Smad Phospho-Isoforms for Hepatocellular Carcinoma Risk Assessment in Patients with Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) sometimes occurs in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is lower than and less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is implicated in fibrosis and carcinogenesis. TGF-βtype I receptor (TβRI) and c-Jun N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3, resulting in 2 distinct phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). In mature hepatocytes, oncogenic signaling via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. We immunohistochemically examined domain-specific Smad3 phosphorylation in liver biopsy specimens from 30 NASH patients representing different fibrotic stages and 20 chronically infected hepatitis C patients as controls, correlating Smad3 phosphorylation with clinical course. HCC occurred during follow-up in 11 of 12 NASH patients with abundant pSmad3L and limited pSmad3C but in only 2 of 18 with limited pSmad3L. In contrast, HCC developed in 12 of 15 NASH patients with limited pSmad3C but only 1 of 15 with abundant pSmad3C. Two of fourteen NASH patients with mild fibrosis developed HCC, their hepatocytic nuclei showed abundant pSmad3L and limited pSmad3C. Five of sixteen patients with severe fibrosis did not develop HCC, their hepatocytic nuclei showed limited pSmad3L and abundant pSmad3C. Smad phospho-isoforms may represent important biomarkers predicting HCC in NASH and potential therapeutic targets for preventing NASH-related HCC.


Exacerbation of gefitinib-induced liver injury by glutathione reduction in mice.

Gefitinib (GEF) is the first selective tyrosine kinase inhibitor of epidermal growth factor receptor. It is associated with the occurrence of clinical drug-induced liver injury. Although GEF is metabolized to chemically reactive metabolites by cytochrome P450 3A and 1A enzymes and then conjugated to glutathione (GSH), whether these reactive metabolites contribute to GEF-induced toxicity remains unknown. In this study, we investigated whether GSH depletion can sensitize mice to liver injury caused by GEF. Male C57BL/6J mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO) at 700 mg/kg to inhibit GSH synthesis and then orally administered GEF at 500 mg/kg every 24 hr for 4 consecutive days. The coadministration of BSO and GEF increased plasma alanine aminotransferase (ALT) levels to approximately 700 U/L and 1600 U/L at 72 and 96 hr after the first administration, respectively, whereas the increase in plasma ALT levels in mice receiving GEF at 500 mg/kg alone was limited, suggesting that GSH plays a protective role in GEF-induced liver injury. Histological examination showed nuclear karyorrhexis and sporadic single hepatocyte death in the livers of BSO+GEF coadministered mice. In these mice, the hepatic expression levels of heme oxygenase 1 (Hmox1) and metallothionein 2 (Mt2) mRNA, caspase 3/7 enzymatic activity, and the amounts of 2-thiobarbiuric acid reactive substances were significantly increased, suggesting the presence of oxidative stress, which may be associated with hepatocellular death. Together, these results show that oxidative stress as well as the reactive metabolites of GEF are involved in GEF-induced liver injury in GSH-depleted mice.


Pharmacological Characterization of a Novel Mouse Model of Cholestatic Pruritus.

receptor antagonist) did not affect scratching. These results suggested that partial obstruction of bile secretion in mice induced anti-histamine-resistant itching and that central opioid system is involved in cholestatic itching.


Dietary fat, cholesterol, and cholic acid affect the histopathologic severity of nonalcoholic steatohepatitis in Sprague-Dawley rats

Understanding of the pathogenesis of nonalcoholic steatohepatitis (NASH)-associated fibrosis has been hampered by the lack of a comprehensive and physiological small animal model of NASH with fibrosis. Feeding a high-fat and high-cholesterol (HFC) diet supplemented with cholic acid to rats is known to replicate human NASH pathology, and it induces fibrosis earlier than with an HFC diet alone. In the present study, physiological and histopathological observations from 65 Sprague-Dawley (SD) rats fed an HFC diet with or without cholic acid for 9 or 18 weeks in our laboratory between January 2013 and February 2018 were retrospectively reviewed. The liver weight/body weight ratio at the end of the rearing period was higher in rats fed an HFC diet than in rats fed a normal diet in a cholesterol dose-, cholic acid dose-, or rearing period dependent manner. Dietary fat, cholesterol and/or cholic acid and rearing period affected the histopathologic severity of NASH. Overall, 56 (86.2%) of 65 SD rats fed an HFC diet for 9 or 18 weeks developed histopathologically proven NASH. It is noted that the SD rats fed an HFC diet supplemented with 2% (w/w) cholic acid for 18 weeks frequently developed advanced fibrosis, including cirrhosis. Thus, this diet-induced NASH rat model is likely to be a highly reproducible.


Hepatic Campylobacter jejuni infection in patients with Castleman-Kojima disease (idiopathic multicentric Castleman disease with thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome).

Castleman-Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD-TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD-TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD-TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD-TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD-TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD-TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD-TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross-reactive autoantibodies.


The challenges of primary biliary cholangitis: What is new and what needs to be done.

Primary Biliary Cholangitis (PBC) is an uncommon, chronic, cholangiopathy of autoimmune origin and unknown etiology characterized by positive anti-mitochondrial autoantibodies (AMA), female preponderance and progression to cirrhosis if left untreated. The diagnosis is based on AMA- or PBC-specific anti-nuclear antibody (ANA)-positivity in the presence of a cholestatic biochemical profile, histologic confirmation being mandatory only in seronegative cases. First-line treatment is ursodeoxycholic acid (UDCA), which is effective in preventing disease progression in about two thirds of the patients. The only approved second-line treatment is obeticholic acid. This article summarizes the most relevant conclusions of a meeting held in Lugano, Switzerland, from September 23rd-25th 2018, gathering basic and clinical scientists with various background from around the world to discuss the latest advances in PBC research. The meeting was dedicated to Ian Mackay, pioneer in the field of autoimmune liver diseases. The role of liver histology needs to be reconsidered: liver pathology consistent with PBC in AMA-positive individuals without biochemical cholestasis is increasingly reported, raising the question as to whether biochemical cholestasis is a reliable disease marker for both clinical practice and trials. The urgent need for new biomarkers, including more accurate markers of cholestasis, was also widely discussed during the meeting. Moreover, new insights in interactions of bile acids with biliary epithelia in PBC provide solid evidence of a role for impaired epithelial protection against potentially toxic hydrophobic bile acids, raising the fundamental question as to whether this bile acid-induced epithelial damage is the cause or the consequence of the autoimmune attack to the biliary epithelium. Strategies are needed to identify difficult-to-treat patients at an early disease stage, when new therapeutic approaches targeting immunologic pathways, in addition to bile acid-based therapies, may be effective. In conclusion, using interdisciplinary approaches, groundbreaking advances can be expected before long in respect to our understanding of the etiopathogenesis of PBC, with the ultimate aim of improving its treatment.


Age-related alterations of nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet.

Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), has a potentially progressive course that can lead to liver cirrhosis. Age is strongly associated with the development and progression of NAFLD/NASH, but the natural history of pediatric NAFLD is still not fully understood. Here, we evaluated the age-related alterations of NASH in 5-, 9- and 13-wk-old male Sprague-Dawley rats that were fed a high-fat and high-cholesterol diet (30% fat, 1.25% cholesterol and 0.5% sodium cholate, w/w) for 9 wk (6 rats/group). Our results showed that the cumulative energy intake, body weight gain and food efficacy during the 9-wk rearing period were highest in the youngest group and lowest in the oldest group. Serologically, almost all parameters including the serum triglyceride and total cholesterol were similar regardless of age. Histopathological findings, such as hepatic steatosis, lobular inflammation and hepatocyte ballooning, were also similar regardless of age, but hepatic fibrosis was more evident in the oldest group. Also, the mRNA expression levels of some fibrogenic, inflammatory, oxidative stress and cholesterol or lipid metabolism-related genes in the liver were highest in the oldest group and lowest in the youngest group, although the difference was not statistically significant. These results indicated that aging is likely associated with the development of NASH. Because the cumulative energy intake and daily food intake/body weight were not similar among groups in the present study, further studies designed with an equivalent daily food intake/body weight among groups are needed in order to interpret the exact nutritional effect.

Journal of Nutritional Science and Vitaminology


Establishment of a mouse model of troglitazone-induced liver injury and analysis of its hepatotoxic mechanism.

