV elimination was confirmed at 24 weeks after cessation of DAA treatment. Acute hepatitis B developed at 15 weeks post- sustained virological response without any symptoms and physical examination findings. Diagnoses This case is speculated to represent HBV reactivation induced by DAA treatment in a patient with previously resolved HBV, based on virologic and clinical status. Genome sequencing revealed the HBV genotype as A2. Interventions The patient was treated with nucleotide analog for HBV reactivation once a day. Outcomes Serum HBV-DNA levels decreased, and serum liver enzymes improved following initiation of nucleotide analog treatment. Also, adverse events of nucleotide analog treatment were not observed. Lessons Although the risk may be very low, DAA therapy can cause HBV reactivation in chronic hepatitis C patients with prior HBV infection. Thus, those patients must be closely monitored for serum HBV DNA levels during and after DAA treatment.

Aim Glycosylation changes induce various types of biological phenomena in human diseases. N-Acetylglucosaminyltransferase V (GnT-V) is one of the most important glycosyltransferases involved in cancer biology. Recently, many researchers have challenged studies of lipid metabolism in cancer. To elucidate the relationships between cancer and lipid metabolism more precisely, we investigated the effects of GnT-V on lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on hepatic triglyceride production. Methods We compared lipid metabolism in GnT-V transgenic (Tg) mice with that of wild-type (WT) mice fed with normal chow or a choline-deficient amino acid-defined (CDAA) diet in vivo. HepG2 cells and GnT-V transfectants of Hep3B cells were used in an in vitro study. Results Serum triglyceride levels and hepatic very low-density lipoprotein (VLDL) secretion in Tg mice were significantly elevated compared with that of WT mice. Hepatic lipogenic genes (Lxrα, Srebp1, Fas and Acc) and VLDL secretion-related gene (Mttp1) were significantly higher in Tg mice. Expression of these genes was also significantly higher in GnT-V transfectants than in mock cells. Knockdown of GnT-V decreased, while both epidermal growth factor and transforming growth factor-β1 stimulation increased LXRα gene expression in HepG2 cells. Finally, we found that the blockade of VLDL secretion by CDAA diet induced massive hepatic steatosis in Tg mice. Conclusion Our study demonstrates that enhancement of hepatic GnT-V activity accelerates triglyceride synthesis and VLDL secretion. Glycosylation modification by GnT-V regulation could be a novel target for a therapeutic approach to lipid metabolism.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 ( Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Background & Aims Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression. Recently, a natural Wnt5a inhibitor, secreted frizzled-related protein 5 (Sfrp5), was identified as a novel adipocytokine that has reduced expression in obese adipose tissue in both rodents and human. In addition, hepatic gene expression of Wnt5a and its receptor frizzled 2 (Fz2) is elevated during fibrosis progression. Therefore, Sfrp5 could have biological significance in liver fibrosis. Methods We first investigated the effects of Sfrp5 on primary cultured mouse hepatic stellate cells (HSCs) in vitro. Next, to elucidate the roles of Sfrp5 in liver fibrosis, we investigated a carbon-tetrachloride (CCl4)-induced liver fibrosis model using Sfrp5 knockout (KO) and wild type (WT) mice in vivo. Each mouse was injected intraperitoneally with CCl4 (0.5 ml/kg) or olive oil as a single dose (acute liver injury model), or twice a week for 6 weeks (liver fibrosis model). Results In in vitro studies, Wnt5a enhanced both proliferation and migration of HSCs, and these effects could be completely blocked by Sfrp5. Moreover, siRNA knockdown of Fz2 in HSCs could block the effects of Wnt5a on both HSC proliferation and migration. In in vivo studies, there were no differences in the CCl4-induced liver injury between KO and WT mice. Hepatic Wnt5a gene expression and plasma Wnt5a levels significantly increased after a single CCl4 injection in both mice. Sfrp5 knockout significantly enhanced CCl4-induced liver fibrosis. Conclusions Our findings demonstrate that Sfrp5 may ameliorate mouse liver fibrosis through inhibition of Wnt5a/Fz2 signalling.

