Mammary tissue microenvironment determines T cell-dependent breast cancer-associated inflammation.

Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis-prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis-prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor-b (NF-B) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF-B/Luc2 reporter gene (4T1 NF-B cells), we observed significantly increased bioluminescence approximately 7 days after metastasis-prone orthotopic mammary fat-pad inoculation but not ectopic s.c. inoculation of 4T1 NF-B cells. Such in vivo NF-B activation within the fat-pad 4T1 tumor was diminished in immune-deficient SCID or nude mice, or T cell-depleted mice, suggesting the requirement of host T cell-mediated immune responses. Given the fat-pad 4T1 tumor expressed higher inflammatory mediators in a T cell-dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.


Bone metastasis in gastrointestinal stromal tumors preferentially occurs in patients with original tumors in sites other than the stomach.

Bone metastases are rare in gastrointestinal stromal tumors (GISTs) and data on the clinicopathological profiles are lacking. The purpose of this report was to identify the clinicopathological profiles of this rare clinical setting by evaluating 23 cases, four of which were our own and the additional 19 were from the relevant English literature. In 18 cases, the primary GISTs occurred in sites other than the stomach, although a high proportion of these tumors do arise in the stomach. All tumors at the disease presentation had more than a low risk of recurrence, with most tumors either at a high risk or initially malignant with liver metastasis. In four cases, bone metastasis was the primary metastatic manifestation. Although rare in GISTs, bone metastasis should be considered in patients with primary tumors at a high risk for recurrence or in initially malignant tumors with liver metastasis, especially with primary tumors in sites other than the stomach.

International Journal of Clinical and Experimental Pathology


Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

Several chemokines/chemokine receptors such as CXCL12, CCL3, CXCR4 and CCR1 attract multiple myelomas to specific microenvironments. In the present study, we investigated whether the CX3CL1/CX3CR1 axis is involved in the interaction of the multiple myeloma cells with their microenvironment. The expression of CX3CR1 (also known as fractalkine) was detected in three of the seven human myeloma cell lines. CX3CL1-induced phosphorylation of Akt and ERK1/2 was detected in the CX3CR1-positive cell lines, but not in the CX3CR1-negative cell lines. In addition, CX3CL1-induced cell adhesion to fibronectin and vascular cell adhesion molecule-1 (VCAM-1) in the human myeloma RPMI-8226 cell line. We also investigated whether a relationship existed between myeloma cells and osteoclasts that may function via the CX3CL1/CX3CR1 axis. Conditioned medium from CX3CL1-stimulated RPMI-8226 cells drastically increased the osteoclast differentiation. Collectively, the results from the present study support the concept of the CX3CL1-mediated activation of the progression of the multiple myeloma via CX3CR1. Thus, CX3CR1 may represent a potential therapeutic target for the treatment of multiple myeloma in a bone microenvironment.


Establishment of a mouse model for amiodarone-induced liver injury and analyses of its hepatotoxic mechanism.

Drug-induced liver injury (DILI) is the most frequent cause of post-marketing warnings and withdrawals. Amiodarone (AMD), an antiarrhythmic, presents a risk of liver injury in humans, and its metabolites, formed by cytochrome P450 3A4, are likely more toxic to hepatocytes than AMD is. However, it remains to be clarified whether the metabolic activation of AMD is involved in liver injury in vivo. In this study, to elucidate the underlying mechanisms of AMD-induced liver injury, mice were administered AMD [1000 mg kg(-1) , per os (p.o.)] after pretreatment with dexamethasone [DEX, 60 mg kg(-1) , intraperitoneal (i.p.)], which induces P450 expression, once daily for 3 days. The plasma alanine aminotransferase (ALT) levels were significantly increased by AMD administration in the DEX-pretreated mice, and the liver concentrations of desethylamiodarone (DEA), a major metabolite of AMD, were correlated with the changes in the plasma ALT levels. Cytochrome c release into the hepatic cytosol and triglyceride levels in the plasma were increased in DEX plus AMD-administered mice. Furthermore, the ratio of reduced glutathione to oxidized glutathione disulfide in the liver significantly decreased in the DEX plus AMD-administered mice. The increase of ALT levels was suppressed by treatment with gadolinium chloride (GdCl3 ), which is an inhibitor of Kupffer cell function. From these results, it is suggested that AMD and/or DEA contribute to the pathogenesis of AMD-induced liver injury by producing mitochondrial and oxidative stress and Kupffer cell activation. This study proposes the mechanisms of AMD-induced liver injury using an in vivo mouse model. Copyright © 2015 John Wiley & Sons, Ltd.


Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats.

Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.


Primary non-gestational pure choriocarcinoma arising in the ovary: A case report and literature review.

Pure non-gestational choriocarcinoma is a primary germ cell neoplasm that has been defined as a tumor without other germ cell elements. The current study presents an extremely rare case of non-gestational pure choriocarcinoma in a postmenarcheal young female and describes details of the tumor, including the clinicopathological findings. The patient was a 10-year-old female who underwent salpingo-oophorectomy. Histologically, the extensive hemorrhagic tumor was composed of choriocarcinoma without additional germ cell tumor components. The tumor cells were immunohistochemically positive for epithelial markers, including cytokeratins and epithelial membrane antigens, and there was a positive cytoplasmic reaction for -human chorionic gonadotropin in the syncytiotrophoblasts. Furthermore, numerous tumor cells were immunohistochemically positive for the 2-microglobulin antibody. The patient received adjuvant cisplatin, etoposide and bleomycin chemotherapy, and is currently disease-free without evidence of recurrence or metastasis subsequent to 62 months of follow-up.


Effect of keishibukuryogan on genetic and dietary obesity models.

Obesity has been recognized as one of the most important risk factors for a variety of chronic diseases, such as diabetes, hypertension/cardiovascular diseases, steatosis/hepatitis, and cancer. Keishibukuryogan (KBG, Gui Zhi Fu Ling Wan in Chinese) is a traditional Chinese/Japanese (Kampo) medicine that has been known to improve blood circulation and is also known for its anti-inflammatory or scavenging effect. In this study, we evaluated the effect of KBG in two distinct rodent models of obesity driven by either a genetic (SHR/NDmcr-cp rat model) or dietary (high-fat diet-induced mouse obesity model) mechanism. Although there was no significant effect on the body composition in either the SHR rat or the DIO mouse models, KBG treatment significantly decreased the serum level of leptin and liver TG level in the DIO mouse, but not in the SHR rat model. Furthermore, a lower fat deposition in liver and a smaller size of adipocytes in white adipose tissue were observed in the DIO mice treated with KBG. Importantly, we further found downregulation of genes involved in lipid metabolism in the KBG-treated liver, along with decreased liver TG and cholesterol level. Our present data experimentally support in fact that KBG can be an attractive Kampo medicine to improve obese status through a regulation of systemic leptin level and/or lipid metabolism.

Evidence-Based Complementary and Alternative Medicine : eCAM


Emerging roles of IL-33 in inflammation and immune regulation

Interleukin-33 (IL-33) belongs to the IL-1 family of cytokines and has been reported to play multiple roles in host defense, allergies and chronic inflammation. Constitutive expression of IL-33 in epithelial cells ensures rapid immune responses against invading pathogens such as parasites and viruses. Tissue damage caused by pathogens results in the release of extracellular IL-33 that in turn alerts a variety of immune cells such as group 2 innate lymphoid cells (ILC2s), eosinophils, basophils and mast cells. These cells mediate T helper type 2 (Th2) immune responses to destroy pathogens. IL-33 also plays central roles in mediating allergic diseases including asthma and atopic rhinitis. Although the functions of IL-33 in host defense and allergies initially received most attention, focus is turning to its roles in chronic inflammatory diseases. Recent advances, however, have led to problematic results. In addition to Th2 responses, IL-33 also promotes Th1 responses and there have been positive and negative roles reported for IL-33 in inflammatory diseases. This mini-review will summarize IL-33 biology and discuss issues regarding previously unrecognized roles of IL-33.


CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF--induced apoptosis by tumor-associated macrophages.

Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. CXCL16 expression enhanced TNF--induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-B-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF--induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF--induced apoptosis through the induction of M1 macrophages, which released TNF-. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.


Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-RII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-RII mice with either C57BL/6 or dnTGF-RII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-RII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-RII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-RII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.