Drug-induced liver injury is a major problem in drug development and clinical drug therapy. Troglitazone (TGZ), a thiazolidinedione antidiabetic drug for the treatment of type II diabetes mellitus, was found to induce rare idiosyncratic severe liver injury in patients, which led to its withdrawal in 2000. However, in normal experimental animals in vivo TGZ has never induced liver injury. To explore TGZ hepatotoxic mechanism, we established a novel mouse model of TGZ-induced liver injury. Administration of BALB/c female mice with a single intraperitoneal TGZ dose (300 mg/kg) significantly elevated alanine aminotransferase and aspartate aminotransferase levels 6 hours after the treatment. The ratio of oxidative stress marker glutathione/disulfide glutathione was significantly decreased. The increased hepatic mRNA levels of inflammation- and oxidative stress-related factors were observed in TGZ-treated mice. Subsequently, hepatic transcriptome profiles of TGZ-exposed liver were compared with those of non-hepatotoxic rosiglitazone. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was activated in TGZ-induced liver injury. The activation of the JAK/STAT pathway promoted phosphorylation of STAT3 in TGZ-treated mice. Consequently, upregulation of STAT3 activation increased mRNA levels of its downstream genes. In conclusion, a single intraperitoneal dose of TGZ exposure could induce liver injury in BALB/c female mice and, by a hepatic transcriptomic analysis, we found that the activation of JAK/STAT pathway might be related to TGZ-induced hepatotoxicity.


Experimental Evidence of Liver Injury by BSEP-Inhibiting Drugs With a Bile Salt Supplementation in Rats.

The bile salt export pump (BSEP, ABCB11) mediates bile acid efflux from hepatocytes into bile. Although the inhibition of BSEP has been implicated as an important mechanism of drug-induced liver injury (DILI), liver injury caused by BSEP-inhibiting drugs is rarely reproduced in experimental animals, probably due to species differences in bile acid composition between humans and rodents. In this study, we tested whether supplementation with chenodeoxycholic acid (CDCA) sodium, a hydrophobic bile salt, could sensitize rats to liver injury caused by a BSEP-inhibiting drug. A potent BSEP inhibitor, ketoconazole (KTZ), which is associated with clinical DILI, was intragastrically administered simultaneously with CDCA at a nontoxic dose once a day for 3 days. Plasma transaminase levels significantly increased in rats receiving CDCA+KTZ, whereas neither treatment with CDCA alone, KTZ alone nor a combination of CDCA and miconazole, a safe analog to KTZ, induced liver injury. In CDCA+KTZ-treated rats, most bile acid species in the liver significantly increased compared with treatment with vehicle or CDCA alone, suggesting that KTZ administration inhibited bile acid excretion. Furthermore, hepatic mRNA expression levels of a bile acid synthesis enzyme, Cyp7a1, and a basolateral bile salt influx transporter, Ntcp, decreased, whereas a canalicular phosphatidylcholine flippase, Mdr2, increased in the CDCA+KTZ group to compensate for hepatic bile acid accumulation. In conclusion, we found that oral CDCA supplementation predisposed rats to KTZ-induced liver injury due to the hepatic accumulation of bile acids. This method may be useful for assessing the potential of BSEP-inhibiting drugs inducing liver injury in vivo.


Inhibition of EP2/EP4 prostanoid receptor-mediated signaling suppresses IGF-1-induced proliferation of pancreatic cancer BxPC-3 cells via upregulating γ-glutamyl cyclotransferase expression.

signaling via EP2/EP4 prostanoid receptors suppresses Insulin-like growth factor (IGF)-1-induced proliferation of pancreatic cancer BxPC-3 cells. To better understand the mechanism of EP2/EP4 signaling for controlling cell proliferation, we performed metabolome analyses in BxPC-3 cells treated with IGF-1 alone or IGF-1 plus EP2/EP4 inhibitors. These analyses revealed increased g-aminobutyric acid and 5-oxoproline production following the addition of EP2/EP4 inhibitors to IGF-1-treated cells. The expression of a 5-oxoproline-catalyzing enzyme, γ-glutamylcyclotransferase (GGCT), was also upregulated by IGF-1 treatment and further enhanced by the addition of EP2/EP4 inhibitors. Knockdown of GGCT expression resulted in the loss of suppressive effects of EP2/EP4 inhibitors on IGF-1-induced BxPC-3 cell proliferation, whereas GGCT overexpression repressed the basal proliferation of BxPC-3 cells but did not affect the suppressive effects of EP2/EP4 inhibitors. To summarize, we propose a role for EP2/EP4 signaling in regulating IGF-1-induced cell proliferation, in which EP2/EP4 signaling represses IGF-1-induced GGCT expression, which mediates and whose amount controls a branch of IGF-1 signaling to promote cell proliferation via extracellular signal-regulated kinase phosphorylation.

Biochemical and Biophysical Research Communications


Analysis of amino acid profiles of blood over time and biomarkers associated with non-alcoholic steatohepatitis in STAM mice.

The changes in free amino acid (AA) levels in blood during the progression from non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) are unclear. We investigated serum AA levels, along with biochemical and histological events, in a mouse model of NASH. We induced NASH in male C57BL/6J mice with a streptozotocin injection and high-fat diet after 4 weeks of age (STAM group). We chronologically (6, 8, 10, 12, and 16 weeks, n=4-12 mice/group) evaluated the progression from steatohepatitis to HCC by biochemical and histological analyses. The serum AA levels were determined using an AA analyzer. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in the STAM group than in the normal group (non-NASH-induced mice). Histological analysis revealed that STAM mice had fatty liver, NASH, and fibrosis at 6, 8, and 10 weeks, respectively. Moreover, the mice exhibited fibrosis and HCC at 16 weeks. The serum branched-chain AA levels were higher in the STAM group than in the normal group, especially at 8 and 10 weeks. The Fischer ratio decreased at 16 weeks in the STAM group, with increasing aromatic AA levels. These results suggested that this model sequentially depicts the development of fatty liver, NASH, cirrhosis, HCC, and AA metabolism disorders within a short experimental period. Additionally, serum amyloid A was suggested to be a useful inflammation biomarker associated with NASH. We believe that the STAM model will be useful for studying AA metabolism and/or pharmacological effects in NASH.


Macrophage-specific hypoxia-inducible factor-1α deletion suppresses the development of liver tumors in high-fat diet-fed obese and diabetic mice.

The activation of macrophage HIF-1α might play important roles in the development of liver cancer associated with diet-induced obesity and diabetes.

Development of liver cancer after HFD feeding was 45% less in KO mice than in wild-type littermates mice. Phosphorylation of extracellular signal-regulated kinase 2 was also lower in the liver of KO mice. Those effects of HIF-1α deletion in macrophages were not observed in normal chow-fed mice. Furthermore, the size of liver tumors did not differ between KO and wild-type littermates mice, even those on a HFD. These results suggest that the activation of macrophage HIF-1α by HFD is involved not in the growth, but in the development of liver cancer with the enhanced oncogenic extracellular signal-regulated kinase 2 signaling in hepatocytes.


Because the association between sphingosine 1-phosphate (S1P)/apolipoprotein M (ApoM) and chronic kidney diseases has not been established, we investigated the involvement of S1P/ApoM in the phenotypes of IgA nephropathy in hyper-IgA (HIGA) mice. The overexpression of ApoM in adenoviral gene transfer ameliorated the phenotypes of IgA nephropathy in HIGA mice, whereas the knockdown of ApoM with siRNA caused deterioration. When ApoM-overexpressing HIGA mice were treated with VPC23019, an antagonist against S1P receptor 1 (S1P1) and 3 (S1P3), we observed that the protective effects of ApoM were reversed, whereas JTE013, an antagonist against S1P2, did not inhibit the effects. We also found that S1P bound to albumin accelerated the proliferation of MES13 cells and the fibrotic changes of HK2 cells, which were inhibited by JTE013, whereas S1P bound to ApoM suppressed these changes, which were inhibited by VPC23019. These results suggest that S1P bound to ApoM possesses properties protective against the phenotypes of IgA nephropathy through S1P1 and S1P3, whereas S1P bound to albumin exerts deteriorating effects through S1P2. ApoM may be useful as a therapeutic target to treat or retard the progression of IgA nephropathy.-Kurano, M., Tsuneyama, K., Morimoto, Y., Nishikawa, M., Yatomi, Y. Apolipoprotein M suppresses the phenotypes of IgA nephropathy in hyper-IgA mice.


Development of a novel aortic dissection mouse model and evaluation of drug efficacy using in-vivo assays and database analyses.

