The effect of heat-killed Lactobacillus brevis SBL88 on improving selective hepatic insulin resistance in non-alcoholic fatty liver disease mice without altering the gut microbiota.

BACKGROUND AND AIM: There have been several reports that some probiotics improve non-alcoholic fatty liver disease (NAFLD); however, many studies have involved cocktail therapies. We evaluated whether heat-killed Lactobacillus brevis SBL88 (L. brevis SBL88) monotherapy improves the clinical features of NAFLD. METHODS: The NAFLD model was induced in mice fed a high-fat diet (HFD) (HFD mice) or HFD + 1% heat-killed L. brevis SBL88 (SBL mice) for 16 weeks. Histopathological liver findings were analyzed. To evaluate the gut microbiota, a modified terminal restriction fragment length polymorphism analysis of the feces was performed. RNA sequencing in the liver was performed with Ion Proton™. To investigate the direct effects of heat-killed L. brevis SBL88, an in vitro study was performed. RESULTS: Histopathological findings revealed that fat droplets in the liver were significantly reduced in SBL mice; however, terminal restriction fragment length polymorphism did not show alterations in the gut microbiota between HFD mice and SBL mice. RNA sequencing and pathway analysis revealed that the regulation of lipid and insulin metabolism was affected. The mRNA expression of insulin receptor substrate 2 (IRS-2) was significantly higher in SBL mice, whereas the expression of IRS-1 was not significantly different. Phospho-IRS-2 expression was also significantly increased in SBL mice. In addition, an in vitro study revealed significant alterations in IRS-2 and forkhead box protein O1 expression levels. CONCLUSION: SBL mice exhibited partially improved selective hepatic insulin resistance. Our data suggest that heat-killed L. brevis SBL88 could attenuate the clinical features of NAFLD that are not mediated by alterations in the gut microbiota.

Treatment of hepatocellular carcinoma with autologous platelets encapsulating sorafenib or lenvatinib: A novel therapy exploiting tumor-platelet interactions.

Hepatocellular carcinoma (HCC) activates platelets through the action of adjacent sinusoidal cells. Activated platelets bind to tumor-associated endothelial cells and release growth factors that promote tumor progression. We hypothesized that platelets encapsulated with tumor inhibitors would function as drug carriers for tumor therapy. We propose a therapeutic strategy for HCC using autologous platelets encapsulating multiple tyrosine kinase inhibitors in a rat chemically induced HCC model. Sorafenib or lenvatinib was encapsulated in platelets isolated from tumor-bearing rats in vitro. The rats were divided into groups that received repeated intravenous injections (twice a week for 10 weeks) of the following materials: placebo, sorafenib (SOR), lenvatinib (LEN), autologous platelets, autologous platelets encapsulating sorafenib (SOR-PLT) and autologous platelets encapsulating lenvatinib (LEN-PLT). The therapeutic effect was then analyzed by ultrasonography (US) and histopathological analysis. Histopathological and US analysis demonstrated extensive tumor necrosis in the tumor tissue of SOR-PLT or LEN-PLT, but not in other experimental groups. By liquid chromatography-mass spectrometry, more abundant sorafenib was detected in tumor tissues after SOR-PLT administration than in surrounding normal tissues, but no such difference in sorafenib level was observed with SOR administration. Therefore, the use of autologous platelets encapsulating drugs might be a novel therapeutic strategy for HCC.

Non-alcoholic fatty liver disease is a potential risk factor for liver injury caused by immune checkpoint inhibitor.

BACKGROUND AND AIM: Because of their survival benefits, immune checkpoint inhibitors (ICIs) are widely administered to patients with various advanced-stage malignancies. During ICI treatment, drug-induced liver injury (DILI) occasionally occurs. In particular, hepatic immune-related adverse events (irAEs) are rare but serious and fatal. In patients with hepatic irAEs, immediate steroid treatment is generally recommended; however, the risk factors for ICI-associated DILI remain unknown. In the present study, we identified a risk factor for ICI-associated DILI. METHODS: We retrospectively analyzed 135 patients treated with anti-programmed cell death-1 (PD-1) antibodies, such as nivolumab and pembrolizumab, at Asahikawa Medical University Hospital. We investigated grade ≥ 2 hepatotoxic AEs during anti-PD-1 therapy, and PD-1 inhibitor-associated DILI was then diagnosed according to the Digestive Disease Week Japan (DDW-J) 2004 scale. The risk factors for PD-1 inhibitor-associated DILI were identified by Cox hazard analysis. RESULTS: Thirty-six patients developed grade ≥ 2 hepatic AEs during anti-PD-1 therapy. Among them, eight patients were diagnosed with PD-1 inhibitor-associated DILI based on the DDW-J 2004 scale. Cox hazard analysis revealed that non-alcoholic fatty liver disease (NAFLD) was a risk factor for PD-1 inhibitor-associated DILI. In addition, we revealed that the outcomes of patients with the DDW-J 2004 score = 3 were improved without steroid treatment. CONCLUSIONS: NAFLD is a potential risk factor for PD-1 inhibitor-associated DILI based on the DDW-J 2004 scale. The DDW-J 2004 scale might be useful for determining whether steroid treatment is required in patients with PD-1 inhibitor-associated DILI.