Role of cytochrome P450-mediated metabolism and identification of novel thiol-conjugated metabolites in mice with phenytoin-induced liver injury

Phenytoin, 5,5-diphenylhydantoin (DPH), is widely used as an anticonvulsant agent. Severe hepatic injury rarely occurs in patients who received DPH. The development of liver injury is thought to be caused by reactive metabolites; however, the metabolites suggested to contribute to hepatotoxicity have not yet been detected in vivo and their effect on developing the liver injury is largely unknown. We recently demonstrated that DPH treatment decreased hepatic glutathione (GSH) contents, and GSH-depleted condition exacerbated DPH-induced liver injury in mice. The aim of the present study was to identify the reactive metabolite and to investigate the role of P450-mediated metabolisms in DPH-induced liver injury. We identified a novel GSH-conjugated (GS)-DPH, a conjugate of putative electrophilic arene oxide intermediate with GSH, in the bile of mice with DPH-induced liver injury. In plasma, cysteine- or N-acetylcysteine-conjugated DPH was detected, and these thiol conjugates levels were correlated with the plasma alanine aminotransferase (ALT) levels. These changes were significantly reduced by pretreatment with P450 inhibitor. Furthermore, the increases of hepatic P450 activities were in parallel with elevation of plasma thiol conjugates levels. These findings suggest that the arene oxide intermediate, which can be converted to thiol conjugates, is involved in DPH-induced liver injury.


Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation.

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-B activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1 production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1 and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases.


Clinicopathological analysis of CD8-positive lymphocytes in the tumor parenchyma and stroma of hepatocellular carcinoma.

Tumor-infiltrating lymphocytes may be a manifestation of antitumor immunity. In the present study, hepatocellular carcinoma (HCC) and pericancerous non-tumor liver tissues samples were obtained from 86 surgical patients who had not received preoperative treatment. The cellular expression levels of CD4 and CD8 were immunohistochemically examined in the two tissue groups using tissue microarrays, to evaluate their clinicopathological relevance. Immunohistochemically, CD4 and CD8 T cells were observed in the tumor parenchyma and tumor stroma, and the intensity of CD4 and CD8 immunoreactivity was homogeneous in all HCC samples examined. Morphometrically, the average numbers of CD4- and CD8-positive T cells were significantly increased in the tumor stroma, compared with those in the tumor parenchyma (tumor stroma versus tumor parenchyma: 22±3.6 versus 7.4±0.9 in CD4, 32.8±4.2 versus 16±2.5 in CD8; both P<0.01). Furthermore, the average numbers of CD8-positive T cells in the tumor parenchyma and stroma were significantly increased, compared with the average numbers of CD4-positive cells (P<0.05). In addition, in the tumor parenchyma and stroma, the average numbers of CD8 T cells were significantly higher in patients with tumor diameters 5 cm compared with those in patients with tumor diameters >5 cm (diameter 5 cm versus diameter >5 cm: 18.1±3.3 versus 12.2±3.8 in tumor parenchyma, 36.5±4.8 versus 21.9±8.9 in tumor stroma; both P<0.05). In addition, CD8 expression was significantly enhanced in patients with chronic hepatitis and cirrhosis, compared with paired tumor parenchymal tissues (P<0.01). Furthermore, a significant positive correlation was observed between CD4 and CD8 expression in the tumor parenchyma and stroma (both P<0.001). These observations suggest that tumor parenchyma- or stroma-infiltrating CD8 T cells may be involved in HCC tumor diameter control.


Neonatal monosodium glutamate treatment causes obesity, diabetes, and macrovesicular steatohepatitis with liver nodules in DIAR mice.

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome (MS). Monosodium glutamate (MSG)-treated ICR mice is a useful model of MS and NASH, but it shows the different patterns of steatosis from human NASH. Because inbred aged DIAR (ddY, Institute for Animal Reproduction) mice spontaneously show the similar pattern of steatosis as NASH, we analyzed their liver pathology after administering MSG. MSG-treated DIAR mice (DIAR-MSG) and untreated DIAR mice (DIAR-controls) were sacrificed and assessed histopathologically at 29, 32, 40, 48, and 54 weeks of age. The NASH activity score, body mass index, blood glucose level, and oral glucose tolerance test were also assessed. The body mass index and blood glucose levels of DIAR-MSG were significantly higher than controls. The oral glucose tolerance test revealed a type 2 diabetes pattern in DIAR-MSG. The livers of DIAR-MSG mice showed macrovesicular steatosis, lobular inflammation with neutrophils, and ballooning degeneration after 29 weeks. At 54 weeks, mild fibrosis was observed in 5/6 DIAR-MSG and 2/5 DIAR-control mice. In imaging mass spectrometry analysis, cholesterol as well as triglyceride accumulated in the liver of DIAR-MSG mice. Atypical liver nodules were also observed after 32 weeks in DIAR-MSG, some with cellular and structural atypia mimicking human hepatocellular carcinoma. The NASH activity score of DIAR-MSG after 29 weeks was higher than that of control mice, suggesting the development of NASH. DIAR-MSG had NASH-like liver pathology and liver nodules typically associated with MS symptoms. DIAR-MSG provides a valuable animal model to analyze NASH pathogenesis and carcinogenesis.

Journal of Gastroenterology and Hepatology


Differential modulation by IL-17A of Cholangitis versus Colitis in IL-2R deleted mice.

IFN- is a signature Th1 cell associated cytokine critical for the inflammatory response in autoimmunity with both pro-inflammatory and potentially protective functions. IL-17A is the hallmark of T helper 17 (Th17) cell subsets, produced by T, CD8+ T, NK and NKT cells. We have taken advantage of our colony of IL-2R-/- mice that spontaneously develop both autoimmune cholangitis and inflammatory bowel disease. In this model CD8+ T cells mediate biliary ductular damage, whereas CD4+ T cells mediate induction of colon-specific autoimmunity. Importantly, IL-2R-/- mice have high levels of interferon (IFN-), and interleukin-17A (IL-17A). We produced unique double deletions of mice that were either IL-17A-/-IL-2R-/- or IFN--/-IL-2R-/- to specifically address the precise role of these two cytokines in the natural history of autoimmune cholangitis and colitis. Of note, deletion of IL-17A in IL-2R-/- mice led to more severe liver inflammation, but ameliorated colitis. In contrast, there were no significant changes in the immunopathology of double knock-out IFN--/- IL-2R-/- mice, compared to single knock-out IL-2R-/- mice with respect to cholangitis or colitis. Furthermore, there was a significant increase in pathogenetic CD8+ T cells in the liver of IL-17A-/-IL-2R-/- mice. Our data suggest that while IL-17A plays a protective role in autoimmune cholangitis, it has a pro-inflammatory role in inflammatory bowel disease. These data take on particular significance in the potential use of anti-IL-17A therapy in humans with primary biliary cirrhosis.


Animal models of primary biliary cirrhosis.

Within the last decade, several mouse models that manifest characteristic features of primary biliary cirrhosis (PBC) with antimitochondrial antibodies (AMAs) and immune-mediated biliary duct pathology have been reported. Here, the authors discuss the current findings on two spontaneous (nonobese diabetic autoimmune biliary disease [NOD.ABD] and dominant negative transforming growth factor- receptor II [dnTGFRII]) and two induced (chemical xenobiotics and microbial immunization) models of PBC. These models exhibit the serological, immunological, and histopathological features of human PBC. From these animal models, it is evident that the etiology of PBC is multifactorial and requires both specific genetic predispositions and environmental insults (either xenobiotic chemicals or microbial), which lead to the breaking of tolerance and eventually liver pathology. Human PBC is likely orchestrated by multiple factors and hence no single model can fully mimic the immunopathophysiology of human PBC. Nevertheless, knowledge gained from these models has greatly advanced our understanding of the major immunological pathways as well as the etiology of PBC.


Neonatal streptozotocin treatment causes type 1 diabetes and subsequent hepatocellular carcinoma in DIAR mice fed a normal diet.

Hepatocellular carcinoma (HCC) represents a major health challenge because of its increasing morbidity and mortality. The establishment of useful models of HCC can significantly contribute to unveiling its pathophysiology. We developed a novel mouse model of HCC based on type 1 diabetes and reported its histopathological features. Newborn male ddY, Institute for Animal Reproduction (DIAR) mice were divided into two groups on the basis of streptozotocin treatment, which induces type 1 diabetes. Streptozotocin was subcutaneously injected (60 mg/g) into the treated group (DIAR-nSTZ mice), whereas physiologic solution was injected into the control group (DIAR-control mice) at 1.5 days after birth. All mice were fed a normal diet and histopathologically assessed at 6, 8, 10, 12, 19, and 27 weeks of age. At 8 weeks, small hepatocytic nodules with mild to moderate cellular atypia were observed in the livers of DIAR-nSTZ mice, which progressed to large hepatocytic nodules with cellular atypia and infiltrating growth at 12 weeks, identical to those in well-differentiated human HCC. At 19 and 27 weeks, moderately differentiated HCC was observed in all DIAR-nSTZ mice. Conversely, no neoplastic findings were evident in DIAR-control mice. No steatosis or fibrosis was evident in either group. Immunohistochemical analysis revealed that all nodules observed in DIAR-nSTZ mice were positive for glutamine synthetase. In DIAR-nSTZ mice, the development of HCC with similarity to human HCC and high reproducibility can be achieved using a short and simple protocol. We believe that this model will be useful for studying liver carcinogenesis.


