Published papers

To improve the tolerability and maintain sufficient efficacy of regorafenib, we conducted a phase II study of regorafenib with a lower starting dose of 120 mg/day for patients with refractory metastatic colorectal cancer. Using this dose-escalation strategy, the disease control rate for the 68 evaluable patients was lower than that in our statistical hypothesis, and a relationship of unbound exposure with toxicity was observed.

Background & aims: Osteosarcopenic obesity (OSO) encompassing obesity, sarcopenia and osteopenia, is due to redistribution or infiltration of fat into muscle and bone. This cross-sectional study evaluated the association between OSO and non-alcoholic fatty liver disease (NAFLD). Methods: Obesity, sarcopenia and osteopenia was defined using the percentage of body fat mass, reduced muscle mass, and the percentage of young adult mean < 80%, measured by dual-energy x-ray absorptiometry, respectively. Non-obese and obese NAFLD was diagnosed by ultrasound and body mass index cut-off point (25 kg/m2). A total of 619 subjects ≥ 50 years who completed health checkups were divided into obesity group including OSO and sarcopenic obesity (SO) alone phenotype, and non-obesity group that did not belong to any phenotype, including standard (St). Results: Overall osteopenia and OSO were detected in only 10% and 1% in males, compared with 45% and 9% in females, respectively. Multivariate analysis for females demonstrated a significant association of OSO with non-obese NAFLD (odds ratio = 3.737, 95% confidence interval = 1.365–10.233, P = 0.010), while the association between SO alone and non-obese NAFLD was equivocal. The OSO phenotype had a significantly higher proportion of slower walking speed and weaker grip strength, compared to the St phenotype. The proportion of OSO increased with age in contrast to constant prevalence of non-obese NAFLD. Conclusion: Non-obese NAFLD had a significant association with OSO in females, independent of plausible confounders. These results suggest that non-obese NAFLD might be an independent risk factor for OSO.

Background & aims: Non-alcoholic fatty liver disease (NAFLD) is significantly related to sarcopenia as well as obesity and its associated comorbidities. This cross-sectional study aims to examine the association between four body composition phenotypes (standard, obesity alone, sarcopenia alone, sarcopenic obesity) and non-obese NAFLD, or obese NAFLD. Methods: Reduced muscle mass and high percentage of body fat mass was measured by dual-energy x-ray absorptiometry, and body composition phenotypes were determined, according to Asian criteria for sarcopenia. Based on body mass index (BMI) cut-off point (25 kg/m2) and hepatic steatosis on ultrasound, 748 subjects who underwent a health checkup were enrolled and divided into three groups: non-obese NAFLD, obese NAFLD, and no steatosis. Results: Of 563 subjects (64.1 ± 13.0 years) without secondary causes for steatosis, the overall prevalence of non-obese NAFLD and obese NAFLD were 17% and 16%, respectively. The former prevalence remained relatively constant at around 20% from the 50s to 80's, while the proportion of sarcopenic obesity in all subjects increased gradually with age, reaching 18% in the 80's. Multivariate analysis demonstrated a significant association between sarcopenic obesity and non-obese NAFLD after adjusting for confounders (odds ratio = 2.367, 95% confidence interval = 1.317–4.254, P = 0.004). On the other hand, no significant association was found between obesity alone and obese NAFLD, when BMI and visceral adipose tissue were added as confounders, although 91% of obese NAFLD was included in obesity alone phenotype. Conclusion: Non-obese NAFLD had a significant association with sarcopenic obesity, independent of metabolic confounders. Early treatment intervention for non-obese NAFLD could suppress the deterioration of sarcopenic obesity because non-obese NAFLD might be a risk factor for sarcopenic obesity.