Aortic dissection is a life-threatening disease. At present, the only therapeutic strategies available are surgery and antihypertensive drugs. Moreover, the molecular mechanisms underlying the onset of aortic dissection are still unclear. We established a novel aortic dissection model in mice using pharmacologically induced endothelial dysfunction. We then used the Japanese Adverse Drug Event Report database to investigate the role of pitavastatin in preventing the onset of aortic dissection. To induce endothelial dysfunction, Nω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, was administered to C57BL/6 mice. Three weeks later, angiotensin II (Ang II) and β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, were administered with osmotic mini-pumps. False lumen formation was used as the pathological determinant of aortic dissection. The incidences of aortic dissection and death from aneurysmal rupture were significantly higher in the Nω-nitro-L-arginine methyl ester, Ang II, and BAPN (LAB) group than they were in the Ang II and BAPN (AB) group.Pitavastatin was administered orally to LAB mice. It significantly lowered the incidences of dissection and rupture. It also decreased inflammation and medial degradation, both of which were exacerbated in the LAB group. The Japanese Adverse Drug Event Report database analysis indicated that there were 113 cases of aortic dissection out of 95 090 patients (0.12%) not receiving statins but only six cases out of 16 668 patients receiving statins (0.04%) (odds ratio: 0.30; P = 0.0043). Our results suggest that endothelial dysfunction is associated with the onset of aortic dissection and pitavastatin can help prevent this condition.


Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G.

Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.


Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

= 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.


Effect of coffee or coffee components on gut microbiome and short-chain fatty acids in a mouse model of metabolic syndrome.

We previously showed that male Tsumura Suzuki obese diabetes (TSOD) mice, a spontaneous mouse model of metabolic syndrome, manifested gut dysbiosis and subsequent disruption of the type and quantity of plasma short-chain fatty acids (SCFAs), and daily coffee intake prevented nonalcoholic steatohepatitis in this mouse model. Here, we present a preliminary study on whether coffee and its major components, caffeine and chlorogenic acid, would affect the gut dysbiosis and the disrupted plasma SCFA profile of TSOD mice, which could lead to improvement in the liver pathology of these mice. Three mice per group were used. Daily intake of coffee or its components for 16 wk prevented liver lobular inflammation without improving obesity in TSOD mice. Coffee and its components did not repair the altered levels of Gram-positive and Gram-negative bacteria and an increased abundance of Firmicutes in TSOD mice but rather caused additional changes in bacteria in six genera. However, caffeine and chlorogenic acid partially improved the disrupted plasma SCFA profile in TSOD mice, although coffee had no effects. Whether these alterations in the gut microbiome and the plasma SCFA profile might affect the liver pathology of TSOD mice may deserve further investigation.


Gut microbiota translocation promotes autoimmune cholangitis.

mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFβRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.


Neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced FGF-2 in a mouse model of asthma with mixed inflammation.

The IL-17/neutrophil axis may play an important role in airway remodelling by contributing to smooth muscle hypertrophy/hyperplasia in mixed allergic inflammation and accordingly represents an attractive therapeutic target for severe asthma.


Comparative hepatic transcriptome analyses revealed possible pathogenic mechanisms of fasiglifam (TAK-875)-induced acute liver injury in mice.

Fasiglifam (TAK-875), a G protein-coupled receptor 40 (GPR40) agonist, was a drug candidate for type 2 diabetes. However, its development was terminated in phase 3 trials due to liver safety concerns. Although TAK-875 was reported to inhibit hepatobiliary transporters and disturb bile acid disposition, pathogenic mechanisms of TAK-875-induced liver injury are not fully understood. In this study, we sought to identify the mechanisms with a hepatic genome-wide transcriptomic analysis in a murine model. We demonstrated that, among the three GPR40 agonists, TAK-875, AMG-837, and TUG-770, only TAK-875 induced acute liver injury in mice. Transcriptome profiles of TAK-875-exposed liver was compared with those of non-hepatotoxic analogues AMG-837 and TUG-770 as negative controls and those of classical hepatotoxicants concanavalin A and carbon tetrachloride as positive controls. The comparative hepatic transcriptome analyses revealed the enrichment of genes involved in inflammation, endoplasmic reticulum (ER) stress, apoptosis, and hepatic lipid accumulation, suggesting that these events play pathophysiologic roles in the development of TAK-875-induced liver injury. These results were validated by quantitative PCR with significant changes in chemokines, danger signals, ER stress mediators, proapoptotic factors, and hepatic steatosis markers only in TAK-875-exposed liver. Pretreatment of TAK-875-administered mice with an ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated the liver injury. Consistent with the in vivo study, pretreatment of HepG2 cells with 4-PBA significantly improved the decrease of cell viability induced by TAK-875. In conclusion, by a comprehensive transcriptomic analysis, we found multiple possible processes that contribute to TAK-875-induced acute liver injury in mice.


Peptides Obtained by Enzymatic Decomposition of Mackerel Prevent Nonalcoholic Steatohepatitis in Sprague-Dawley Rats fed a High-fat and High-cholesterol Diet


A case of pulmonary pleomorphic carcinoma with malignant phenotypes induced by ZEB1-associated epithelial-mesenchymal transition.

A 60-year-old man was admitted to our hospital with non-small cell lung cancer (NSCLC). Imaging and pathological studies revealed NSCLC, not otherwise specified (NOS), at clinical stage T3N1M0 stage IIIA. We started radiotherapy alone because of obstructive pneumonia and end-stage renal disease, but the tumors progressed rapidly and resulted in death due to air obstruction by pharyngeal metastasis. The cancer was diagnosed as pleomorphic carcinoma in an autopsy. Viable lung tumor cells, which were resistant to radiotherapy, and the pharyngeal metastasis had mesenchymal phenotypes and expressed ZEB1 but not SNAI1. These observations indicated that ZEB1-associated epithelial-mesenchymal transition has malignant features including resistance to radiotherapy and aggressive metastatic potential. ZEB1-associated EMT may be an important mechanism to understand the pathophysiology of pleomorphic carcinoma.


Application of 2-Picolylamine Derivatized Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry for Determination of Short-chain Fatty Acids in Feces Samples.


Detection of aberrant crypt foci with image-enhanced endoscopy.

Conventional detection of aberrant crypt foci (ACF) with dye-spraying and magnifying observation is labor- and skill-intensive. We performed a prospective non-inferiority study to investigate the utility of image-enhanced endoscopy (IEE) for detection of ACF. Patients with a history of colorectal neoplasm were eligible. The number of ACF in the lower rectum was counted first using IEE magnification with narrow-band imaging (NBI) or blue-laser imaging (BLI), and subsequently using the methylene blue method. The primary endpoint was the ACF detection rate with IEE, i. e., the number of ACF detected with IEE relative to the number of ACF detected with methylene blue. The secondary endpoints were bowel preparation time, ACF detection time, and the detection rate with NBI or BLI. A total of 40 patients were enrolled (NBI 20 and BLI 20). The overall detection rate for ACF with IEE was 81.7 % (503/616; 95 %CI 78.8 - 84.6 %), meeting the primary endpoint. The detection rate for ACF with BLI (84.9 %, 258/304) was significantly higher than with NBI (78.5 %, 245/312; < 0.05). Both bowel preparation time and ACF detection time were significantly shorter with IEE versus the methylene blue method ( < 0.01, respectively). The detection rates for dysplastic and non-dysplastic ACF with IEE were 84.4 % (27/32) and 80.3 % (469/584), respectively. IEE is able to detect ACF during colonoscopy with sensitivity non-inferior to that of the conventional methylene blue method. IEE is simpler than the methylene blue method and is therefore a potentially useful new tool for ACF detection.


Tissue resident memory CD8 T cells mediate salivary gland damage in a murine model of Sjögren's syndrome.

Although a role for CD4 T cells in the pathology of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8 T cells is unclear. The aim of this study is to investigate the role of CD8 T cells in the development of SS. Flow cytometry and immunofluorescence were utilized to detect T cell infiltration within the labial glands of patients with primary SS. In parallel p40 CD25 mice were used as a murine model of SS. In addition CD4, CD8a and IFN-γ KO mice were crossed with p40 CD25 mice respectively to study the pathogenic significance of specific lineage subpopulations including functional salivary, histopathological and serological data. CD8 T cell specific depletion antibody was used to evaluate this potential therapeutic strategy. CD8 T cells with tissue resident memory phenotype outnumbered CD4 T cells in the labial gland of SS patients and were primarily co-localized with saliva duct epithelial cells and acinar cells. Furthermore, infiltrated CD8 T cells with resident phenotype CD69 CD103 with a significant elevation of IFN-γ production were dominant in the submandibular gland of this SS murine model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFN-γ decreased CD8 T cell infiltration and gland destruction. More importantly, depletion of CD8 T cells fully protected mice from SS even after the onset of disease. Our data reveal pathogenic significance of CD8 T cells in the salivary gland pathology in SS. Treatment directed against CD8 T cells may be a rational therapy for human SS. This article is protected by copyright. All rights reserved.