High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague-Dawley rats.

The development of fibrosis is considered an important phase in the progress of non-alcoholic steatohepatitis (NASH) towards the end stage of liver disease, including cirrhosis. However, few small animal models can display NASH-associated fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis using genetically normal rats. Nine-week-old male Sprague-Dawley rats were fed with normal, high-fat (HF), or two types of high-fat and high-cholesterol (HFC) diets for 9 weeks (n = 5 each). All HFC diets contained 1.25% or 2.5% cholesterol. The rats fed with the HF diet developed mild steatosis and inflammation without fibrosis at 18 weeks of age, whereas all rats given the HFC diet developed obvious steatosis and inflammation with hepatocyte ballooning and fibrosis. Two of five (40%) rats given the HFC diet containing 2.5% cholesterol progressed to liver cirrhosis. Hepatic total cholesterol levels were significantly higher in rats given the HFC, than the normal or HF diets. The HFC diet significantly and dose-dependently decreased microsomal triglyceride transfer protein expression. Cholesterol tended to suppress carnitine palmitoyltransferase activity and adenosine triphosphate-binding cassette transporter G5 expression. Adding cholesterol to the HF diet modified hepatic lipid metabolism at the molecular level. The HFC diet induced hepatic features of NASH and eventually progressed cirrhosis in Sprague-Dawley rats within 9 weeks.


Changes in the expression of miRNAs at the pericentral and periportal regions of the rat liver in response to hepatocellular injury: comparison with the changes in the expression of plasma miRNAs.

MicroRNAs (miRNAs) in body fluids have received attention as potential biomarkers of organ damage because miRNAs that are highly or specifically expressed in a given organ are likely released into body fluids as a result of damage to that organ. We previously determined that the plasma miRNA profile in rats was dramatically changed due to acetaminophen (APAP)-induced pericentral necrosis and methapyrilene (MP)-induced periportal necrosis in the liver. The purpose of this study was to examine whether the expression of hepatic miRNAs is differentially modulated at different zones due to injury and to examine the relationship of the hepatic miRNA profile with the changes in the plasma miRNA expression profile. Through the laser microdissection of the periportal and periportal regions of the liver and TaqMan microRNA Array analysis, we found that 49 miRNAs are differentially expressed between the pericentral and periportal regions of control rats. In both APAP- and MP-treated rats, the miRNAs that presented decreased expression dominated in both the injured and non-injured areas compared with the miRNAs that exhibited increased expression. The changes in miRNA expression in each region of the liver were compared with those observed in the plasma. Of the 301 plasma miRNAs with expression that was changed as a result of APAP administration, only 21% were changed in the injured area of the liver. Of the 263 plasma miRNAs with expression that was changed due to MP administration, only 24% were changed in the injured area of the liver. Thus, the miRNA expression profiles in the plasma do not merely reflect the release of miRNAs from the damaged cells in the liver. This report provides the first demonstration of zonal miRNA expression in the liver and of the relationship of the miRNA expression profile in a tissue with the plasma miRNA profile.


Induction of insulin secretion by apolipoprotein M, a carrier for sphingosine 1-phosphate.

High-density lipoprotein (HDL) has been proposed to enhance -cell functions. Clinical studies have suggested that apolipoprotein M (apoM), which rides mainly on HDL, is involved in diabetes; however, the underlying mechanism has not yet been elucidated. Recently, apoM was shown to be a carrier for sphingosine 1-phosphate (S1P), a bioactive lipid mediator. In the present study, we investigated the modulation of insulin secretion by apoM through the action of S1P. We overexpressed apoM in the livers of C57BL6 mice using adenovirus gene transfer and found that the blood glucose levels under ad libitum feeding conditions were lower in the apoM-overexpressing mice. While an insulin tolerance test revealed that insulin sensitivity was not significantly affected, a glucose tolerance test revealed that apoM-overexpressing mice had a better glucose tolerance because of enhanced insulin secretion, a phenomenon that was reversed by treatment with VPC 23019, an antagonist against S1P1 and S1P3 receptor. In vitro experiments with MIN6 cells also revealed that apoM-containing lipoproteins enhanced insulin secretion, which was again inhibited by VPC 23019. ApoM retarded the degradation of S1P, and an increase in Pdx1 expression, the attenuation of endoreticulum stress, and the phosphorylation of Akt, AmpK, and Erk were observed as possible underlying mechanisms for the effect of S1P, maintained at a high concentration by apoM, on the increase in insulin secretion. ApoM augmented insulin secretion by maintaining the S1P concentration under both in vivo and in vitro conditions.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids


A case of progressing focal nodular hyperplasia and its molecular expression pattern.

We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although imaging studies suggested that the tumor was a classical FNH, the tumor biopsy showed glutamine synthetase expression and heat shock protein 70 in part of the tumor. As we could not definitely distinguish this case of FNH from early hepatocellular carcinoma (HCC), we performed laparoscopic partial hepatectomy. The surgical resected specimen showed that the tumor had a central scar with vascular and cholangiolar proliferation, which is compatible with FNH. Immunohistochemical analysis showed that the molecular expression pattern was compatible with FNH in the center of the tumor, whereas it partly resembled early HCC in the periphery of the tumor. FNH progression is occasionally found, and the molecular pattern of the progressing area in FNH might resemble that of early HCC due to morphologic and phenotypic changes induced by the regenerative mechanism and the alteration of blood flow. We should carefully observe progressing FNH.


IL-12/Th1 and IL-23/Th17 biliary microenvironment in primary biliary cirrhosis: implications for therapy.

The interleukin (IL)-12/IL-23-mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, interferon-gamma (IFN-), IL-12RB2, IL-23p40, IL-23p19, IL-17, and IL-23R using liver from PBC (n = 51) and non-PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN- expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN- production that contribute to PBC pathology. Our data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.


Reversible phospho-Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection.

Transforming growth factor (TGF)-, type I receptor (TRI) and c-Jun N-terminal kinases (JNK) phosphorylate Smad3 differentially to create 2 isoforms phosphorylated (p) at the COOH-terminus (C) or at the linker region (L) and regulate hepatocytic fibrocarcinogenesis. This study aimed to compare the differences between how hepatitis B virus (HBV) infection affected hepatocytic Smad3 phosphorylated isoforms before and after anti-viral therapy. To clarify the relationship between Smad3 phosphorylation and liver disease progression, we studied 10 random patients in each stage of HBV-related fibrotic liver disease (F1-4) and also 10 patients with HBV-associated HCC. To examine changes in phosphorylated Smad3 signalling before and after anti-HBV therapies, we chose 27 patients with chronic hepatitis B who underwent baseline and follow-up biopsies at 52 weeks from the start of nucleoside analogue treatments (Lamivudine 100 mg daily or Telbivudine 600 mg daily). Fibrosis stage, inflammatory activity and phosphorylated Smad3 positivity in the paired biopsy samples were compared. Hepatocytic pSmad3C signalling shifted to fibrocarcinogenic pSmad3L signalling as the livers progressed from chronic hepatitis B infection to HCC. After nucleoside analogue treatment, serum alanine aminotransferase (ALT) and HBV-DNA levels in 27 patients with HBV-related chronic liver diseases were decreased dramatically. Decrease in HBV-DNA restored pSmad3C signalling in hepatocytes, while eliminating prior fibrocarcinogenic pSmad3L signalling. Oral nucleoside analogue therapies can suppress fibrosis and reduce HCC incidence by successfully reversing phosphorylated Smad3 signalling; even liver disease progressed to cirrhosis in chronic hepatitis B patients.


Molecular mechanisms of hyperthermia-induced apoptosis enhanced by docosahexaenoic acid: implication for cancer therapy.