Dysregulation of cell metabolism is a hallmark of cancer. The mevalonate pathway in lipid metabolism has been implicated as a potential target of cancer therapy for hepatocellular carcinoma (HCC). The role of the Forkhead Box M1 (FoxM1) transcription factor in HCC development has been well documented, however, its involvement in cancer metabolism of HCC has not been fully determined. Here, we hypothesized that FoxM1 is involved in the mevalonate pathway of cholesterol biosynthesis in HCC. Inhibition of the mevalonate pathway by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), resulted in reduced expression of FoxM1 and increased cell death in human hepatoma cells. Re-exposure of mevalonate, a product of HMGCR, restored these effects. Likewise, knockdown of HMGCR reduced FoxM1 expression, indicating that FoxM1 expression was regulated by the mevalonate pathway in HCC. Mechanistically, protein geranylgeranylation was found to be responsible for FoxM1 expression and geranylgeranylated proteins, including RhoA, Rac1 or Cdc42, were shown to be involved in this process. In surgically resected human HCC tissues, the gene expression of FoxM1 had a positive correlation with that of the mevalonate pathway-related genes, such as HMGCR or sterol regulatory element-binding protein 2 (SREBP2). Furthermore, the gene expression of FoxM1 along with that of HMGCR or SREBP2 defined prognosis of HCC patients, suggesting the clinical significance of the mevalonate-FoxM1 pathway in human HCC. Our data indicate that FoxM1 links the mevalonate pathway to oncogenic signals in HCC. Thus, we propose a novel therapeutic approach to inhibit FoxM1 by targeting the mevalonate pathway for HCC.

Aims: We assessed the validity of a next-generation sequencing protocol using in-solution hybridization-based enrichment to identify NF1 mutations for the diagnosis of 86 patients with a prototypic genetic syndrome, neurofibromatosis type 1. In addition, other causative genes for classic genetic syndromes were set as the target genes for coverage analysis. Results: The protocol identified 30 nonsense, 19 frameshift, and 8 splice-site mutations, together with 10 nucleotide substitutions that were previously reported to be pathogenic. In the remaining 19 samples, 10 had single-exon or multiple-exon deletions detected by a multiplex ligation-dependent probe amplification method and 3 had missense mutations that were not observed in the normal Japanese SNP database and were predicted to be pathogenic. Coverage analysis of the genes other than the NF1 gene included on the same diagnostic panel indicated that the mean coverage was 115-fold, a sufficient depth for mutation detection. Conclusions: The overall mutation detection rate using the currently reported method in 86 patients who met the clinical diagnostic criteria was 92.1% (70/76) when 10 patients with large deletions were excluded. The results validate the clinical utility of this next-generation sequencing-based method for the diagnosis of neurofibromatosis type 1. Comparable detection rates can be expected for other genetic syndromes, based on the results of the coverage analysis.

Deficiency of tetrahydrobiopterin (BH4) in the vascular tissue contributes to endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O2−) generation in the insulin-resistant state. We investigated the effects of atorvastatin, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor; amlodipine, a calcium antagonist; and their combination on blood pressure, arterial relaxation and contraction, and vascular oxidative stress in aortas of high fructose–fed rats. Oral administration of atorvastatin for 8 weeks did not significantly lower blood pressure, but normalized angiotensin II–induced vasoconstriction and endothelial function in the fructose-fed rats. Atorvastatin treatment of fructose-fed rats increased vascular BH4 content, which was associated with an increase in endothelial NO synthase activity as well as a reduction in endothelial O2− production. On the other hand, administration of amlodipine did not affect the angiotensin II–induced vasoconstriction and endothelial function, but normalized the elevated blood pressure in the fructose-fed rats. The combined treatment did not show synergistic but additive beneficial effects. The present study suggests that combined therapy of HMG-CoA reductase inhibitors and calcium antagonists prevents functional vascular disorders in the insulin-resistant state, possibly resulting in the protection against or delay of development of hypertension, vascular dysfunction in diabetes, and thereafter atherosclerosis. Keywords:: 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor, calcium antagonist, nitric oxide, tetrahydrobiopterin, insulin resistance

Purpose Mac-2 binding protein (Mac-2bp) is one of the major fucosylated glycoproteins, which we identified with glycol-proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac-2bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker. Experimental design Serum Mac-2bp levels were determined with our developed ELISA kit. Our cohort of 127 patients with NAFLD had liver biopsy to make a histological diagnosis of NASH and simple fatty liver. Results Mac-2bp levels were significantly elevated in NASH patients compared with non-NASH (simple steatosis) patients (2.132 ± 1.237 vs. 1.103 ± 0.500 μg/mL, p < 0.01). The area under the receiver-operating characteristic curve for predicting NASH by Mac-2bp was 0.816. Moreover, multivariate logistic regression analyses showed Mac-2bp levels could predict the fibrosis stage and the presence of ballooning hepatocytes in NAFLD patients. Conclusions and clinical relevance These results support the potential usefulness of measuring Mac-2bp levels in clinical practice as a biomarker for NASH.