Polymorphism of receptor-type tyrosine-protein phosphatase delta gene in the development of non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Some single-nucleotide polymorphisms (SNPs) are associated with the development of non-alcoholic fatty liver disease (NAFLD). As one of the genetic factors, PNPLA3 rs738409 (I148M) is important to associate with pathogenesis of NAFLD. Because other SNPs remain unclear in Japan, we performed a high-throughput sequencing that targeted more than 1000 genes to identify a novel genetic variant in Japanese patients with NAFLD. METHODS: The present study in 36 NAFLD patients and 27 healthy volunteers was performed. A high-throughput sequencer was used to detect the gene variations. Candidate genes were validated by TaqMan SNP genotyping assay in 53 NAFLD patients and 41 healthy volunteers. To investigate the function of candidate gene, we performed biochemical analyses in cultured hepatocytes and liver tissues. RESULTS: EXO1 rs1047840, PTPRD rs35929428, IFNAR2 rs2229207, CPOX rs1131857, IL23R rs1884444, IL10RA rs2228055, and FAM3B rs111988437 were identified as candidate genetic variants, and PTPRD rs35929428 was only extracted as a SNP predicting to cause protein dysfunction. In validation analysis, PTPRD rs35929428 associated with the development of NAFLD (P = 0.015, odds ratio = 5.00, 95% confidence interval: 1.33-18.70). In addition, PTPRD rs35929428 was associated with Fib-4 index and with hepatic fat droplets. Biochemical analyses indicated that PTPRD rs35929428 promoted dephosphorylation of tyrosine 705 signal transducer and activator of transcription 3 (Tyr 705) in hepatocytes. CONCLUSION: PTPRD rs35929428 was a novel SNP in patients with NAFLD. Through exacerbation of the dephosphorylation of signal transducer and activator of transcription 3 (Tyr 705) in hepatocytes, PTPRD rs35929428 might play a role in hepatic lipid accumulation and fibrosis, followed by the development of NAFLD.

Daikenchuto (TU-100) Suppresses Tumor Development in the Azoxymethane and APCmin/+ Mouse Models of Experimental Colon Cancer.

Chemopreventative properties of traditional medicines and underlying mechanisms of action are incompletely investigated. This study demonstrates that dietary daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper effectively suppresses intestinal tumor development and progression in the azoxymethane (AOM) and APCmin/+ mouse models. For the AOM model, TU-100 was provided after the first of six biweekly AOM injections. Mice were sacrificed at 30 weeks. APCmin/+ mice were fed diet without or with TU-100 starting at 6 weeks, and sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In APC min/+ mice, the number of small intestinal tumors was significantly decreased. In the AOM model, both TU-100 and Japanese ginseng decreased colon tumor numbers. Decreased Ki-67 and ß-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression were observed. EGF receptor expression and stimulation/phosphorylation in vitro were investigated in C2BBe1 cells. TU-100, ginger, and 6-gingerol suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol inhibited EGF ERK1/2 activation. TU-100 and some of its components and metabolites of these components inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation. Copyright © 2016 John Wiley & Sons, Ltd.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines.

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells.

Augmented hepatic Toll-like receptors by fatty acids trigger the pro-inflammatory state of non-alcoholic fatty liver disease in mice.