To develop a non-toxic enhancer for hyperthermia-induced cell death as a potential cancer treatment, we studied the effect and mechanism of docosahexaenoic acid (DHA) on hyperthermia-induced apoptosis. Treatment with 20M DHA and 44°C for 10min induced significant apoptosis, increased intracellular reactive oxygen species (ROS), and caspase-3 activation in U937 cells, but heat or DHA alone did not induce notable apoptosis. Decreased mitochondrial transmembrane potentials were dramatically increased by the combined treatment, accompanied by increased pro-apoptotic Bcl-2 family protein tBid, and decreased anti-apoptotic Bcl-2 and Bcl-xL. Combined hyperthermia-DHA treatment induced significant phosphorylation of protein kinase C (PKC)- (p-PKC-), and apoptosis in a DHA dose-dependent manner. Using both 20M DHA and 44°C for 10min induced significant PKC- cleavage and its translocation to mitochondria. These results were also seen in HeLa cells. However, MAPKs and Akt were not affected by the treatment. In conclusion, DHA enhances hyperthermia-induced apoptosis significantly via a mitochondria-caspase-dependent pathway; its underlying mechanism involves elevated intracellular ROS, mitochondria dysfunction, and PKC- activation.


Social isolation stress-induced fear memory deficit is mediated by down-regulated neuro-signaling system and Egr-1 expression in the brain.

We previously reported that social isolation (SI) rearing of rodents not only elicits a variety of behavioral abnormalities including attention deficit hyperactivity disorder-like behaviors, but also impairs fear memory in mice. This study aimed to clarify a putative mechanism underlying SI-induced conditioned fear memory deficit. Mice were group-housed (GH) or socially isolated for 2 weeks or more before the experiments. SI animals acquired contextual and auditory fear memory elucidated at 90 min and 4 h after training, respectively; however, they showed significantly impaired contextual and auditory memory performance at 24 h and 4 days after the training, respectively, indicating SI-induced deficit of the consolidation process of fear memory. Neurochemical studies conducted after behavioral tests revealed that SI mice had a significantly down-regulated level of Egr-1 but not Egr-2 in the hippocampal and cortical cytosolic fractions compared with those levels in the GH control animals. Moreover, in the SI group, phosphorylated levels of synaptic plasticity-related signaling proteins in the hippocampus, NR1 subunit of N-methyl-D-aspartate receptor, glutamate receptor 1, and calmodulin-dependent kinase II but not cyclic AMP-responsive element binding protein were significantly down-regulated compared with those levels in GH animals, whereas non-phosphorylated levels of these proteins were not affected by SI. These findings suggest that dysfunctions of Egr-1 and neuro-signaling systems are involved in SI-induced deficits of fear memory consolidation in mice.


Malignant transformation of hyperplastic gastric polyps: An immunohistochemical and pathological study of the changes of neoplastic phenotype.

In spite of the evidence that the malignant transformation of gastric hyperplastic polyps (HPs) is a rare event, it must always be taken into account during diagnosis. The aim of the current study was to clarify the mechanism of the malignant transformation of gastric hyperplasia polyps, with focus on phenotypic expression, cell proliferation and p53 overexpression. Immunohistochemistry for mucin phenotypic markers, including MUC1, MUC2, MUC5AC, MUC6, tight junction factors (claudin-3, -4 and -18), an intestinal phenotypic marker [caudal type homeobox 2 (Cdx2)], Ki-67 proliferative index and p53 overexpression, was performed on archival specimens of gastric polyps excised from six patients. Histologically, the intermingled components of several lesions were present in these polyps. Furthermore, the cancer components were predominantly differentiated adenocarcinoma. Immunohistochemically, all hyperplastic components expressed MUC5AC, but did not exhibit positivity for MUC2. Additionally, the majority of hyperplastic components were immunonegative for claudin-3, while claudin-3 positivity was observed in the majority of areas of dysplasia and carcinoma. Expression of claudin-4 was also observed in the majority of cases and claudin-18 was preserved in the hyperplastic, dysplastic and adenocarcinomatous lesions of all cases. Nuclear accumulation of Cdx2 was detected in almost all the samples with dysplasia and carcinoma, while nuclear p53 was detected in 24-80% of the dysplastic areas and >85% of the cancer components. The Ki-67 labeling index appeared to correlate with neoplastic progression. The observations provided evidence that the mechanism underlying malignant transformation of gastric HPs may occur by multistep carcinogenesis, such as the hyperplasia-adenoma (dysplasia)-adenocarcinoma sequence, and these neoplastic cells may acquire various phenotypes during this process.


A dietary restriction influences the progression but not the initiation of MSG-Induced nonalcoholic steatohepatitis.

The metabolic syndrome is a major worldwide health care issue and a dominant risk factor for cardiovascular disease. The liver manifestations of this syndrome include nonalcoholic fatty liver disease (NAFLD) and its progressive variant nonalcoholic steatohepatitis (NASH). Although significant research has been performed, the basic pathogenesis of NAFLD/NASH remains controversial and effective treatments are still unavailable. We have previously reported on a murine model of NASH induced by the neonatal injection of monosodium glutamate (MSG), which includes the clinical manifestations of central obesity, diabetes, hyperlipidemia, and ultimately liver inflammation, fibrosis, and cancer. Although MSG is considered a safe food additive, its administration to pregnant rats increases the voracity and growth hormone levels in the offspring. To further understand the biology of this model, we have investigated the influence of the calorie intake on these clinical manifestations by feeding animals a restrictive diet. MSG-treated animals fed a restrictive diet continue to manifest obesity and early stage NASH but have improvements in serum lipid profiles. At 12 months of age, mice had manifestations of obesity, whether animals were fed a restricted or control diet, but animals fed a restrictive diet had a reduction in the progression of NASH. In conclusion, MSG appears to be a critical factor in the initiation of obesity, whereas calorie intake may modulate the progression of disease.


A simple and rapid decalcification procedure of skeletal tissues for pathology using an ultrasonic cleaner with D-mannitol and formic acid.

Decalcification procedures are required in order to prepare histopathological preparations of hard tissues such as bone and teeth. Decalcification is usually performed by immersing the hard tissue in different decalcification fluids with various properties. These decalcification fluids typically include inorganic and organic acids, a neutral fluid containing a chelating agent, or a mixture of solutions. Unfortunately, there is no universal decalcification fluid that satisfies all the requirements of pathologists such as rapid decalcification, easy handling, and minimal tissue damage. Techniques involving use of microwaves (MW) or ultrasonic apparatus (US) have been shown to be useful for shortening the time for decalcification procedures. In the present study, we investigated a unique decalcification procedure that uses a common commercial ultrasonic cleaner and a decalcification fluid (formic acid) containing a free-radical scavenger (D-mannitol). The time required to complete the procedure is approximately half of that required to complete a standard decalcification procedure. In addition, tissue morphology and antigenicity is fairly well preserved after decalcification. The procedure is quick, easy to perform, and achieves decalcification of hard tissue with minimal tissue damage.


Escherichia coli infection induces autoimmune cholangitis and anti-mitochondrial antibodies in non-obese diabetic (NOD).B6 (Idd10/Idd18) mice.

Several epidemiological studies have demonstrated that patients with primary biliary cirrhosis (PBC) have a higher incidence of urinary tract infections (UTI) and there is significant homology of the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), between mammals and bacteria. Previous work has demonstrated that non-obese diabetic (NOD).B6 Idd10/Idd18 infected with Novosphingobium aromaticivorans developed liver lesions similar to human PBC. It was postulated that the biliary disease was dependent upon the presence of the unique N. aro glycosphingolipids in activating natural killer T (NK T) cells. To address this issue, we infected NOD.B6 Idd10/Idd18 mice with either Escherichia coli, N. aro or use of a phosphate-buffered saline (PBS) vehicle control and serially followed animals for the appearance of liver pathology and anti-mitochondrial autoantibodies (AMA). Of striking importance, the biliary disease of E. coli-infected mice was more severe than N. Aro-infected mice and the titre of AMA was higher in E. coli-infected mice. Furthermore, the immunopathology did not correlate with the ability of bacterial extracts to produce antigen-dependent activation of NK T cells. Our data suggest that the unique glycosphingolipids of N. aro are not required for the development of autoimmune cholangitis. Importantly, the data highlight the clinical significance of E. coli infection in a genetically susceptible host, and we suggest that the appearance of autoimmune cholangitis is dependent upon molecular mimicry. These data highlight that breach of tolerance to PDC-E2 is probably the first event in the natural history of PBC in genetically susceptible hosts.


Histopathological analysis for osteomalacia and tubulopathy in itai-itai disease.