Background: The pituitary homeobox 1 (PITX1) protein is a member of the bicoid-related homeobox transcription factors and has essential roles in human development. Recently, the PITX1 gene has been considered as a tumor suppressor gene in various human cancers. Objective: This study examined the expression of PITX1 in the development and progression of human cutaneous malignant melanoma. Materials & Methods: Immunohistochemical and/or immunofluorescence analyses were performed to examine the histological expression of PITX1 in healthy skin and 40 cutaneous malignant melanoma cases, including 10 melanoma in situ cases. Results: Expression of PITX1 was shown in nuclei of melanocytes in normal skin. PITX1 expression was positive (labeling index: ≥10%) in 21 (52.5%) cases and negative (labeling index

Background and aim Impaired fatty liver regeneration has already been reported in many genetic modification models. However, in diet-induced simple hepatic steatosis, which showed similar phenotype with clinical pathology, whether liver regeneration is impaired or not remains unclear. In this study, we evaluated liver regeneration in mice with diet-induced simple hepatic steatosis, and focused on excess lipid accumulation occurring during liver regeneration. Methods Mice were fed high fat diet (HFD) or control diet for 9–10 weeks. We analyzed intrahepatic lipid accumulation, DNA replication, and various signaling pathways including cell proliferation and ER stress during liver regeneration after partial hepatectomy. In addition, some of mice were pretreated with tauroursodeoxycholic acid (TUDCA), a chemical chaperone which alleviates ER stress, and then we estimated TUDCA effects on liver regeneration. Results The peak of hepatocyte BrdU incorporation, the expression of proliferation cell nuclear antigen (PCNA) protein, and the expressions of cell cycle-related genes were observed in delayed time in HFD mice. The expression of phosphorylated Erk1/2 was also delayed in HFD mice. The amounts of liver triglyceride were at least twofold higher in HFD mice at each time point. Intrahepatic palmitic acid was increased especially in HFD mice. ER stress induced during liver regeneration was significantly higher in HFD mice. In HFD mice, pretreatment with TUDCA reduced ER stress and resulted in improvement of delayed liver regeneration. Conclusion In simple hepatic steatosis, lipid overloading occurring during liver regeneration might be caused ER stress and results in delayed hepatocyte DNA replication.

In Japan, there are more than 25,000 strawberry farms totaling 6360 ha. Over 95% of the acreage is in an annual hill culture system and covered with polyethylene plastic to force June-bearing culti...

Background Patients with steroid-resistant bullous pemphigoid (BP) require an appropriate treatment option. Objective A multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of high-dose intravenous immunoglobulin (IVIG; 400 mg/kg/day for 5 days) in BP patients who showed no symptomatic improvement with prednisolone (≥0.4 mg/kg/day) administered. Methods We evaluated the efficacy using the disease activity score on day15 (DAS15) as a primary endpoint, and changes in the DAS over time, the anti-BP180 antibody titer, and safety for a period of 57 days as secondary endpoints. Results We enrolled 56 patients in this study. The DAS15 was 12.5 points lower in the IVIG group than in the placebo group (p = 0.089). The mean DAS of the IVIG group was constantly lower than that of the placebo group throughout the course of observation, and a post hoc analysis of covariance revealed a significant difference (p = 0.041). Furthermore, when analyzed only in severe cases (DAS ≥ 40), the DAS15 differed significantly (p = 0.046). The anti-BP180 antibody titers showed no difference between the two groups. Conclusion IVIG provides a beneficial therapeutic outcome for patients with BP who are resistant to steroid therapy.

Aim Many studies indicate an accelerated progression of non-alcoholic steatohepatitis (NASH) in postmenopausal women. Very recently, we reported that estrogen deficiency enhanced the progression of steatohepatitis in mice fed a high fat and high cholesterol (HFHC) diet. Hypercholesterolemia is often observed in postmenopausal women, and recent studies indicate it to be an important risk factor for the progression of NASH. Statins can slow NASH progression in the estrogen-deficient state but the precise mechanisms of their effects are still unclear. Methods We investigated the effects of pitavastatin on steatohepatitis progression using ovariectomized (OVX) mice fed a HFHC diet or HFHC + pitava diet (containing 5 p.p.m. pitavastatin) for 6 weeks. Results Serum alanine aminotransferase and cholesterol levels significantly decreased in mice fed the HFHC + pitava diet compared with mice fed the HFHC diet. Real-time reverse transcription polymerase chain reaction representing hepatic inflammatory gene expressions significantly decreased in mice fed the HFHC + pitava diet compared with the HFHC-fed mice. Pitavastatin treatment also decreased both hepatic macrophage infiltration and hepatocyte chemokine (C-C motif) ligand 2 expression and improved the liver fibrosis condition when compared with the mice fed the HFHC diet. In addition, the enhanced spleen monocyte chemokine (C-C motif) receptor 2 expression in ovariectomized mice fed the HFHC diet was also decreased by pitavastatin administration. Conclusion Our study demonstrated that the exacerbated steatohepatitis progression in OVX mice fed a HFHC diet could be attenuated by pitavastatin treatment at least through inhibition of hepatic macrophage infiltration. We concluded that statins should be useful for treating NASH in postmenopausal women.