AIM: There is considerable evidence that intestinal microbiota are involved in the development of metabolic syndromes and, consequently, with the development of non-alcoholic fatty liver disease (NAFLD). Toll-like receptors (TLRs) are essential for the recognition of microbiota. However, the induction mechanism of TLR signals through the gut-liver axis for triggering the development of non-alcoholic steatohepatitis (NASH) or NAFLD remains unclear. In this study, we investigated the role of palmitic acid (PA) in triggering the development of a pro-inflammatory state of NAFLD. METHODS: Non-alcoholic fatty liver disease was induced in mice fed a high fat diet (HFD). The mice were killed and the expression of TLRs, tumor necrosis factor (TNF), interleukin (IL)-1ß, and phospho-interleukin-1 receptor-associated kinase 1 in the liver and small intestine were assessed. In addition, primary hepatocytes and Kupffer cells were treated with PA, and the direct effects of PA on TLRs induction by these cells were evaluated. RESULTS: The expression of inflammatory cytokines such as TNF, IL-1ß, and TLR-2, -4, -5, and -9 was increased in the liver, but decreased in the small intestine of HFD-fed mice in vivo. In addition, the expression of TLRs in primary hepatocytes and Kupffer cells was increased by treatment with PA. CONCLUSION: In the development of the pro-inflammatory state of NAFLD, PA triggers the expression of TLRs, which contribute to the induction of inflammatory cytokines through TLR signals by intestinal microbiota.

Contrast-enhanced Ultrasonography Features for Diagnosing Pseudoprogression of Hepatocellular Carcinoma with Immunotherapy: A Case Report of the Response after Pseudoprogression.

A male patient in his 70s with recurrent hepatocellular carcinoma (HCC) after surgery received atezolizumab plus bevacizumab (Atezo+Bev) therapy. Initial computed tomography (CT) revealed tumor growth along with an increase in tumor markers, and contrast-enhanced ultrasonography (CEUS) showed multiple round avascular areas within the nodules with an appearance similar to a slice of Swiss cheese. Continuation of immunotherapy with consideration of the potential for pseudoprogression produced a dramatic response. Although it is difficult to distinguish between true progression and pseudoprogression, the Swiss cheese-like appearance on CEUS may be important for the early diagnosis of pseudoprogression.

A Successful Case of Hepatocellular Carcinoma Treated with Atezolizumab Plus Bevacizumab with Multisystem Immune-related Adverse Events.

A 63-year-old man with hepatitis C was treated with atezolizumab plus bevacizumab for unresectable diffuse hepatocellular carcinoma (HCC). After four cycles of atezolizumab plus bevacizumab, the diffuse HCC markedly shrank; however, he complained of general fatigue, loss of appetite, and slight loss of muscle strength in the lower legs. He was diagnosed with isolated adrenocorticotropic hormone deficiency (IAD), hypothyroidism, and myopathy, suggesting multisystem immune-related adverse events (irAEs). After administration of hydrocortisone, the clinical symptoms rapidly disappeared. Patients with multisystem irAEs can have favorable outcomes; thus, to continue immune-checkpoint inhibitors therapy, a correct diagnosis and management of multisystem irAEs are important.

Repeated Perforation of the Gallbladder in a Patient with Hepatocellular Carcinoma Receiving Lenvatinib.

A 59-year-old man who was receiving lenvatinib as a third-line tyrosine kinase inhibitor to treat hepatocellular carcinoma and multiple bone metastases complained of general fatigue four months after starting lenvatinib. A blood examination showed unexpectedly elevated serum C-reactive protein (CRP) levels. Computed tomography (CT) revealed rupture of the gallbladder wall, indicating gallbladder perforation. After conservative treatment, the patient received lenvatinib again under informed consent; however, one month later, CT revealed repeated rupture of the gallbladder wall. Gallbladder perforation had again been induced by lenvatinib. For this reason, lenvatinib is strongly considered a causative drug for gallbladder perforation.

Skeletal muscle mass is associated with toxicity, treatment tolerability, and additional or subsequent therapies in patients with hepatocellular carcinoma receiving sorafenib treatment.