Cadmium (Cd) is a widespread environmental contaminant that causes both renal tubulopathy and osteomalacia. Osteomalacia is thought to be a result of renal tubulopathy, but there are few studies about the histopathological relationship between the two pathoses. Therefore, in the present study, we examined specimens from cases of itai-itai disease (IID), the most severe form of chronic cadmium poisoning, to evaluate the relationship between them. We analyzed kidney and bone specimens of 61 IID cases and the data regarding Cd concentration in kidney and bone. Tubulopathy was graded on the basis of a three-step scale (mild, moderate, and severe) using the following three items: the degree of proximal tubular defluxion, thickness of renal cortex, and weight of the kidney. Osteomalacia was evaluated using the relative osteoid volume (ROV). There were 15 cases of mild, 19 cases of moderate, and 27 cases of severe tubulopathy. The average ROV was 24.9 ± 2.0%. ROV tended to increase as tubulopathy advanced in severity, and ROV was significantly higher in cases with severe tubulopathy than those with mild or moderate tubulopathy. ROV had a negative correlation with Cd concentration in the kidney but no correlation with that in the bone. Our results suggest that the development of osteomalacia was related to the development of tubulopathy.


Splenic lymph follicles generate immunoglobulin M-producing B cells in primary biliary cirrhosis.

To reveal the site of immunoglobulin (Ig)M production in primary biliary cirrhosis (PBC) we performed immunohistochemical analysis on spleens collected from patients with PBC. Splenic tissue samples were collected at the time of the autopsy from patients with hepatic failure. Immunostaining for IgM, CD21 and CXCL13 were performed using the splenic tissue samples. The samples from five out of eight cases with PBC but not in eight cases of chronic hepatitis C virus infection showed accumulation of IgM positive cells in CD21 positive lymph follicles. The CXCL13 positive cells also accumulated in the center of the lymph follicles where the IgM positive cells accumulated. The present results suggest that excess IgM is produced from the spleen of PBC. Furthermore, it was suggested that CXCL13 positive follicular dendritic cells possibly contribute to this process.


Abdominal subcutaneous adipose tissue accumulation is positively correlated with hepatic steatosis in Sprague-Dawley rats

The precise roles of visceral (VAT) or subcutaneous adipose tissue (SAT) on hepatic fat accumulation have not been fullyelucidated. In this report, we examined the correlation between VAT or SAT volume and severity of hepatic fat accumulation. Inthe present study, Sprague-Dawley (SD) rats were fed a standard diet containing 10% fat or a high-fat diet containing 45% or 60%fat for 16 weeks. Abdominal VAT and SAT volume, as well as fat percentage of the liver were measured by computed tomography(CT). Hepatic triglyceride (TG) content and histopathological findings of hepatic steatosis were also examined. Abdominal SATweight/body weight ratio was positively and strongly correlated with abdominal VAT weight/body weight ratio. Fat percentage ofthe liver by CT evaluation, hepatic TG content, and hepatic steatosis score by histopathological evaluation showed positive correlationswith one another. Fat percentage of the liver by CT evaluation and hepatic TG content was positively correlated withboth the abdominal VAT weight/body weight ratio and SAT weight/body weight ratio, respectively. Furthermore, hepatic TG contentwas negatively correlated with the abdominal VAT weight/SAT weight ratio. Our data suggest that abdominal SAT accumulationis positively correlated with hepatic steatosis in SD rats, rather than abdominal VAT accumulation. Further investigations areneeded in order to clarify the precise mechanisms of SAT and VAT effects on the development of hepatic fat accumulation.


Introduction and utility of liquid-based cytology on aspiration biopsy of peripheral nodular lesions of the lung.

In the present study, aspiration biopsy cytology (ABC) was used for the diagnosis of peripheral nodular lesions in the lung (PNLL), and liquid-based cytology (LBC) was carried out on the material collected to evaluate it in comparison with the conventional method (CM). The subjects comprised 130 cases that underwent computed tomography (CT)-guided ABC for PNLL. A total of 73 cases received a tumor resection, with a diagnosis based on the pathology, while 57 cases were followed up, as the tumor showed no change on the radiological examinations. Biopsy samples from these patients and lavage fluid from the aspiration needles were used for analysis. Cellular material was obtained by centrifugation of the lavage fluid, and samples were prepared by two methods, direct smearing and LBC according to the ThinPrep method. The samples were categorized into three diagnoses: i) Benign, ii) suspicion of malignancy and iii) malignant. Appropriate samples were collected in 72% of cases by LBC, but only in 36% of cases by the CM. There was no marked difference in cellular images between the two methods, with the exception of a few specific cases. LBC on its own provided sensitivity at 68%, specificity at 61% and accuracy at 65%, while a combination of LBC and biopsy markedly improved these figures to 94, 81 and 84%, respectively. The introduction of LBC is considered useful for the cytopathological diagnosis of PNLL by CT-guided ABC. LBC enables the examination of appropriate samples rich in cellular components and supports a biopsy-based diagnosis. A combination of these two methods provides even higher diagnostic accuracy, and LBC is considered an excellent method to evaluate these pathological samples.


Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor receptor II (dnTGFRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.


Involvement of oxidative stress and immune- and inflammation-related factors in azathioprine-induced liver injury.

Drug-induced liver injury (DILI) is a growing concern in the fields of drug development and clinical drug therapy because numerous drugs have been linked to hepatotoxicity. However, it is difficult to predict DILI in humans due to the lack of experimental animal models. Although azathioprine (AZA), which is a widely used immunosuppressive drug, is generally well tolerated, a small number of patients prescribed AZA develop severe hepatitis. However, the mechanism underlying this process has not yet been elucidated. In this study, we developed a mouse model of AZA-induced liver injury and investigated the mechanisms responsible for the hepatotoxicity of AZA. Female BALB/c mice were orally administered AZA. After AZA administration, the plasma levels of alanine aminotransferase and aspartate aminotransferase were increased, and liver damage was confirmed through a histological evaluation. In addition, the hepatic glutathione levels and superoxide dismutase activity were significantly decreased. The plasma levels of reactive oxygen species were significantly increased during the early phase of AZA-induced liver injury, and the hepatic mRNA levels of immune- and inflammation-related factors were also significantly changed. In conclusion, oxidative stress and the subsequently activated immune- and inflammation-related factors are involved in AZA-induced liver injury.


Identification of potential cytokine pathways for therapeutic intervention in murine primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN- prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN- is significantly decreased in the liver of IL-23p19(-/-), IL-17A(-/-) and IL-22(-/-) mice compared with controls. However, the ability of T cells to produce IFN- was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN- producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN- has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN--mediated immunopathology.


A novel mouse model for phenytoin-induced liver injury: involvement of immune-related factors and P450-mediated metabolism.

Drug-induced liver injury is an important issue for drug development and clinical drug therapy; however, in most cases, it is difficult to predict or prevent these reactions due to a lack of suitable animal models and the unknown mechanisms of action. Phenytoin (DPH) is an anticonvulsant drug that is widely used for the treatment of epilepsy. Some patients who are administered DPH will suffer symptoms of drug-induced liver injury characterized by hepatic necrosis. DPH-induced liver injury occurs in 1 in 1000 or 1 in 10 000 patients. Clinically, 75% of patients who develop liver injury develop a fever and 63% develop a rash. In this study, we established a mouse model for DPH-induced liver injury and analyzed the mechanisms for hepatotoxicity in the presence of immune-related or inflammation-related factors and metabolic activation. Female C57BL/6 mice were administered DPH for 5 days in combination with L-buthionine-S,R-sulfoximine. Then, the plasma alanine aminotransferase (ALT) levels were increased, hepatic lesions were observed during the histological evaluations, the hepatic glutathione levels were significantly reduced, and the oxidative stress marker levels were significantly increased. The inhibition of cytochrome P450-dependent oxidative metabolism significantly suppressed the elevated plasma ALT levels and depleted hepatic glutathione. Among the innate immune factors, the hepatic mRNA levels of NACHT, LRR, pyrin domain-containing protein 3, interleukin-1, and damage-associated molecular patterns were significantly increased. Prostaglandin E1 treatment ameliorated the hepatic injury caused by DPH. In conclusion, cytochrome P450-dependent metabolic activation followed by the stimulation of the innate immune responses is involved in DPH-induced liver injury.


STAT3-mediated attenuation of CCl4-induced mouse liver fibrosis by the protein kinase inhibitor sorafenib.