Comprehensive analysis of serum microRNAs in hepatic sinusoidal obstruction syndrome (SOS) in rats: implication as early phase biomarkers for SOS.

Sinusoidal obstruction syndrome (SOS) is a liver injury caused by clinical chemotherapy, of which pathogenesis is associated with the damage in liver sinusoidal endothelial cells (LSEC). The unavailability of appropriate specific biomarkers for the early diagnosis of SOS may potentially overlook SOS patients. In this study, we sought to find serum microRNAs (miRNAs) as non-invasive biomarkers for investigating SOS in rats. Male Sprague-Dawley rats were orally administered monocrotaline, and then, their livers and sera were collected after 0.25, 0.5, 1, 2, 4, and 7 days. The rats showed a typical SOS phenotype including LSEC damage as early as day 0.25, followed by severe hepatocyte damage on day 2, and developed hepatic fibrosis from days 4 to 7. The miRNA microarray showed that 65 serum miRNAs were increased in their levels on day 0.25, when LSEC damage was observed, while hepatocyte damage was absent. Among the increased serum miRNAs on days 0.25-1, miR-511-3p was enriched in normal LSECs and miR-21-5p was in both LSECs and hepatocytes, suggesting that they were released into blood from the damaged LSECs. The miR-122-5p, miR-192-5p, and miR-101b-3p, which were enriched in hepatocytes, reached the highest levels in serum on day 2, suggesting their utility as indicators for hepatocyte damage. No miRNA showing an increasing trend from days 4 to 7 was found as a biomarker for fibrosis. In conclusion, we found that LSEC-derived miR-21-5p and especially miR-511-3p in serum would serve as early phase biomarkers for SOS in response to LSEC damage.


Apolipoprotein M Protects Lipopolysaccharide-Treated Mice from Death and Organ Injury.

These results suggest that apoM possesses protective properties against LPS-induced organ injuries and could potentially be introduced as a novel therapy for the severe conditions that are consequent to sepsis.


Establishment of a drug-induced rhabdomyolysis mouse model by co-administration of ciprofloxacin and atorvastatin.

Rhabdomyolysis is one of the serious side effects of ciprofloxacin (CPFX), a widely used antibacterial drug; and occasionally, acute kidney injury (AKI) occurs. Often, rhabdomyolysis has occurred in patients taking CPFX co-administered with statins. The purpose of this study is to establish a mouse model of drug-induced rhabdomyolysis by co-administration of CPFX and atorvastatin (ATV) and to clarify the mechanisms of its pathogenesis. C57BL/6J mice treated with L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, were orally administered with CPFX and ATV for 4 days. Plasma levels of creatinine phosphokinase (CPK) and aspartate aminotransferase (AST) were significantly increased in the CPFX and ATV-co-administered group. Histopathological examination of skeletal muscle observed degeneration in gastrocnemius muscle and an increased number of the satellite cells. Expressions of skeletal muscle-specific microRNA and mRNA in plasma and skeletal muscle, respectively, were significantly increased. The area under the curve (AUC) of plasma CPFX was significantly increased in the CPFX and ATV-co-administered group. Furthermore, cytoplasmic vacuolization and a positively myoglobin-stained region in kidney tissue and high content of myoglobin in urine were observed. These results indicated that AKI was induced by myoglobin that leaked from skeletal muscle. The established mouse model in the present study would be useful for predicting potential rhabdomyolysis risks in preclinical drug development.


Tsumura-Suzuki obese diabetic mice-derived hepatic tumors closely resemble human hepatocellular carcinomas in metabolism-related genes expression and bile acid accumulation.

Our results strongly support the significance of TSOD mice as a model of spontaneously developing HCC.


A Novel Theranostic Combination of Near-infrared Fluorescence Imaging and Laser Irradiation Targeting c-KIT for Gastrointestinal Stromal Tumors.

It is difficult to distinguish gastrointestinal stromal tumors (GISTs) from other types of submucosal tumors under conventional gastrointestinal endoscopy. We aimed to detect GISTs by molecular fluorescence imaging using a near-infrared (NIR) photosensitizer (IR700)-conjugated anti-c-KIT antibody and to treat GISTs by photoimmunotherapy with NIR irradiation as a non-invasive theranostic procedure. We also investigated the therapeutic mechanisms. Human GIST cell lines GIST-T1 and GIST-882M were incubated with IR700-conjugated anti-c-KIT antibody, IR700-12A8, and observed by confocal laser microscopy. Mice with GIST-T1 xenografts or rats with orthotopic xenografts were injected with IR700-12A8 or AF488-conjugated antibody, and observed under IVIS or autofluorescence imaging (AFI) endoscopy. GIST cells were treated with IR700-12A8 and NIR light and vivo, and cell viability, histology and apoptosis were evaluated. Strong red fluorescence of IR700-12A8 was observed on the cell membrane of GIST cells and was gradually internalized into the cytoplasm. Tumor-specific accumulation of IR700-12A8 was observed in GIST-T1 xenografts in mice. Under AFI endoscopy, a strong fluorescence signal was observed in orthotopic GIST xenografts in rats through the normal mucosa covering the tumor. The percentage of dead cells significantly increased in a light-dose-dependent manner and both acute necrotic and late apoptotic cell death was observed with annexin/PI staining. Cleaved PARP expression was significantly increased after IR700-12A8-mediated NIR irradiation, which was almost completely reversed by NaN. All xenograft tumors (7/7) immediately regressed and 4/7 tumors completely disappeared after IR700-12A8-mediated NIR irradiation. Histologic assessment and TUNEL staining revealed apoptosis in the tumors. NIR fluorescence imaging using IR700-12A8 and subsequent NIR irradiation could be a very effective theranostic technology for GIST, the underlying mechanism of which appears to involve acute necrosis and supposedly late apoptosis induced by singlet oxygen.


A Patient with Non-alcoholic Steatohepatitis Complicated by Multiple Myeloma.

A 68-year-old woman with liver dysfunction was diagnosed with nonalcoholic steatohepatitis (NASH) stage 1. Three years later, she showed massive ascites and jaundice. A trans-jugular liver biopsy confirmed advanced cirrhosis, suggesting that her liver fibrosis had progressed rapidly. At the same time, she was diagnosed with multiple myeloma (MM). In this case, the plasma levels of osteopontin (OPN), a proinflammatory cytokine that promotes liver fibrosis progression through the hedgehog pathway and is increased in patients with MM, were increased. This increased OPN expression was accompanied by the upregulation of the hedgehog pathway in this patient, suggesting that the MM-associated increase in OPN had promoted the progression of liver fibrosis through the hedgehog pathway. The progression of liver fibrosis should be monitored in patients with NASH if other diseases, such as MM, are present.


Clinical features of autoimmune hepatitis with acute presentation: a Japanese nationwide survey.

AIH with acute presentation is a newly recognized disease entity for which diagnostic hallmarks, such as ALT, fibrosis, and ANA, are needed. Further investigation is also required on the mechanisms of this disorder. Clinicians should be mindful of disease relapse during patient care.

Seventy-five patients were female and 11 were male. Patient age ranged from adolescent to over 80 years old, with a median age of 55 years. Median alanine transaminase (ALT) was 776 U/L and median immunoglobulin G (IgG) was 1671 mg/dL. There were no significant differences between genders in terms of ALT (P = 0.27) or IgG (P = 0.51). The number of patients without and with histopathological fibrosis was 29 and 57, respectively. The patients with fibrosis were significantly older than those without (P = 0.015), but no other differences in clinical or histopathological findings were observed. Moreover, antinuclear antibody (ANA)-positive (defined as × 40, N = 63) and -negative (N = 23) patients showed no significant differences in clinical or histopathological findings or disease outcomes. Twenty-five patients experienced disease relapse and two patients died during the study period. ALP ≥ 500 U/L [odds ratio (OR) 3.20; 95% confidence interval (CI) 1.12-9.10; P < 0.030] and GGT ≥ 200 U/L (OR 2.98; 95% CI 1.01-8.77; P = 0.047) were identified as independent risk factors of disease relapse.


miRNA in Rat Liver Sinusoidal Endothelial Cells and Hepatocytes and Application to Circulating Biomarkers that Discern Pathogenesis of Liver Injuries.