Background: Adrenomedullin (AM) is a bioactive peptide having many pleiotropic effects, including mucosal healing and immunomodulation. AM has shown beneficial effects in rodent models and in preliminary study for patients with ulcerative colitis (UC). We performed a clinical trial to investigate the efficacy and safety of AM in patients with UC. Methods: This was a multi-center, double-blind, placebo-controlled phase-2a trial evaluating 28 patients in Japan with steroid-resistant UC. Patients were randomly assigned to four groups and given an infusion of 5, 10, 15 ng/kg/min of AM or placebo for 8 h per day for 14 days. The primary endpoint was the change in Mayo scores at 2 weeks. Main secondary endpoints included the change in Mayo scores and the rate of clinical remission at 8 weeks, defined as a Mayo score 0. Results: No differences in the primary or secondary endpoints were observed among the four groups at 2 weeks. Despite the insufficient tracking rate, the Mayo score at 8 weeks was only significantly decreased in the high-dose AM group (15 ng/kg/min) compared with the placebo group (− 9.3 ± 1.2 vs. − 3.0 ± 2.8, P = 0.035), with its rate of clinical remission at 8 weeks being significantly higher (3/3, 100% vs. 0/2, 0%, P = 0.025). We noted mild but no serious adverse events caused by the vasodilatory effect of AM. Conclusions: In this double-blind randomized trial, we observed the complete remission at 8 weeks in patients with steroid-resistant UC receiving a high dose of AM. Clinical trial registry: JAPIC clinical trials information; Japic CTI-205255 (200410115290). https://www.clinicaltrials.jp/cti-user/trial/Search.jsp.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes. Gastroenteropancreatic neuroendocrine neoplasms are a rare but lethal cancer with a scarcity of clinically relevant models. Kawasaki et al. establish and characterize 25 organoid lines to identify molecular subtypes with genotype-phenotype mapping.

Abstract Imbalance in gut bacterial composition provokes host proinflammatory responses causing diseases such as colitis. Colonization with a mixture of Clostridium species from clusters IV and XIVa was shown to suppress colitis through the induction of IL-10-producing regulatory T (Treg) cells. We demonstrate that a distinct Clostridium strain from cluster I, Clostridium butyricum (CB), prevents acute experimental colitis in mice through induction of IL-10, an anti-inflammatory cytokine. However, while CB treatment had no effect on IL-10 production by T cells, IL-10-producing F4/80(+)CD11b(+)CD11c(int) macrophages accumulated in the inflamed mucosa after CB treatment. CB directly triggered IL-10 production by intestinal macrophages in inflamed mucosa via the TLR2/MyD88 pathway. The colitis-preventing effect of CB was negated in macrophage-specific IL-10-deficient mice, suggesting that induction of IL-10 by intestinal macrophages is crucial for the probiotic action of CB. Collectively, CB promotes IL-10 production by intestinal macrophages in inflamed mucosa, thereby preventing experimental colitis in mice.

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. CONCLUSION: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

BACKGROUND: Interleukin (IL)-22-producing RORγt innate lymphoid cells (ILCs) play a pivotal role in intestinal immunity. Recent reports demonstrated that ILCs contribute to mucosal protection and intestinal inflammation in mice. In humans, numbers of RORγt ILCs are significantly increased in the intestine of patients with Crohn's disease (CD), suggesting that ILCs may be associated with intestinal inflammation in CD. However, the mechanism by which ILCs are regulated in the intestine of patients with CD is poorly understood. This study aimed to determine the activation mechanism of intestinal ILCs in patients with CD. METHODS: CD45 lineage marker ILCs were isolated from intestinal lamina propria of patients with CD. ILCs were then subdivided into 4 distinct populations based on the expression of CD56 and CD127. Purified ILC subsets were cocultured with intestinal CD14 macrophages, and IL-22 production was evaluated. RESULTS: CD127CD56 and CD127CD56 ILC, but not CD127CD56 or CD127CD56 ILC, subsets expressed RORγt and produced IL-22. IL-22 production by these ILC subsets was enhanced when ILCs were cocultured with intestinal macrophages. IL-23 or cell-to-cell contact was required for macrophage-mediated activation of ILCs. IL-22 production by ILCs was perturbed in inflamed mucosa compared with noninflamed mucosa. IL-22 induced the expression of Reg1α and Claudin-1 in human intestinal epithelial organoids. CONCLUSIONS: RORγt ILCs might enhance mucosal barrier function through the upregulation of Reg1α through production of IL-22. Although CD14 macrophages augment intestinal inflammation in patients with CD, macrophages also promote a negative feedback pathway through the activation of IL-22 production by RORγt ILCs.