There have been major advances in defining the immunological events associated with fibrosis in various chronic liver diseases. We have taken advantage of this data to focus on the mechanisms of action of a unique multi-kinase inhibitor, coined sorafenib, on CCl4-induced murine liver fibrosis, including the effects of this agent in models of both acute and chronic CCl4-mediated pathology. Importantly, sorafenib significantly attenuated chronic liver injury and fibrosis, including reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including -smooth muscle actin, collagen, matrix metalloproteinases and the tissue inhibitor of metalloproteinase-1. Furthermore, sorafenib treatment resulted in translocation of cytoplasmic STAT3 to the nucleus in its active form. Based on this observation, we used hepatocyte-specific STAT3 knockout (STAT3(Hep-/-)) mice to demonstrate that hepatic STAT3 was critical for sorafenib-mediated protection against liver fibrosis, and that the upregulation of STAT3 phosphorylation was dependent on Kupffer cell-derived IL-6. In conclusion, these data reflect the clinical potential of the multi-kinase inhibitor sorafenib for the prevention of fibrosis as well as the treatment of established liver fibrosis and illustrate the immunological mechanisms that underlie the protective effects of sorafenib.


Studies of efficacy and liver toxicity related to adeno-associated virus-mediated RNA interference.

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.


Autoimmune features in metabolic liver disease: a single-center experience and review of the literature.

Non-alcoholic steatohepatitis (NASH) is the progressive phenotype of non-alcoholic fatty liver disease associated with the metabolic syndrome. The existence of autoimmune features in NASH has been reported, but its significance remains unclear. We herein report the autoantibody profile of 54 patients with histologically proven NASH and further determined the development of autoimmunity in three different murine NASH models (monosodium glutamate, CDAA (choline-deficient L-amino acid-defined), and TSOD (Tsumura Suzuki, Obese Diabetes)) at 48 weeks of age. Forty-eight percent (26/54) of NASH cases were positive for antinuclear (ANA) or antimitochondrial antibody and manifested histological signs of overlap with autoimmune hepatitis and primary biliary cirrhosis, respectively. These patients were significantly older (60 ± 10 versus 50 ± 16 years), more frequently women (81 % versus 43 %), and with more severe portal inflammatory infiltrate compared with patients without autoimmunity. In one third of mice, regardless of the model, we observed a marked lymphoid infiltrate with non-suppurative cholangitis, and several cases were ANA-positive, but none AMA-positive. Our data suggest that autoimmunity may share some pathogenetic traits with the chronic inflammation of NASH, possibly related to advanced age.

Clinical Reviews in Allergy & Immunology


A case of primary biliary cirrhosis that progressed rapidly after treatment involving rituximab.

Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are recommended. Rituximab, an anti-CD20 monoclonal antibody, is expected to be a useful therapeutic regimen for PBC. Previous studies indicated biochemical and immunological improvement in PBC after rituximab treatment. Although rituximab shows therapeutic potential for PBC, few cases have been reported and histological improvement and long-term outcome remain uncertain. Here, we report a case of PBC in a 66-year-old Japanese female patient who presented with a gastric lymphoma and who had been treated with a regimen containing rituximab for incidental malignant lymphoma. She showed biochemical and immunological improvements, and liver histology before and after rituximab treatment confirmed a decrease in liver inflammation. However, she developed liver cirrhosis a short time after rituximab treatment without biochemical or immunological worsening. Rituximab treatment for PBC might be considered and careful observation is required after treatment.


Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.


The liver in itai-itai disease (chronic cadmium poisoning): pathological features and metallothionein expression.

Cadmium (Cd) is a highly hepatotoxic heavy metal, which is widely dispersed in the environment. Acute Cd hepatotoxicity has been well studied in experimental animals; however, effects of prolonged exposure to Cd doses on the liver remain unclear. In the present study, to evaluate chronic Cd hepatotoxicity, we examined specimens from cases of itai-itai disease, the most severe form of chronic Cd poisoning. We compared 89 cases of itai-itai disease with 27 control cases to assess Cd concentration in organs. We also examined 80 cases of itai-itai disease and 70 control cases for histopathological evaluation. In addition, we performed immunohistochemistry for metallothionein, which binds and detoxifies Cd. Hepatic Cd concentration was higher than Cd concentration in all other organs measured in the itai-itai disease group, whereas it was second highest following renal concentration in the control group. In the liver in the itai-itai disease group, fibrosis was observed at a significantly higher rate than that in the control group. Metallothionein expression was significantly higher in the itai-itai disease group than that in the control group. Prolonged exposure to low doses of Cd leads to high hepatic accumulation, which can then cause fibrosis; however, it also causes high expression of metallothionein, which is thought to reduce Cd hepatotoxicity.


Phosphorylated Smad2 and Smad3 signaling: Shifting between tumor suppression and fibro-carcinogenesis in chronic hepatitis C.

Insight into hepatic fibrogenesis and carcinogenesis (fibro-carcinogenesis) caused by hepatitis C virus (HCV) infection has come from recent analyses of transforming growth factor (TGF)- signaling. TGF- type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. This study aimed to elucidate how HCV infection affected hepatic fibro-carcinogenesis, particularly via phospho-Smad signaling. We first studied phospho-Smad2/3 positivity of 100 patients in different stages of HCV-related chronic liver disease. To examine changes in phospho-Smad2/3 after HCV clearance, we analyzed 32 paired liver biopsy samples obtained before and after sustained virological response (SVR), dividing patients into two groups: 20 patients not developing hepatocellular carcinoma (HCC) after attaining SVR (non-HCC group), and 12 patients who developed HCC despite SVR (HCC group). Hepatocytic tumor-suppressive pSmad3C signaling shifted to carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling as liver diseases progressed. In the non-HCC group, 13 patients (65%) displayed fibrotic regression and inflammation reduction after SVR. Interestingly, SVR restored cytostatic pSmad3C signaling in hepatocytes, while eliminating prior carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling. In the HCC group, seven patients (58%) displayed unchanged or even progressed fibrosis despite smoothened inflammatory activity, reflecting persistently high numbers of hepatocytes with pSmad3L- and pSmad2L/C-signaling and low pSmad3C-signaling. HCV clearance limits fibrosis and reduces HCC incidence by switching inflammation-dependent phospho-Smad signaling from fibro-carcinogenesis to tumor suppression. However, progression to HCC would occur in severely fibrotic livers if an inflammation-independent fibro-carcinogenic process has already begun before HCV clearance.


Deletion of interleukin (IL)-12p35 induces liver fibrosis in dominant-negative TGFβ receptor type II mice.

Mice with a dominant-negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40(-/-) dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFβRII mice, resulting in an IL-12p35(-/-) dnTGFβRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40(-/-) mice, the IL-12p35(-/-) mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35(-/-) mice also demonstrated a distinct cytokine profile characterized by a shift from a T-helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35(-/-) mice. In conclusion, IL-12p35(-/-) dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC.


Therapeutic effect of cytotoxic T lymphocyte antigen 4/immunoglobulin on a murine model of primary biliary cirrhosis.

Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T-cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA-4). Germane to the studies reported herein, signaling by CTLA-4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well-defined murine model of PBC, in which mice are immunized with 2-octynoic acid coupled to bovine serum albumin (2OA-BSA), leading to the production of high-titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA-4-Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA-4-Ig treatment, begun 1 day before 2OA-BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T-cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA-4-Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T-cell infiltrates and biliary cell damage, although AMA levels were not altered. These data suggest that an optimized regimen with CTLA-4-Ig has the potential to serve as an investigative therapeutic tool in patients with PBC.


A novel type of selective immunoglobulin m deficiency in a patient with autoimmune liver cirrhosis with recurrent hepatocellular carcinoma: a case report and review of the literature.

A 64-year-old female with advanced liver cirrhosis who had never experienced severe infections presented in 2004 with general malaise. At the time, her serum showed low levels of immunoglobulin (Ig) M (11 mg/dl) with high levels of both IgG (2,942 mg/dl) and IgA (808 mg/dl). Because serum levels of IgG and IgA in previous cases of selective IgM deficiency were normal, this case could have a novel immunological mechanism. By 2006, serum IgM was undetectable (<5 mg/dl). Liver biopsy showed liver cirrhosis from autoimmune hepatitis. She had no other autoimmune diseases or hematological malignancies. She developed hepatocellular carcinoma (HCC) several times and died of liver failure. Immunological analyses performed before the first diagnosis of HCC showed polyclonal -globulin elevation, normal chromosome and normal gene rearrangement of immunoglobulin heavy chain Cµ. Peripheral blood showed low count B cells with few surface IgM-positive B lymphocytes, but the percentages of T cell subsets were normal. Expression of activation-induced cytidine deaminase (AID), which plays a critical role in immunoglobin class switching, was found to be overexpressed in the HCC tissue and B cells in bone marrow. This phenomenon could account for the clinical and immunological features of this case. In conclusion, we propose a novel type of selective IgM deficiency in association with the overexpression of AID.