Sinusoidal obstruction syndrome is a serious liver injury caused by toxic injury to liver sinusoidal endothelial cells (LSECs) during clinical chemotherapy. Although circulating miRNAs, such as hepatocyte-specific miR-122-5p and miR-192-5p, have been proposed as potential noninvasive biomarkers of hepatocellular liver injury, these miRNAs may not be specific to damage to other hepatic cell types, including LSECs. We characterized miRNA expression in LSECs and hepatocytes and investigated whether cell type-specific miRNAs in plasma can discern pathogenesis of liver injuries in rats. Comprehensive miRNA expression analyses found that 66 and 12 miRNAs were highly expressed in LSECs and hepatocytes isolated from nontreated rats, respectively. An LSEC-enriched miR-511-3p was relatively liver specific according to public data. For establishing LSEC and hepatocyte injury models, rats were orally treated with monocrotaline and thioacetamide, respectively. In monocrotaline-treated rats, a sinusoidal obstruction syndrome model, LSEC damage was observed 6 hours after dosing, whereas hepatocellular damage was observed after 48 hours. Interestingly, the level of miR-511-3p in plasma was increased as early as 6 hours after monocrotaline dosing, followed by an increase of miR-122-5p after 24 hours. In the thioacetamide-induced hepatocellular injury model, the level of miR-511-3p was not altered in plasma, whereas miR-122-5p levels were increased after 6 hours. In conclusion, we identified miR-511-3p in plasma as a possible biomarker for LSEC damage.


Paradoxical development of polymyositis-like autoimmunity through augmented expression of autoimmune regulator (AIRE).

Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.


Analysis of the gut microbiome and plasma short-chain fatty acid profiles in a spontaneous mouse model of metabolic syndrome.

Male Tsumura Suzuki obese diabetes (TSOD) mice spontaneously develop obesity and obesity-related metabolic syndrome. Gut dysbiosis, an imbalance of gut microbiota, has been implicated in the pathogenesis of metabolic syndrome, but its mechanisms are unknown. Short-chain fatty acids (SCFAs) are the main fermentation products of gut microbiota and a link between the gut microbiota and the host's physiology. Here, we investigated a correlation among gut dysbiosis, SCFAs, and metabolic syndrome in TSOD mice. We detected enriched levels of Gram-positive bacteria and corresponding decreases in Gram-negative bacteria in 24-wk-old metabolic syndrome-affected TSOD mice compared with age-matched controls. The abundance of Bacteroidetes species decreased, the abundance of Firmicutes species increased, and nine genera of bacteria were altered in 24-wk-old TSOD mice. The total plasma SCFA level was significantly lower in the TSOD mice than in controls. The major plasma SCFA-acetate-decreased in TSOD mice, whereas propionate and butyrate increased. TSOD mice had no minor SCFAs (valerate and hexanoate) but normal mice did. We thus concluded that gut dysbiosis and consequent disruptions in plasma SCFA profiles occurred in metabolic syndrome-affected TSOD mice. We also propose that the TSOD mouse is a useful model to study gut dysbiosis, SCFAs, and metabolic syndrome.


Neutrophil depletion protects against zomepirac-induced acute kidney injury in mice.

Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs have been implicated in drug toxicity. Zomepirac (ZP) is a non-steroidal anti-inflammatory drug that was withdrawn from the market because of anaphylactic reactions and renal injury. We recently established a novel mouse model of ZP-induced kidney injury by increasing zomepirac acyl-glucuronide (ZP-AG) concentration via pretreatment with tri-O-tolyl phosphate, a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine, a glutathione synthesis inhibitor. Although we have shown that ZP-AG is responsible for ZP-induced kidney injury in mice, the exact pathogenic mechanisms of ZP-induced kidney injury have not been investigated yet. In this study, we aimed to investigate the role of immune cells in the pathogenesis of ZP-induced kidney injury, as a representative of AG toxicity. We found that the counts of neutrophils and inflammatory monocytes increased in the blood of mice with ZP-induced kidney injury. However, clodronate liposome- or GdCl3-induced monocyte and/or macrophage depletion did not affect blood urea nitrogen and plasma creatinine levels in mice with ZP-induced kidney injury. Neutrophil infiltration into the kidneys was observed in mice with ZP-induced kidney injury, whereas anti-lymphocyte antigen 6 complex, locus G (Ly6G) antibody pretreatment prevented the renal neutrophil infiltration and partially protected against ZP-induced kidney injury. The mRNA expression of neutrophil-infiltrating cytokines and chemokines, interleukin-1 and macrophage inflammatory protein-2, increased in mice with ZP-induced kidney injury, whereas pretreatment with anti-Ly6G antibody resulted in a marked reduction of their expression. These results suggest that ZP-AG might be involved in kidney injury, partly via induction of neutrophil infiltration. Therefore, this study may provide an important understanding on toxicological role of ZP-AG in vivo that helps to understand toxicity of AG metabolites.


Mode of tolerance induction and requirement for Aire are governed by the cell types that express self-antigen and those that present antigen

Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.


Clinicopathological Study of Autoimmune Hepatitis Cases That Were Difficult to Differentiate from Drug-Induced Liver Injury.

This study showed that DDW-J scale was useful for differentiating AIH from DILI in cases with a DDW-J scale score of 5. The histologic features of AIH were characterized by cobblestone hepatocellular change, interface hepatitis, and plasma cell infiltration of the portal region.


Animal models for analyzing metabolic syndrome-associated liver diseases.

Metabolic syndrome (MS) is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases affecting the entire body. The hepatic manifestations of MS include nonalcoholic fatty liver disease (NAFLD) and its progressive variant, nonalcoholic steatohepatitis (NASH). NASH is known to progress to liver cirrhosis and hepatocellular carcinoma (HCC). Excellent animal models for determining the mechanism of pathogenesis and establishing therapeutic treatment of NASH/HCC are strongly required worldwide. We recently reported that two previously established mouse models of obesity and diabetes mellitus, namely, Tsumura-Suzuki Obese Diabetes (TSOD) mice and MSG mice, developed MS-associated NASH and that their clinical course and pathological characteristics closely mimicked those of human MS-NASH patients. Interestingly, most of the mice developed HCC with advancing age, and the pathological and functional characteristics of this condition were identical to those of human HCC. We further established a novel mouse model of HCC based on type 1 diabetes (DIAR-nSTZ mice) and reported its histopathological features. By comparing various aspects of these mouse models, specific and useful characteristics in a suitable model of MS-associated liver diseases, including hepato-carcinogenesis, can be highlighted.


The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity.

We have reported a murine model of autoimmune cholangitis, generated by altering the AU rich element by deletion of the IFN- 3'UTR region (coined ARE-Del(-/-) ), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the gender bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del(-/-) mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del(-/-) Ifnar1(-/-) mice dramatically reduces liver pathology and abrogated gender bias. More importantly, female ARE-Del(-/-) mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del(-/-) Ifnar1(-/-) mice corrects these GC abnormalities, including abnormal follicular structure. In conclusion, our data implicate type I IFN signaling as a necessary component of the gender bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. This article is protected by copyright. All rights reserved.


Facilitatory effect of insulin treatment on hepatocellular carcinoma development in diabetes.

Insulin treatment promoted, rather than inhibited, the progression of liver carcinogenesis in DIAR-nSTZ mice. Hyperinsulinemia rather than hyperglycemia can accelerate the progression of HCC via insulin signaling.

DIAR-nSTZ mice had significantly lower body weight and higher blood glucose levels than DIAR-control mice, whereas no significant differences were observed between DIAR-nSTZ/INS mice and control mice. At 12 weeks of age, lower weight of paratesticular fat and higher levels of total cholesterol, triglyceride, and free fatty acids were observed in DIAR-nSTZ mice compared to DIAR-control mice, whereas there were no significant differences between DIAR-nSTZ/INS mice and DIAR-control mice. In the livers of DIAR-nSTZ mice, HCC was observed in 15% of cases, and dysplastic nodules were observed in 77% of cases. In the livers of DIAR-nSTZ/INS mice, HCC was observed in 39% of cases and dysplastic nodules were observed in 61% of cases (p = 0.011). Moreover, the average tumor size was significantly larger in STZ/INS-treated mice than in STZ-treated mice. Immunohistochemical analysis demonstrated that the expression of ERK1/2, downstream substrates of insulin signaling that activate cell proliferation, was significantly higher in STZ/INS-treated mice compared to STZ-treated mice.


CD206(+) M2-like macrophages regulate systemic glucose metabolism by inhibiting proliferation of adipocyte progenitors.

Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGF signaling pathway. We show that adipose tissue CD206(+) cells are primarily M2-like macrophages, and ablation of CD206(+) M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206(+) M2-like macrophages show a down-regulation of TGF signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206(+) M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity.Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-1.


A fermented mixed tea made with camellia (Camellia japonica) and third-crop green tea leaves prevents nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet.

Established treatments for non-alcoholic steatohepatitis (NASH) are few, thus it is imperative to develop novel dietary strategies that can prevent NASH. A fermented mixed tea (FMT) made with Camellia japonica (Japanese camellia) and third- crop green tea leaves by tea-rolling processing was reported to reduce body weight and adipose tissue weight in Sprague-Dawley (SD) rats. Because visceral fat is one of the most important factors for the development of hepatic steatosis, this FMT supplementation can be a candidate dietary strategy for the prevention of NASH. Nine-week-old male SD rats were fed a high-fat and high-cholesterol (HFC) diets with or without FMT (camellia and third-crop green tea leaves at ratios of 1:5, 1:2 and 1:1) for 9 weeks (n=6-7/group). Histopathology, serology and expressions of fibrogenetic, proinflammatory, oxidative stress and lipid metabolism-related genes in the liver were evaluated. Histologically, HFC diet with FMT at a ratio of 1:5 dramatically reduced NASH progression (14%) compared to the HFC diet without FMT (100%). FMT at a ratio of 1:5 reduced hepatic steatosis due to the activation of microsomal triglyceride transfer protein, and FMT at a ratio of 1:2 reduced mRNA levels of some proinflammatory, lipid metabolism-related, fibrogenic and oxidative stress marker genes. Our data suggest that FMT at a ratio of 1:5 or 1:2 likely possesses a preventive effect on NASH progression.


Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis.

Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.


Ovarian Large Cell Neuroendocrine Carcinoma Associated with Serous Carcinoma: Correlation of Pathology with MR Imaging


Peritumoral Hyperplasia in Hepatic Sclerosed Hemangioma.

Peritumoral hyperplasia (PTH) is a hyperplastic lesion located around hypervascular tumors. Hepatic sclerosed hemangioma is a very rare form of hemangioma with sclerotic changes and is distinct from sclerosing hemangioma. We present a patient with non-alcoholic steatohepatitis-induced cirrhosis who presented with a hypervascular tumor. The tumor showed atypical findings of hemangioma and was treated with surgical resection because hepatic malignancy could not be ruled out. Histopathologic examination revealed the tumor was a sclerosed hemangioma with PTH. Lesions with carcinogenic potential were found in the PTH lesion. Sclerosed hemangioma should be observed and managed carefully.


Acute presentation of autoimmune hepatitis: a multicentre study with detailed histological evaluation in a large cohort of patients.

The acute presentation of AIH represents the entire histological spectrum of acute hepatitis and chronic hepatitis with various activity grades and fibrosis stages that clinically correspond to acute-onset AIH and acute exacerbation of classic AIH, respectively. Although there are no pathognomonic features for the pathological diagnosis, the prominent presence of lobular and perivenular necroinflammatory activity, pigmented macrophages and cobblestone appearance of hepatocytes in addition to the classic AIH features, such as plasma cell infiltration and emperipolesis, are useful for the pathological diagnosis of the acute presentation of AIH.


Prolonged activation of IL-5-producing ILC2 causes pulmonary arterial hypertrophy.

IL-33 is one of the critical cytokines that activates group 2 innate lymphoid cells (ILC2s) and mediates allergic reactions. Accumulating evidence suggests that IL-33 is also involved in the pathogenesis of several chronic inflammatory diseases. Previously, we generated an IL-5 reporter mouse and revealed that lung IL-5-producing ILC2s played essential roles in regulating eosinophil biology. In this study, we evaluated the consequences of IL-33 administration over a long period, and we observed significant expansion of ILC2s and eosinophils surrounding pulmonary arteries. Unexpectedly, pulmonary arteries showed severe occlusive hypertrophy that was ameliorated in IL-5- or eosinophil-deficient mice, but not in Rag2-deficient mice. This indicates that IL-5-producing ILC2s and eosinophils play pivotal roles in pulmonary arterial hypertrophy. Administration of a clinically used vasodilator was effective in reducing IL-33-induced hypertrophy and repressed the expansion of ILC2s and eosinophils. Taken together, these observations demonstrate a previously unrecognized mechanism in the development of pulmonary arterial hypertrophy and the causative roles of ILC2 in the process.


Daily Coffee Intake Inhibits Pancreatic Beta Cell Damage and Nonalcoholic Steatohepatitis in a Mouse Model of Spontaneous Metabolic Syndrome, Tsumura-Suzuki Obese Diabetic Mice.

A daily habit of drinking coffee could possibly play a role in the prevention of metabolic syndrome.

Metabolic Syndrome and Related Disorders


Human -Defensin 2 in Primary Sclerosing Cholangitis.

Our data show that HBD2 serum levels and tissue expression are increased in PSC subjects, suggesting that this arm of innate immunity may be important in the etiopathogenesis of PSC.

Mean serum levels of HBD2 were significantly greater in PSC (1,086±1,721 ng/μl) compared with primary biliary cholangitis (544±754 ng/μl), ulcerative colitis (417±506 ng/μl), and healthy controls (514±731 ng/μl) (P=0.02). However, no significant differences between the frequencies of high DEFB4 gene copy number, defined by >4 copies, and PSC were found in the US, Italian, or combined cohorts. Importantly, a high number of biliary ducts were found immunopositive in PSC samples compared with controls.

Clinical and Translational Gastroenterology


Establishment of a mouse model of enalapril-induced liver injury and investigation of the pathogenesis.

Drug-induced liver injury (DILI) is a major concern in drug development and clinical drug therapy. Since the underlying mechanisms of DILI have not been fully understood in most cases, elucidation of the hepatotoxic mechanisms of drugs is expected. Although enalapril (ELP), an angiotensin-converting enzyme inhibitor, has been reported to cause liver injuries with a low incidence in humans, the precise mechanisms by which ELP causes liver injury remains unknown. In this study, we established a mouse model of ELP-induced liver injury and analyzed the mechanisms of its hepatotoxicity. Mice that were administered ELP alone did not develop liver injury, and mice that were pretreated with a synthetic glucocorticoid dexamethasone (DEX) and a glutathione synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) exhibited liver steatosis without significant increase in plasma alanine aminotransferase (ALT). In mice pretreated with DEX and BSO, ALT levels were significantly increased after ELP administration, suggesting that hepatic steatosis sensitized the liver to ELP hepatotoxicity. An immunohistochemical analysis showed that the numbers of myeloperoxidase-positive cells that infiltrated the liver were significantly increased in the mice administered DEX/BSO/ELP. The levels of oxidative stress-related factors, including hepatic heme oxygenase-1, serum hydrogen peroxide and hepatic malondialdehyde, were elevated in the mice administered DEX/BSO/ELP. The involvement of oxidative stress in ELP-induced liver injury was further supported by the observation that tempol, an antioxidant agent, ameliorated ELP-induced liver injury. In conclusion, we successfully established a model of ELP-induced liver injury in DEX-treated steatotic mice and demonstrated that oxidative stress and neutrophil infiltration are involved in the pathogenesis of ELP-induced liver injury.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8(+) T cell activation.

CXC Chemokine Receptor 3 (CXCR3) is functionally pleiotropic and not only plays an important role in chemotaxis, but also participates in T cell differentiation and may play a critical role in inducing and maintaining immune tolerance. These observations are particularly critical for autoimmune cholangitis in which CXCR3 positive T cells are found around the portal areas of both humans and mouse models of primary biliary cholangitis (PBC). Herein, we investigated the role of CXCR3 in the pathogenesis of autoimmune cholangitis. We have taken advantage of a unique CXCR3 knockout dnTGFRII mouse to focus on the role of CXCR3, both by direct observation of its influence on the natural course of disease, as well as through adoptive transfer studies into Rag-/- mice. We report herein that not only do CXCR3 deficient mice develop an exacerbation of autoimmune cholangitis associated with an expanded effector memory T cell number, but also selective adoptive transfer of CXCR3 deficient CD8(+) T cells induces autoimmune cholangitis. In addition, gene microarray analysis of CXCR3 deficient CD8(+) T cells reveal an intense pro-inflammatory profile. Our data suggests that the altered gene profiles induced by CXCR3 deficiency promotes autoimmune cholangitis through pathogenic CD8(+) T cells. These data have significance for human PBC and other autoimmune liver diseases in which therapeutic intervention might be directed to chemokines and/or their receptors.