BACKGROUND & AIMS: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. METHODS: ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. RESULTS: ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. CONCLUSIONS: ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. KEYWORDS: Acyl-coenzyme A:cholesterol acyltransferase; Bone morphogenetic protein and activin membrane-bound inhibitor; Free cholesterol; Hepatic stellate cell; Toll-like receptor 4; Transforming growth factor-β

Gut-derived microbial antigens trigger the innate immune system during acute liver injury. During recovery, regulatory immunity plays a role in suppressing inflammation; however, the precise mechanism underlying this process remains obscure. Here, we find that recruitment of immune-regulatory classical dendritic cells (cDCs) is crucial for liver tolerance in concanavalin A-induced acute liver injury. Acute liver injury resulted in enrichment of commensal Lactobacillus in the gut. Notably, Lactobacillus activated IL-22 production by gut innate lymphoid cells and raised systemic IL-22 levels. Gut-derived IL-22 enhanced mucosal barrier function and promoted the recruitment of regulatory cDCs to the liver. These cDCs produced IL-10 and TGF-β through TLR9 activation, preventing further liver inflammation. Collectively, our results indicate that beneficial gut microbes influence tolerogenic immune responses in the liver. Therefore, modulation of the gut microbiota might be a potential option to regulate liver tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolism by the gut microbiota affects host physiology beyond the gastrointestinal tract. Here, we find that antibiotic-induced dysbiosis, in particular, overgrowth of Lactobacillus murinus (L. murinus), impaired gut metabolic function and led to the development of alopecia. While deprivation of dietary biotin per se did not affect skin physiology, its simultaneous treatment with vancomycin resulted in hair loss in specific pathogen-free (SPF) mice. Vancomycin treatment induced the accumulation of L. murinus in the gut, which consumes residual biotin and depletes available biotin in the gut. Consistently, L. murinus induced alopecia when monocolonized in germ-free mice fed a biotin-deficient diet. Supplementation of biotin can reverse established alopecia symptoms in the SPF condition, indicating that L. murinus plays a central role in the induction of hair loss via a biotin-dependent manner. Collectively, our results indicate that luminal metabolic alterations associated with gut dysbiosis and dietary modifications can compromise skin physiology. Copyright © 2017. Published by Elsevier Inc.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

BACKGROUND/AIMS: Recent developments in analytical techniques including next-generation sequencing have clarified the correlation between intestinal microbiota and inflammatory bowel disease. Fecal microbiota transplantation (FMT) for patients with ulcerative colitis (UC) is proposed as a potential approach to resolving their dysbiosis; however, its safety and efficacy have not been confirmed. This single-arm, open-label, non-randomized study aimed to evaluate the safety and efficacy of FMT for Japanese patients with UC as the first registered clinical trial in Japan. METHODS: We enrolled 10 patients with active UC despite medical therapy. The donors were the patients' relatives and were carefully screened for infectious diseases. Fecal material was administered via colonoscopy, and the primary endpoint was the presence or absence of serious adverse events related to FMT. The secondary endpoint was a change in partial Mayo score at 12 weeks post-FMT. Scores ≤2 were considered a clinical response. Fecal samples were collected to follow changes in gut microbiota, while extracted complementary DNA were analyzed by a next-generation sequencer. We obtained written informed consent from all patients and donors. This study was approved by our Institutional Review Board and is registered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN 000012814). RESULTS: Five patients with moderate disease and five with severe disease were enrolled. No severe adverse effects were observed. One patient achieved clinical response; however, none of the patients' microbiota diversity recovered to the donor levels. CONCLUSIONS: The use of single FMT for UC was safe; however, we failed to show its clinical efficacy and potential to change the intestinal microbiota.