International Archives of Allergy and Immunology


Endogenous acetylcholine rescues NMDA-induced long-lasting hippocampal cell damage via stimulation of muscarinic M(1) receptors: elucidation using organic hippocampal slice cultures.

This study aimed to investigate a recuing role of cholinergic systems in the excitotoxicity-induced hippocampal cell damage. Organotypic hippocampal slice cultures (OHSCs) were prepared from 7-day-old mice and exposed to N-methyl-d-aspartate (NMDA) for 24h. After washing out the NMDA, OHSCs were incubated in medium containing test drugs for 0-6 days. Hippocampal cell damage was evaluated by propidium iodide staining, immunofluorescence, and Western blotting. NMDA (1-10 M) dose-dependently damaged hippocampal cells. The toxic effect of 3 M NMDA was also observed at 3-6 days, even after washing out NMDA, and was blocked by MK-801 from day 3 to day 6. Post-treatments with tacrine, donepezil, and galantamine reduced the NMDA-induced long-lasting hippocampal cell damage. The effect of tacrine was induced in a manner dependent on the incubation period after NMDA treatment and was confirmed by Nissl staining and immunostaining with NeuN, a marker of mature neurons. The effect of tacrine was attenuated by scopolamine and a muscarinic M(1) receptor antagonist, pirenzepine, but not by a muscarinic M(3) receptor antagonist, darifenacin, or a nicotinic receptor antagonist, mecamylamine. The protein kinase C inhibitor Ro-31-8220 abolished the effect of tacrine. The pretreatment with 3 M NMDA had no effect on the expression level of presynaptic cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter, in OHSCs. These results suggest that a low concentration of NMDA causes long-lasting hippocampal cell damage and that endogenous acetylcholine plays, via muscarinic M(1) receptor, a rescuing role in the excitotoxicity-induced long-lasting hippocampal cell damage.


Study of the effects of monacolin K and other constituents of red yeast rice on obesity, insulin-resistance, hyperlipidemia, and nonalcoholic steatohepatitis using a mouse model of metabolic syndrome

Evidence-Based Complementary and Alternative Medicine : eCAM


Spontaneous onset of nonalcoholic steatohepatitis and hepatocellular carcinoma in a mouse model of metabolic syndrome.

Metabolic syndrome is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases that affect the entire body. The hepatic manifestations of this syndrome include nonalcoholic fatty liver disease (NAFLD) and its progressive variant nonalcoholic steatohepatitis (NASH). The basic pathogenesis of NAFLD/NASH remains controversial because it is difficult to clarify the disease process of NASH on the basis of metabolic syndrome alone. To determine the pathogenesis and effective treatment, an excellent animal model of NASH is required. Tsumura Suzuki obese diabetes (TSOD) male mice spontaneously develop diabetes mellitus, obesity, glucosuria, hyperglycemia, and hyperinsulinemia without any special treatments such as gene manipulation. In this study, we examined the histopathological characteristics of visceral fat and liver of 56 male TSOD mice aged 4-17 months and 9 male Tsumura Suzuki non-obesity (control) mice aged 6-12 months. In the visceral fat, enlargement of adipocytes and perivascular and pericapsular CD8-positive lymphoid aggregation were observed in 4-month-old mice. Abnormal expression of tumor necrosis factor-, interleukin-6, and lipid peroxidation endo products was observed in macrophages. In the liver, microvesicular steatosis, hepatocellular ballooning, and Mallory bodies were observed in 4-month-old mice, with severity worsening with increasing time. These pathological findings in the liver mimic those seen in patients with NASH. Interestingly, small liver nodules with high cellularity and absence of portal tracts were frequently observed after 12 months. Most of them showed nuclear and structural atypia, and mimicked human hepatocellular carcinoma. The degree of steatosis in the non-tumor portions of the liver improved when the liver nodules developed. These findings were not observed in control mice. Here, we report that TSOD male mice spontaneously developed NAFLD without any special treatment, and that these mice are a valuable model for assessing NASH and NASH carcinogenesis owing to metabolic syndrome.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Overexpression of enhancer of zeste homolog 2 and MUC1 may be related to malignant behaviour in intraductal papillary neoplasm of the bile duct.

Intraductal papillary neoplasm of the bile duct (IPNB) usually has a favourable prognosis, but occasionally is associated with invasive carcinoma. Overexpression of the polycomb group protein enhancer of zeste homolog 2 (EZH2) is involved in the progression of malignant tumours. In this study, we examined the significance of EZH2 expression in IPNB and its association with clinicopathological features and the expression of p16(INK4a) , p53 and mucin core proteins. We examined immunohistochemically the expression of EZH2, p16(INK4a) , MUC mucin core proteins and p53 in 15 patients with IPNB without invasion, including the cystic variant [male/female ratio (M/F) = 9/6], and in 19 with IPNB associated with invasive carcinoma (M/F = 13/6). The expression levels of EZH2, p53 and MUC1 were significantly lower (P < 0.01), and of MUC6 were significantly higher (P < 0.05), in IPNB without invasion than in IPNB with invasion. Expression of EZH2 was significantly correlated with expression of MUC1 (P < 0.01) and inversely correlated with expression of MUC6 (P < 0.05). In cholangiocarcinoma cells (HuCTT-1 and TFK-1), knockdown of EZH2 and MUC1 by small interfering RNA decreased invasion and proliferation, whereas knockdown of MUC6 increased invasion. Overexpression of EZH2 may be associated with malignant behaviour in IPNB in parallel with up-regulated MUC1 expression and down-regulated MUC6 expression.


Lymphoma-like T cell infiltration in liver is associated with increased copy number of dominant negative form of TGFβ receptor II.

Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T cell lymphoma with an aggressive course and poor outcome despite multiagent chemotherapy. Contradictory literature, an unknown etiology, and poor response to treatment highlight the need to define the malignant process and identify molecular targets with potential for successful therapeutic interventions. Herein, we report that mice homozygously expressing a dominant negative TGFβRII (dnTGFβRII) under the control of the CD4 promoter spontaneously develop lymphoma-like T cell infiltration involving both spleen and liver. Splenomegaly, hepatomegaly and liver dysfunction were observed in homozygous dnTGFβRII mice between 10 weeks and 10 months of age associated with a predominant infiltration of CD4(-)CD8(-)TCRβ(+)NK1.1(+) or CD8(+)TCRβ(+)NK1.1(-) T cell subsets. Notch 1 and c-Myc expression at the mRNA levels were significantly increased and positively correlated with the cell number of lymphoid infiltrates in the liver of dnTGFβRII homozygous compared to hemizygous mice. Further, 2×10(4) isolated lymphoma-like cells transplant disease by adoptive cell transfers. Collectively, our data demonstrate that increased copy number of dnTGFβRII is critical for development of lymphoma-like T cell infiltration.


Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain.

These results suggest that CTS ameliorates diabetes-induced cognitive deficits by protecting central cholinergic and VEGF/PDGF systems via Akt signaling pathway and that CTS exhibits the anxiolytic effect via neuronal mechanism(s) independent of cholinergic or VEGF/PDGF systems in db/db mice.

BMC Complementary and Alternative Medicine


The immunobiology of colitis and cholangitis in interleukin-23p19 and interleukin-17A deleted dominant negative form of transforming growth factor beta receptor type II mice.

Dominant negative form of transforming growth factor beta receptor type II (dnTGFβRII) mice, expressing a dominant negative form of TGFβ receptor II under control of the CD4 promoter, develop autoimmune colitis and cholangitis. Deficiency in interleukin (IL)-12p40 lead to a marked diminution of inflammation in both the colon and the liver. To distinguish whether IL-12p40 mediates protection by the IL-12 or IL-23 pathways, we generated an IL-23p19(-/-) dnTGFβRII strain deficient in IL-23, but not in IL-12; mice were longitudinally followed for changes in the natural history of disease and immune responses. Interestingly, IL-23p19(-/-) mice demonstrate dramatic improvement in their colitis, but no changes in biliary pathology; mice also manifest reduced T-helper (Th)17 cell populations and unchanged IFN-γ levels. We submit that the IL-12/Th1 pathway is essential for biliary disease pathogenesis, whereas the IL-23/Th17 pathway mediates colitis. To further assess the mechanism of the IL-23-mediated protection from colitis, we generated an IL-17A(-/-) dnTGFβRII strain deficient in IL-17, a major effector cytokine produced by IL-23-dependent Th17 cells. Deletion of the IL-17A gene did not affect the severity of either cholangitis or colitis, suggesting that the IL-23/Th17 pathway contributes to colon disease in an IL-17-independent manner. These results affirm that the IL-12/Th1 pathway is critical to biliary pathology in dnTGFβRII mice, whereas colitis is caused by a direct effect of IL-23.