BACKGROUND AND AIM: Several reports have demonstrated that skeletal muscle mass influences mortality in patients with hepatocellular carcinoma (HCC) receiving sorafenib treatment; however, there is still controversy with regard to whether skeletal muscle and adipose tissue are associated with the prognosis in HCC patients. We examined the relationship between body composition and prognosis in HCC patients. METHODS: We retrospectively analyzed 82 patients with unresectable HCC receiving sorafenib treatment. The skeletal muscle area and adipose tissue area were measured by computed tomography. Patients with low skeletal muscle index (male ≤36.2 cm2/m2, female ≤29.6 cm2/m2) and high visceral to subcutaneous adipose tissue area ratio (VSR) (male ≥ 1.33, female ≥ 0.93) were diagnosed as low skeletal muscle mass (LSMM) and high VSR, respectively. RESULTS: A total of 16 and 34 patients were classified as LSMM and high VSR, respectively. LSMM patients frequently experienced serious adverse events (SAEs) and thus had a shorter duration of sorafenib treatment than non-LSMM patients. High VSR was a significant factor for progression-free survival. LSMM patients less frequently received additional/subsequent therapies combined with sorafenib than non-LSMM patients. Multivariate Cox hazard analysis demonstrated that LSMM was a significant factor for the duration of sorafenib treatment. The treatment duration and receiving of additional/subsequent therapies were significantly associated with overall survival (OS) but not with LSMM or high VSR. CONCLUSION: LSMM was associated with the frequency of SAEs, treatment tolerability, and treatment duration. LSMM patients were less likely to receive additional/subsequent therapies than non-LSMM patients. Thus, LSMM could identify a subgroup of patients with poor OS.

Successful Treatment of Nivolumab-related Cholangitis with Prednisolone: A Case Report and Review of the Literature.

The patient was a 76-year-old man who was treated with nivolumab due to recurrent gastric cancer. A blood examination revealed grade 3 alkaline phosphatase (ALP) elevation. A histopathological examination revealed marked portal infiltration, including eosinophils and CD4+ and CD8+ T lymphocytes, suggesting nivolumab-related cholangitis accompanied by the features of both an immune-related adverse event (irAE) and drug-induced liver injury (DILI) with allergic reaction. The patient's ALP level immediately decreased after the administration of prednisolone. Although nivolumab-related cholangitis, a rare irAE, has been reported to be refractory to steroid therapy, patients with features of irAE and allergic DILI might immediately respond to prednisolone.

Long-term growth of intrahepatic papillary neoplasms: A case report.

BACKGROUND: Intraductal papillary neoplasm of the bile duct (IPNB) is a type of tumor that presents in the intra- or extrahepatic bile ducts. Cystic-type intrahepatic IPNB often mimics simple liver cysts, making the diagnosis difficult. Because the growth of IPNB is slow, careful follow-up and timely therapeutic intervention is recommended. There are few reports with a follow-up period longer than a decade; thus, we report the case of a patient with an IPNB that grew for over 13 years. CASE SUMMARY: A 65-year-old man was diagnosed, 13 years prior with a cystic hepatic tumor with abnormal imaging findings. The targeted tumor biopsy results showed no malignancy. Biannual follow-up examinations were performed because of the potential for malignancy. The cystic lesions showed gradual enlargement over 11 years and a 4 mm papillary proliferation appeared on the cyst wall, which is compatible with IPNB. The tumor was observed for another 2 years because of the patient's wishes. The imaging findings showed enlargement to 8 mm and a new 9 mm papillary proliferation of the cystic tumor. Contrast-enhanced ultrasonography showed hyperenhancement during the arterial phase in both cyst walls, indicating intraductal tumor progression in both tumors. Thus, liver segment 8 subsegmentectomy was performed. The pathological findings indicated that the tumors contained mucin, and high-grade atypia was observed in the papillary lesions, showing IPNB. CONCLUSION: The development of IPNB should be monitored in patients with cystic lesions and ultrasonography are useful tool for the evaluation.

Tegafur-uracil-induced rapid development of advanced hepatic fibrosis.

Tegafur-uracil has been reported to have only minor adverse effects and is associated with liver injury in 1.79% of Japanese patients. The development of tegafur-uracil-induced hepatic fibrosis with portal hypertension is rare. Here, we report a case of a 74-year-old woman with rapidly developing tegafur-uracil-induced hepatic fibrosis. The patient had no history of liver disease and had been treated with tegafur-uracil for 8 mo after breast cancer surgery. The patient was admitted to our hospital for abdominal distension and leg edema associated with liver dysfunction. Computed tomography imaging revealed massive ascites and splenomegaly, and a non-invasive assessment of liver fibrosis indicated advanced fibrosis. The histopathological findings revealed periportal fibrosis and bridging fibrosis with septation. The massive ascites resolved after discontinuing tegafur-uracil. These findings suggest that advanced hepatic fibrosis can develop from a relatively short-term administration of tegafur-uracil and that non-invasive assessment is useful for predicting hepatic fibrosis.