A diet-induced Sprague-Dawley rat model of nonalcoholic steatohepatitis-related cirrhosis.

Certain modified diets containing saturated fatty acids, cholesterol or fructose lead to the development of nonalcoholic steatohepatitis (NASH)-related fibrosis in rodents; however, progression to cirrhosis is rare. Experimental liver cirrhosis models have relied on genetic manipulation or administration of hepatotoxins. This study aimed to establish a reliable dietary model of NASH-related cirrhosis in a relatively short period. Male Sprague-Dawley rats (9 weeks of age) were randomly assigned to normal, high-fat (HF), or two types (1.25% or 2.5% cholesterol) of high-fat and high-cholesterol (HFC) diets for 18 weeks. All HFC diets contained 2% cholic acid by weight. Histopathological analysis revealed that the HFC diets induced obvious hepatic steatosis, inflammation with hepatocyte ballooning and advanced fibrosis (stage 3-4) in all 12 rats at 27 weeks of age. In contrast, all five rats given the HF diet developed mild steatosis and inflammation without fibrosis. The amount of cholesterol in the liver and hepatocellular mitochondrial and microsomal fractions was significantly higher in rats fed the HFC diets than the normal or HF diets. The HFC diets significantly suppressed mRNA levels of microsomal triglyceride transfer protein, adenosine triphosphate binding cassette transporter G5, bile acid CoA: amino acid N-acyltransferase and bile salt export pump, as well as the enzymatic activity of carnitine palmitoyltransferase in the liver. In conclusion, the HFC diets induced liver cirrhosis in conjunction with hepatic features of NASH in Sprague-Dawley rats within 18 weeks, and altered gene expression and enzyme activity to accumulate lipid and bile acid in the liver.


Primary Biliary Cholangitis: Its Pathological Characteristics and Immunopathological Mechanisms.

Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is an organ-specific autoimmune disease that predominantly affects middle-aged women and is characterized by the chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and, ultimately, fibrosis. The serological hallmark of PBC is the presence of anti-mitochondrial autoantibodies (AMA). Several mechanisms have been proposed for immune-mediated bile duct damage in PBC, including the roles of T cells, B cells, other cell phenotypes, and AMA. A sign of fragility of biliary epithelial cells caused by apoptosis, senescence, and autophagy has also been noted. Several complex steps and mechanisms appear to be involved in the induction and progression of cholangitis and biliary degeneration in patients with PBC. J. Med. Invest. 64: 7-13, February, 2017.

The Journal of Medical Investigation : JMI


Preparation and characterization of newly developed matrix using functional γ-Fe2O3 nanoparticles for mass spectrometry in small molecules.


A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression.

Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P .021) and intravesical recurrence-free survival (P .013) were detected between ATBF1+ (n 10) and ATBF1- (n ) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P .008). Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.


Aberrant iron metabolism might have an impact on progression of diseases in Tsumura Suzuki obese diabetes mice, a model of spontaneous metabolic syndrome.

Tsumura Suzuki obese diabetes (TSOD) mice spontaneously develop obesity and type 2 diabetes with aberrant accumulation of excessive iron in the spleen. Aberrantly accumulated iron may cause oxidative stress and result in various symptoms of metabolic syndrome in the mice. We investigated iron metabolism and oxidative stress in TSOD mice. Male TSOD and control mice were killed at 2, 3, 6, and 8 months of age, and blood and tissue samples were collected. The serum levels of ferritin and oxidized low-density lipoprotein (OxLDL) were measured. Total glutathione concentrations of liver and spleen were also measured. Serum ferritin and OxLDL were higher in TSOD mice than in control mice at 2 and 6 months. In addition, the glutathione concentrations in TSOD mice were lower in the liver and higher in the spleen at 3 and 6 months than those in control mice. These results suggest that abnormal iron metabolism and imbalanced oxidative stress occurs in young and old TSOD mice. We propose herein that TSOD mice might be a unique and valuable model for investigating the role of iron metabolism in pathogenesis of metabolic syndrome.


Toxicological role of an acyl glucuronide metabolite in diclofenac-induced acute liver injury in mice.

The acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs are potentially chemically reactive and are suggested to be implicated in toxicity, including hepatotoxicity, nephrotoxicity and drug hypersensitivity reactions. However, it remains unknown whether AG formation is related to toxicity in vivo. In this study, we sought to determine whether AG is involved in the pathogenesis of liver injury using a mouse model of diclofenac (DIC)-induced liver injury. Mice that were administered DIC alone exhibited significantly increased plasma alanine aminotransferase levels, whereas mice that were pretreated with the UDP-glucuronosyltransferase inhibitor (-)-borneol (BOR) exhibited suppressed alanine aminotransferase levels at 3 and 6 h after DIC administration although not significant at 12 h. The plasma DIC-AG concentrations were significantly lower in BOR- and DIC-treated mice than in mice treated with DIC alone. The mRNA expression levels of chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2 and the neutrophil marker CD11b were reduced in the livers of mice that had been pretreated with BOR compared to those that had been administered DIC alone, whereas mRNA expression of the macrophage marker F4/80 was not altered. An immunohistochemical analysis at 12 h samples revealed that the numbers of myeloperoxidase- and lymphocyte antigen 6 complex-positive cells that infiltrated the liver were significantly reduced in BOR- and DIC-treated mice compared to mice that were treated with DIC alone. These results indicate that DIC-AG is partly involved in the pathogenesis of DIC-induced acute liver injury in mice by activating innate immunity and neutrophils. Copyright © 2016 John Wiley & Sons, Ltd.


HIF-1 in Myeloid Cells Promotes Adipose Tissue Remodeling Toward Insulin Resistance.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1 (HIF-1) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1 is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1 deletion that were fed an HFD. Myeloid cell-specific HIF-1 gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1 is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.


Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse).

We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of -catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.


Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-RII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-RII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.


Chronic Expression of Interferon Gamma Leads to Murine Autoimmune Cholangitis with a Female Predominance.

In most autoimmune diseases the serologic hallmarks of disease precede clinical pathology by years. Therefore the use of animal models in defining early disease events becomes critical. Herein we have taken advantage of a "designer" mouse with dysregulation of interferon gamma (IFN) characterized by prolonged and chronic expression of IFN through deletion of the IFN 3' UTR AU-rich element. These mice develop primary biliary cholangitis (PBC) with a female predominance that mimics human disease and is characterized by upregulation of total bile acids, spontaneous production of AMA, and portal duct inflammation. Transfer of CD4 T cells from ARE-Del-/- to B6/Rag1(-/-) mice induced moderate portal inflammation, and parenchymal inflammation, RNA-sequencing of liver gene expression revealed that upregulated genes potentially define early stages of cholangitis. Interestingly, upregulated genes specifically overlap with the gene expression signature of biliary epithelial cells in PBC, implying that IFN may play a pathogenic role in biliary epithelial cells (BEC) in the initiation stage of PBC. Moreover, differentially expressed genes in female mice have stronger Type I and II interferon signaling and lymphocyte-mediated immune responses and thus may drive the female bias of the disease. In conclusion, changes in IFN expression are critical for the pathogenesis of PBC. This article is protected by copyright. All rights reserved.


Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice.

Glucuronidation, an important phase II metabolic route, is generally considered to be a detoxification pathway. However, acyl glucuronides (AGs) have been implicated in the toxicity of carboxylic acid drugs due to their electrophilic reactivity. Zomepirac (ZP) was withdrawn from the market because of adverse effects such as renal toxicity. Although ZP is mainly metabolized to acyl glucuronide (ZP-AG) by UDP-glucuronosyltransferase, the role of ZP-AG in renal toxicity is unknown. In this study, we established a ZP-induced kidney injury mouse model by pretreatment with tri-o-tolyl phosphate (TOTP), a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor. The role of ZP-AG in renal toxicity was investigated using this model. The model showed significant increases in blood urea nitrogen (BUN) and creatinine (CRE), but not alanine aminotransferase. The ZP-AG concentrations were elevated by cotreatment with TOTP in the plasma and liver and especially in the kidney. The ZP-AG concentrations in the kidney correlated with values for BUN and CRE. Upon histopathological examination, vacuoles and infiltration of mononuclear cells were observed in the model mouse. In addition to immune-related responses, oxidative stress markers, such as the glutathione/disulfide glutathione ratio and malondialdehyde levels, were different in the mouse model. The suppression of ZP-induced kidney injury by tempol, an antioxidant agent, suggested the involvement of oxidative stress in ZP-induced kidney injury. This is the first study to demonstrate that AG accumulation in the kidney by TOTP and BSO treatment could explain renal toxicity and to show the in vivo toxicological potential of AGs.