Aim: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. Methods: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. Results: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. Conclusion: This study showed that HB-EGF plays a protective role during acute liver injury.

The present study assessed the direct effects of IFNs on human astrocytes. Human astrocytes were exposed to human recombinant IFNs, and the proliferation of cells was measured. Type I IFN receptor mRNA and protein expression, the phosphoprotein levels of signaling molecules including JNK, ERK1/2, IκB, p38MAPK, Stat3, and the expression of cytokines were determined respectively. In addition, cellular glucose consumption was measured as well as Glut-1 protein and activation of GSK-3β/mTOR signal were determined. The expression of Type I IFN receptor was detected in cultured human astrocytes. 2 IU/ml IFNα2a and IFNα2b significantly decreased the proliferation of human astrocytes respectively, compared to control. IFNβ had no significant effect on the proliferation of the cells. The phosphorylation of JNK stimulated by all IFNs detected was more pronounced and sustained than ERK1/2 and IκB. No effects were observed on the activation of p38MAPK and Stat3. Moreover, Treatment with IFNα, especially with IFNα2b, decreased glucose consumption and stimulated phosphorylation of GSK-3β and mTOR, but decreased the expression of Glut-1. In contrast, IFNβ had no significant effect on either glucose consumption or activation of GSK-3β/mTOR signals. INFα2b significantly decreased the levels of IL-8 whereas the levels of GM-CSF were increased. The present study demonstrates direct inhibitory effects of IFNα on cell proliferation, cell signaling and glucose utilization in human astrocytes.

A 57-year-old Japanese man presented with a 10-year history of a plaque on his face. The lesion had gradually increased in volume. Physical examination revealed a brownish-grey plaque, approximately 5 × 5 cm in size, with ulceration on the right nasolabial area (Fig. 1a). Dermoscopy showed a milky-red area with some milia-like cysts and dilated vessels on the surface (not shown). Histo pathological examination revealed a poorly circumscribed tumour invading deeply into the dermis and subcutis. Tumour nests composed of atypical basaloid cells were embedded in a desmoplastic stroma. Some keratinous cysts and cystic glands were seen in the mid-dermis. Small ductal or glandular structures were also seen in the deep dermis (Fig. 1 b, c). Immunohistochemically, most of tumour cells were positive for carcinoembryonic antigen (CEA).

Recent studies indicate an accelerated progression of nonalcoholic steatohepatitis (NASH) in postmenopausal women. Hypercholesterolemia, an important risk factor for NASH progression, is often observed after menopause. This study examined the effects of estrogen on NASH in ovariectomized (OVX) mice fed a high-fat and high-cholesterol (HFHC) diet. To investigate the effects of estrogen deficiency, OVX mice and sham-operated (SO) mice were fed normal chow or HFHC diet for 6 wk. Next, to investigate the effects of exogenous estrogen replenishment, OVX mice fed with HFHC diet were treated with implanted hormone release pellets (containing 17β-estradiol or placebo vehicle) for 6 wk. OVX mice on the HFHC diet showed enhanced liver injury with increased liver macrophage infiltration and elevated serum cholesterol levels compared with SO-HFHC mice. Hepatocyte monocyte chemoattractant protein-1 (MCP1) protein expression in OVX-HFHC mice was also enhanced compared with SO-HFHC mice. In addition, hepatic inflammatory gene expressions, including monocytes chemokine (C-C motif) receptor 2 (CCR2), were significantly elevated in OVX-HFHC mice. Estrogen treatment improved serum cholesterol levels, liver injury, macrophage infiltration, and inflammatory gene expressions in OVX-HFHC mice. Moreover, the elevated expression of liver CCR2 and MCP1 were decreased by estrogen treatment in OVX-HFHC mice, whereas low-density lipoprotein dose dependently enhanced CCR2 expression in THP1 monocytes. Our study demonstrated that estrogen deficiency accelerated NASH progression in OVX mice fed HFHC diet and that this effect was improved by estrogen therapy. Hypercholesterolemia in postmenopausal women would be a potential risk factor for NASH progression.