An elderly man with syncytial giant cell hepatitis successfully treated by immunosuppressants.

Here, we report an elderly man with acute-on-chronic hepatitis accompanied by massive ascites. He showed elevated serum transaminase and anti-nuclear antibody (ANA) levels. Liver biopsy showed diffuse multinucleated giant hepatocytes with interface hepatitis, and he recovered with administration of azathioprine in addition to corticosteroids. Follow-up liver biopsy after recovery showed improvement of hepatic inflammation and reduction of giant hepatocyte formation. The patient is receiving low-dose corticosteroid maintenance therapy and he has remained healthy for 8 years to date. Active immunosuppressive treatment may be beneficial in patients with adult syncitial giant cell hepatitis (AGCH).


Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver.

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.


[A case of gastrointestinal stromal tumor of the stomach mimicking a primary tumor of the omentum minus due to the extramural growth].

We report a case of gastrointestinal stromal tumor (GIST) of the stomach mimicking a primary tumor of the omentum minus. The tumor presented as an isolated mass in the omentum minus without any adhesion to the stomach. Microscopic examination revealed that the tumor pseudocapsule on the gastric side included a small smooth muscle tissue component. The patient was given a diagnosis of a gastric GIST that showed extensive extramural growth. GISTs should not be defined by the localization of the tumor.

The Japanese Journal of Gastro-enterology


Mechanisms of the hepatoprotective effects of tamoxifen against drug-induced and chemical-induced acute liver injuries.

Although estrogen receptor (ER)α agonists, such as estradiol and ethinylestradiol (EE2), cause cholestasis in mice, they also reduce the degree of liver injury caused by hepatotoxicants as well as ischemia-reperfusion. The functional mechanisms of ERα have yet to be elucidated in drug-induced or chemical-induced liver injury. The present study investigated the effects of an ERα agonist, selective ER modulators (SERMs) and an ER antagonist on drug-induced and chemical-induced liver injuries caused by acetaminophen, bromobenzene, diclofenac, and thioacetamide (TA). We observed hepatoprotective effects of EE2, tamoxifen (TAM) and raloxifene pretreatment in female mice that were exposed to a variety of hepatotoxic compounds. In contrast, the ER antagonist did not show any hepatoprotective effects. DNA microarray analyses suggested that monocyte to macrophage differentiation-associated 2 (Mmd2) protein, which has an unknown function, is commonly increased by TAM and RAL pretreatment, but not by pretreatment with the ER antagonist. In ERα-knockout mice, the hepatoprotective effects of TAM and the increased expression of Mmd2 mRNA were not observed in TA-induced liver injury. To investigate the function of Mmd2, the expression level of Mmd2 mRNA was significantly knocked down to approximately 30% in mice by injection of siRNA for Mmd2 (siMmd2). Mmd2 knockdown resulted in a reduction of the protective effects of TAM on TA-induced liver injury in mice. This is the first report of the involvement of ERα in drug-induced or chemical-induced liver injury. Upregulation of Mmd2 protein in the liver was suggested as the mechanism of the hepatoprotective effects of EE2 and SERMs.


Metabolic activation and inflammation reactions involved in carbamazepine-induced liver injury.

Drug-induced liver injury is a major safety concern in drug development and clinical pharmacotherapy; however, advances in the understanding of the mechanisms of drug-induced liver injury are hampered by the lack of animal models. Carbamazepine (CBZ) is a widely used antiepileptic agent. Although the drug is generally well tolerated, only a small number of patients prescribed CBZ develop severe hepatitis. In the present study, we developed a mouse model of CBZ-induced liver injury and elucidated the mechanisms accounting for the hepatotoxicity of CBZ. Male BALB/c mice were orally administered CBZ for 5 days. The plasma levels of alanine aminotransferase and aspartate aminotransferase were prominently increased, and severe liver damage was observed via histological evaluation. The analysis of the plasma concentration of CBZ and its metabolites demonstrated that 3-hydroxy CBZ may be relevant in CBZ-induced liver injury. The hepatic glutathione levels were significantly decreased, and oxidative stress markers were significantly altered. Mechanistic investigations found that hepatic mRNA levels of toll-like receptor 4, receptor for advanced glycation end products, and their ligands were significantly increased. Moreover, the plasma concentrations of proinflammatory cytokines were also increased. Prostaglandin E(1) administration ameliorated the hepatic injury caused by CBZ. In conclusion, metabolic activation followed by the stimulation of immune responses was demonstrated to be involved in CBZ-induced liver injury in mice.


Immunohistochemical characteristics and malignant progression of hepatic cystic neoplasms in comparison with pancreatic counterparts.

The recent World Health Organization classification for tumors of the digestive system defined grossly and histologically hepatic mucinous cystic neoplasms and intraductal papillary neoplasms of the bile duct separately. In this study, the immunohistochemical features of intraductal papillary neoplasm of the bile duct (19 cases) and hepatic mucinous cystic neoplasm (5 cases) were characterized and compared with those of similar pancreatic lesions, intraductal papillary mucinous neoplasm of the pancreas (12 cases), and pancreatic mucinous cystic neoplasm (6 cases) and with those of other biliary cystic lesions, peribiliary cysts (10 cases). Intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas frequently expressed cytokeratin 7; mucin core proteins 1, 2, 5AC, and 6; trypsin; and amylase. Hepatic and pancreatic mucinous cystic neoplasms frequently expressed cytokeratin 7, mucin core proteins 1 and 5AC, estrogen receptor, progesterone receptor, trypsin, and amylase. Estrogen and progesterone receptors were expressed in the subepithelial stromal cells. The groups with intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas were different from the groups with hepatic and pancreatic mucinous cystic neoplasm with respect to several phenotypes reflecting gastric and intestinal metaplasia and also the lack of expression of estrogen and progesterone receptors. The Ki-67 and p53 labeling indexes increased significantly with the malignant progression of intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas. The p16 labeling index decreased and EZH2 labeling index increased significantly with the malignant progression of intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas. In conclusion, intraductal papillary neoplasm of the bile duct and hepatic mucinous cystic neoplasm might be regarded as biliary counterparts of intraductal papillary mucinous neoplasm of the pancreas and pancreatic mucinous cystic neoplasm, respectively, and the mucinous cystic neoplasm and intraductal papillary neoplasm groups differed from each other. Labeling indexes of Ki-67, p53, p16, and EZH2 were comparable in intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas along with their malignant progression, suggesting a common carcinogenic process of the tumors.


The role of natural killer (NK) and NK T cells in the loss of tolerance in murine primary biliary cirrhosis.

One of the major obstacles in dissecting the mechanism of pathology in human primary biliary cirrhosis (PBC) has been the absence of animal models. Our laboratory has focused on a model in which mice, following immunization with a xenobiotic chemical mimic of the immunodominant autoepitope of the E2 component of pyruvate dehydrogenase complex (PDC-E2), develop autoimmune cholangitis. In particular, following immunization with 2-octynoic acid (a synthetic chemical mimic of lipoic acid-lysine located within the inner domain of PDC-E2) coupled to bovine serum albumin (BSA), several strains of mice develop typical anti-mitochondrial autoantibodies and portal inflammation. The role of innate immune effector cells, such as natural killer (NK) cells and that NK T cells, was studied in this model based on the hypothesis that early events during immunization play an important role in the breakdown of tolerance. We report herein that, following in-vivo depletion of NK and NK T cells, there is a marked suppression of anti-mitochondrial autoantibodies and cytokine production from autoreactive T cells. However, there was no change in the clinical pathology of portal inflammation compared to controls. These data support the hypothesis that there are probably multiple steps in the natural history of PBC, including a role of NK and NK T cells in initiating the breakdown of tolerance. However, the data suggest that adaptive autoimmune effector mechanisms are required for the progression of clinical disease.


The Radioprotective 105/MD-1 complex contributes to diet-induced obesity and adipose tissue inflammation.

Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesity-associated metabolic disorders.