BACKGROUND & AIMS: Although obesity is a risk factor for acute liver failure, the pathogenic mechanisms are not yet fully understood. High cholesterol (HC) intake, which often underlies obesity, is suggested to play a role in the mechanism. We aimed to elucidate the effect of a HC diet on acetaminophen-induced acute liver injury, the most frequent cause of acute liver failure in the USA. METHODS: C57BL/6 Toll-like receptor 9 (TLR9) knockout (Tlr9-/-) mice and their Tlr9+/+ littermates were fed an HC diet for fourweeks and then treated with acetaminophen. Liver sinusoidal endothelial cells (LSECs) were isolated from the mice for in vivo and in vitro analyses. RESULTS: The HC diet exacerbated acetaminophen-induced acute liver injury in a TLR9/inflammasome pathway-dependent manner. LSECs played a major role in the cholesterol loading-induced exacerbation. The accumulation of free cholesterol in the endolysosomes in LSECs enhanced TLR9-mediated signaling, thereby exacerbating the pathology of acetaminophen-induced liver injury through the activation of the TLR9/inflammasome pathway. The accumulation of free cholesterol in LSEC endolysosomes induced a dysfunction of the Rab7 membrane trafficking recycling mechanism, thus disrupting the transport of TLR9 from late endosomes to the lysosomes. Consequently, the level of active TLR9 in the late endosomes increased, thereby enhancing TLR9 signaling in LSECs. CONCLUSIONS: HC intake exaggerated acetaminophen-induced acute liver injury via free cholesterol accumulation in LSECs, demonstrating a novel role of free cholesterol as a metabolic factor in TLR9 signal regulation and pathologies of acetaminophen-induced liver injury. Therapeutic approaches may target this pathway. Lay summary: High cholesterol intake exacerbated acetaminophen-induced acute liver injury via the accumulation of free cholesterol in the endolysosomes of liver sinusoidal endothelial cells. This accumulation enhanced Toll-like receptor 9 signaling via impairment of its membrane trafficking mechanism. Thus, free cholesterol accumulation, as an underlying metabolic factor, exacerbated the pathology of acetaminophen-induced liver injury through activation of the TLR9/inflammasome pathway. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. KEYWORDS: Acetaminophen; Endosome; Free cholesterol; Inflammasome; Liver injury; Liver sinusoidal endothelial cell; Lysosome; Metabolic syndrome; Rab7; Toll-like receptor 9

Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development. Copyright © 2016 Elsevier Inc. All rights reserved.

BACKGROUND AND AIMS: Although several noninvasive and easily accessible biomarkers for inflammatory bowel disease [IBD] are available, their sensitivity and specificity are not adequate to be used as single markers and do not overrule the need for endoscopic evaluation. We previously reported that serum leucine-rich alpha-2 glycoprotein [LRG] was a novel biomarker for rheumatoid arthritis and IBD. We herein investigated whether LRG could indicate endoscopic activity in patients with ulcerative colitis [UC]. METHODS: Serum LRG concentrations were determined by enzyme-linked immunosorbent assay [ELISA] in consecutive 129 patients with UC in two tertiary care hospitals, and associations of LRG with clinical and endoscopic activities were evaluated. Clinical activity index [CAI] < 6 was defined as clinical remission, and mucosal healing [MH] and complete mucosal healing were defined as Matts' endoscopic grades of 1 or 2 and grade of 1, respectively. RESULTS: Serum LRG levels were significantly increased and correlated with clinical and endoscopic activities in patients with UC. LRG levels were associated with both clinical and endoscopic activities even in patients with normal serum C-reactive protein [CRP] levels. Furthermore, LRG levels were significantly lower in patients with complete MH and deep remission. Serial measurements of LRG levels in a subset of patients demonstrated that LRG was significantly elevated during the endoscopically active stage compared with that during the MH stage. CONCLUSIONS: Serum LRG is a novel biomarker for detecting MH during disease course in patients with UC and a surrogate marker of endoscopic inflammation in patients with normal CRP levels.

Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of Apc(Min/+) (Min) mice and subjected them to 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.