Deletion of SIRT1 in myeloid cells impairs glucose metabolism with enhancing inflammatory response to adipose tissue hypoxia


Complete Resolution of Pseudomalignant Erosion in a Reflux Gastroesophageal Polyp with Proton Pump Inhibitor.

Pseudomalignant erosion is a diagnostic pitfall for pathologists in the differential diagnosis of malignant neoplasms. Here, we present a challenging case of a biopsy specimen from the eroded head of a polyp at the esophagogastric junction. A malignant neoplasm could not be ruled out due to the presence of bizarre stromal cells. A second biopsy performed after the administration of a proton pump inhibitor (PPI) for 4 weeks revealed endoscopic resolution of the polyp along with the complete histological resolution of the bizarre stromal cells and led to the diagnosis of pseudomalignant erosion in a reflux gastroesophageal polyp. In conclusion, histological and endoscopic response to PPI therapy is an important clue for the correct diagnosis of reflux gastroesophageal polyps with pseudomalignant erosion.


Immunohistochemical Characterization of the Ectopic Epithelium Devoid of Goblet Cells From a Posttraumatic Iris Cyst Causing Mucogenic Glaucoma.

The ectopic epithelium of an iris cyst causing mucogenic glaucoma was most likely to originate from limbal basal cells, which showed dual direction of differentiation toward both the conjunctival and corneal epithelia. The membrane-bound mucin may have caused mucogenic glaucoma in the absence of goblet cells.


Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy.

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4(-/-)Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN- produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4(-/-)Tg mice with established disease at 8-9 weeks of age with C57BL/6 control mice. Such parabiotic "twins" had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4(-/-)Tg and CD8(-/-) mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8(+) T effector cells in the presence of wild type CD4 cells was noted. In conclusion, "correcting" the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.


Accumulated myeloid-derived suppressor cells demonstrate distinct phenotypes and functions in two non-alcoholic steatohepatitis mouse models.

These results suggest that hepatic fatty changes promote MDSC accumulation, and inflammatory changes induce phenotypic and functional alteration in hepatic MDSCs in NASH mouse models.


Pathogenetic analyses of carbamazepine-induced liver injury in F344 rats focused on immune- and inflammation-related factors.

Drug-induced liver injury is one of the major reasons for a drug to be withdrawn postmarketing. Carbamazepine (CBZ), an anticonvulsant agent, has been reported rarely to cause liver failure in humans. We recently generated a rat model of CBZ-induced liver injury using F344 rats for five consecutive days of CBZ administration combined with a glutathione (GSH) depletor, l-buthionine S,R-sulfoximine, treatment. The involvement of metabolic activation was demonstrated in developing CBZ-induced liver injury, and a difference in metabolic activation reactions between mice and rats was indicated. In this study, we analyzed the pathogenetic mechanism of CBZ-induced liver injury, primarily focusing on immune- and inflammation-related factors using the rat model for CBZ-induced liver injury. After the last CBZ administration, plasma alanine aminotransfearase (ALT) levels were drastically increased. In the histopathological evaluation, time-dependent hepatocellular degeneration and necrosis were observed in the centrilobular region. Different from mice, although hepatic mRNA expression levels of inflammation-related genes were increased, T-helper cell-related genes were not predominantly changed in rats. The number of ED1- and ED2-positive macrophages was increased in injured centrilobular areas in the liver with CBZ-induced liver injury. Treatment with a Kupffer cell depletor, gadolinium chloride, prevented the elevation of plasma ALT levels and an increase in the hepatic mRNA expression levels of inflammation-related genes. Hepatic adenosine triphosphate (ATP) contents were significantly decreased 24h after CBZ administration. Therefore, the Kupffer cells-mediated inflammation was predominant in the development of the CBZ-induced liver injury in rats.


Kupffer cell-mediated exacerbation of methimazole-induced acute liver injury in rats.

Methimazole (MTZ), an anti-thyroid drug, is known to cause liver injury in humans. It has been demonstrated that MTZ-induced liver injury in Balb/c mice is accompanied by T helper (Th) 2 cytokine-mediated immune responses; however, there is little evidence for immune responses associated with MTZ-induced liver injury in rats. To investigate species differences in MTZ-induced liver injury, we administered MTZ with a glutathione biosynthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO), to F344 rats and subsequently observed an increase in plasma alanine aminotransferase (ALT) and high-mobility group box 1 (HMGB1), which are associated with hepatic lesions. The hepatic mRNA expression of innate immune-related genes significantly increased in BSO- and MTZ-treated rats, but the change in Th2-related genes was not much greater than the change observed in the previous mouse study. Moreover, an increase in Kupffer cells and an induction of the phosphorylation of extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) proteins were accompanied by an increase in Toll-like receptor 4 (TLR4) expression, indicating that Kupffer cell activation occurs through HMGB1-TLR4 signaling. To elucidate the mechanism of liver injury in rats, gadolinium chloride, which inactivates the function of Kupffer cells, was administered before BSO and MTZ administration. The gadolinium chloride treatment significantly suppressed the increased ALT, which was accompanied by decreased hepatic mRNA expression related to innate immune responses and ERK/JNK phosphorylation. In conclusion, Kupffer cell-mediated immune responses are crucial factors for the exacerbation of MTZ-induced liver injury in rats, indicating apparent species differences in the immune-mediated exacerbation of liver injury between mice and rats. Copyright © 2015 John Wiley & Sons, Ltd.


Insight into the molecular mechanism of heme oxygenase-1 induction by docosahexaenoic acid in U937 cells.

Heme oxygenase-1 (HO-1) has anti-inflammatory effects on myeloid cells in response to various stimuli. To date, little is known about whether fatty acids can affect HO-1 induction. Here, we report the induction of HO-1 by docosahexaenoic acid (DHA) and the associated molecular mechanisms in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with DHA, eicosapentaenoic acid, palmitic acid or oleic acid, DHA was the most effective inducer of HO-1. The activation of AKT and glycogen synthase kinase-3 did not significantly change after DHA treatment. However, DHA increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not of other mitogen-activated protein kinases such as p38 and JNK. The increase in HO-1 expression was significantly inhibited by U0126, an ERK1/2 inhibitor. Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its binding to the HO-1 promoter significantly increased upon DHA treatment. An increase in intracellular reactive oxygen species was detected by dichlorofluorescein diacetate, but not by hydroethidium or 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid after DHA treatment. Pretreatment with NAC dramatically inhibited the ERK1/2 activation, binding of Nrf-2 to antioxidant response elements (AREs) located in the HO-1 promoter and the induction of HO-1 by DHA. In conclusion, DHA increased HO-1 expression in U937 cells via activation of ERK1/2 and increased Nrf-2 binding to ARE in the HO-1 promoter. These findings will help develop better strategies for treating inflammatory disorders with DHA.


Wild-type ATTR amyloidosis of the ureter in a 56-year-old woman with rheumatoid arthritis and Sjögren's syndrome.

We present a case of acute pyelonephritis with right hydronephrosis in a middle-aged woman, who had suffered from rheumatoid arthritis and Sjögren's syndrome. She had successfully treated with antibiotics, however, ureteral stenosis sustained. She underwent ureteroscopy and stenting of right ureter. Biopsy specimen revealed submucosal amyloid deposition in the interstitium overlying a benign urothelium. Amyloid protein was positive for transthyretin (TTR) by immunohistochemistry and amyloid deposition was not demonstrated in other organs. The patient's TTR genes were wild type and she was diagnosed with wild-type ATTR (ATTR wt) amyloidosis. This is the first report about symptomatic ATTR wt amyloidosis, which was also called 'systemic senile amyloidosis (SSA)' in the ureter. We should aware that SSA can occur at younger age and cause symptomatic ureteral stenosis. Further investigation is needed to clarify the association of autoimmune diseases to develop ATTR wt amyloidosis.

International Journal of Clinical and Experimental Pathology


Gastrointestinal Immune Response to the Shrimp Allergen Tropomyosin: Histological and Immunological Analysis in an Animal Model of Shrimp Tropomyosin Hypersensitivity.

Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN- in intestinal tissue was measured via RT-PCR. In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.

International Archives of Allergy and Immunology


Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co-stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)- receptor II dominant negative (dnTGF-RII) transgene to address the potential therapeutic efficacy of CTLA-4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA-4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA-4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF-RII mice, CTLA-4 Ig treatment has short-term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA-4 Ig will be critical to determining whether such efforts can be applied to human PBC.