Bone morphogenetic protein-binding endothelial regulator of liver sinusoidal endothelial cells induces iron overload in a fatty liver mouse model.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is frequently accompanied by iron overload. However, because of the complex hepcidin-regulating molecules, the molecular mechanism underlying iron overload remains unknown. To identify the key molecule involved in NAFLD-associated iron dysregulation, we performed whole-RNA sequencing on the livers of obese mice. METHODS: Male C57BL/6 mice were fed a regular or high-fat diet for 16 or 48 weeks. Internal iron was evaluated by plasma iron, ferritin or hepatic iron content. Whole-RNA sequencing was performed by transcriptome analysis using semiconductor high-throughput sequencer. Mouse liver tissues or isolated hepatocytes and sinusoidal endothelial cells were used to assess the expression of iron-regulating molecules. RESULTS: Mice fed a high-fat diet for 16 weeks showed excess iron accumulation. Longer exposure to a high-fat diet increased hepatic fibrosis and intrahepatic iron accumulation. A pathway analysis of the sequencing data showed that several inflammatory pathways, including bone morphogenetic protein (BMP)-SMAD signaling, were significantly affected. Sequencing analysis showed 2314 altered genes, including decreased mRNA expression of the hepcidin-coding gene Hamp. Hepcidin protein expression and SMAD phosphorylation, which induces Hamp, were found to be reduced. The expression of BMP-binding endothelial regulator (BMPER), which inhibits BMP-SMAD signaling by binding BMP extracellularly, was up-regulated in fatty livers. In addition, immunohistochemical and cell isolation analyses showed that BMPER was primarily expressed in the liver sinusoidal endothelial cells (LSECs) rather than hepatocytes. CONCLUSIONS: BMPER secretion by LSECs inhibits BMP-SMAD signaling in hepatocytes and further reduces hepcidin protein expression. These intrahepatic molecular interactions suggest a novel molecular basis of iron overload in NAFLD.

Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical ß-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial ß-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.

Daikenchuto (TU-100) shapes gut microbiota architecture and increases the production of ginsenoside metabolite compound K.

Many pharmaceutical agents not only require microbial metabolism for increased bioavailability and bioactivity, but also have direct effects on gut microbial assemblage and function. We examined the possibility that these actions are not mutually exclusive and may be mutually reinforcing in ways that enhance long-term of these agents. Daikenchuto, TU-100, is a traditional Japanese medicine containing ginseng. Conversion of the ginsenoside Rb1 (Rb1) to bioactive compound K (CK) requires bacterial metabolism. Diet-incorporated TU-100 was administered to mice over a period of several weeks. T-RFLP and 454 pyrosequencing were performed to analyze the time-dependent effects on fecal microbial membership. Fecal microbial capacity to metabolize Rb1 to CK was measured by adding TU-100 or ginseng to stool samples to assess the generation of bioactive metabolites. Levels of metabolized TU-100 components in plasma and in stool samples were measured by LC-MS/MS. Cecal and stool short-chain fatty acids were measured by GC-MS. Dietary administration of TU-100 for 28 days altered the gut microbiota, increasing several bacteria genera including members of Clostridia and Lactococcus lactis. Progressive capacity of microbiota to convert Rb1 to CK was observed over the 28 days administration of dietary TU-100. Concomitantly with these changes, increases in all SCFA were observed in cecal contents and in acetate and butyrate content of the stool. Chronic consumption of dietary TU-100 promotes changes in gut microbiota enhancing metabolic capacity of TU-100 and increased bioavailability. We believe these findings have broad implications in optimizing the efficacy of natural compounds that depend on microbial bioconversion in general.

Effective control of relapsing disseminated intravascular coagulation in a patient with decompensated liver cirrhosis by recombinant soluble thrombomodulin.

A 70-year-old Japanese man was hospitalized for expanding purpura and chronic disseminated intravascular coagulation (DIC) caused by decompensated liver cirrhosis. As there are no effective treatments for chronic DIC caused by liver cirrhosis, we decided to administer recombinant human soluble thrombomodulin (rhsTM) after he provided informed consent. The DIC was rapidly improved; however, the purpura and coagulopathy recurred after two months, and repeated rhsTM treatments were required. The rhsTM treatment sufficiently controlled the coagulopathy for two years, without any complications, including bleeding. This is the first report demonstrating that rhsTM can be administered safely and repeatedly to a patient with decompensated liver cirrhosis, and that it appears to be associated with a favorable outcome.