Gut insulin action protects from hepatocarcinogenesis in diabetic mice comorbid with nonalcoholic steatohepatitis.

Diabetes is known to increase the risk of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Here we treat male STAM (STelic Animal Model) mice, which develop diabetes, NASH and HCC associated with dysbiosis upon low-dose streptozotocin and high-fat diet (HFD), with insulin or phlorizin. Although both treatments ameliorate hyperglycemia and NASH, insulin treatment alone lead to suppression of HCC accompanied by improvement of dysbiosis and restoration of antimicrobial peptide production. There are some similarities in changes of microflora from insulin-treated patients comorbid with diabetes and NASH. Insulin treatment, however, fails to suppress HCC in the male STAM mice lacking insulin receptor specifically in intestinal epithelial cells (ieIRKO), which show dysbiosis and impaired gut barrier function. Furthermore, male ieIRKO mice are prone to develop HCC merely on HFD. These data suggest that impaired gut insulin signaling increases the risk of HCC, which can be countered by restoration of insulin action in diabetes.

NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis.

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.

Preparation and culture of bone marrow-derived macrophages from mice for functional analysis.

The assessment of macrophage function has been a topic of intense discussion due to multiple subtypes. This protocol describes the collection of bone marrow cells from the femur and tibia of mice, differentiation into bone marrow-derived macrophages (BMDM cells), and sampling from cultures. This protocol focuses on the efficient preparation of BMDM cells, providing a way to assess the function of macrophages. For complete details on the use and execution of this protocol, please refer to Toda et al. (2020).

Insulin- and Lipopolysaccharide-Mediated Signaling in Adipose Tissue Macrophages Regulates Postprandial Glycemia through Akt-mTOR Activation.

The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.

Lung abscess without sepsis in a patient with diabetes with refractory episodes of spontaneous hypoglycemia: a case report and review of the literature.

INTRODUCTION: Hypoglycemia is a cause of considerable morbidity. Although hypoglycemia has been documented in the setting of septic shock and has been associated with higher mortality, hypoglycemia in infection without sepsis has not been reported in the literature. CASE PRESENTATION: A 72-year-old Japanese woman treated with high-dose glucocorticoids for autoimmune hemolytic anemia, as well as intensive insulin therapy for type 2 diabetes, presented with severe hypoglycemia. A lung abscess was diagnosed by imaging studies and treated with intravenous antibiotics. Hypoglycemia spontaneously recurred during lung abscess exacerbations, despite appropriate de-escalation of antidiabetic therapy. Only mild sporadic episodes of hypoglycemia occurred after the lung abscess was controlled. Infection accompanied with malnutrition and immunosuppression, although in the absence of sepsis, may have contributed to hypoglycemia. CONCLUSIONS: Caution is warranted in the management of hypoglycemia in patients with diabetes with the conditions described here, that is malnutrition and immunosuppression, as infection may be a contributing factor.

The efficacy of ursodeoxycholic acid and bezafibrate combination therapy for primary biliary cirrhosis: A prospective, multicenter study.

AIM: Treatment with ursodeoxycholic acid (UDCA) improves the survival of stage I and II primary biliary cirrhosis (PBC) patients. However, new therapeutic options are needed for patients who are refractory to UDCA and for those whose disease is at an advanced stage. Bezafibrate could be useful in PBC treatment, since it increases phospholipid output into the bile and reduces the cytotoxicity of hydrophobic bile acids, which are increased with cholestasis. METHODS: We conducted two prospective, multicenter randomized open studies in non-cirrhotic patients with PBC to evaluate the efficacy of bezafibrate. One study compared UDCA and bezafibrate monotherapy (study 1: 45 patients [37 females], mean age 55.9 years), and the other evaluated the addition of bezafibrate to patients who were refractory to UDCA (study 2: 21 patients [18 females], mean age 54.1 years). RESULTS: Study 1 demonstrated that bezafibrate monotherapy was as effective as UDCA and study 2 revealed that bezafibrate combined with UDCA was effective in improving and maintaining biliary enzymes where the ineffectiveness of long-term treatment with UDCA was confirmed. CONCLUSION: This multicenter, randomized, open study revealed that combination therapy of bezafibrate and UDCA improved biliary enzymes in non-cirrhotic Japanese patients with PBC refractory to UDCA. Further studies are needed to evaluate whether combination therapy improves histological staging and prognosis.

Outcome and prognostic factors of 391 Japanese patients with primary sclerosing cholangitis.

OBJECTIVES: We performed a national survey in 2003, and demonstrated characteristic features of primary sclerosing cholangitis (PSC) patients in Japan. In this study, we aimed to clarify the outcome and prognostic factors of Japanese PSC patients. METHODS: Questionnaires were sent to gastroenterologists in Japan, and 391 patients with PSC were registered and enrolled in the current study. The median follow-up was 5.3 years (range 0.1-20.8 years). The cumulative incidence for survival was analysed using the Kaplan-Meier method. Univariate and multivariate analyses were performed using the Cox-proportional hazards regression model for determining prognostic variables. RESULTS: The estimated median survival of all patients was 13.1 years, with a 5-year survival rate of 74.5%. Thirty-eight patients (9.7%) who underwent liver transplantation (LT) had a 5-year survival rate of 92.0%. Both univariate and multivariate analysis demonstrated that younger age [below 49 years old; odds ratio (OR)=1.76, 1.12-2.76, P=0.0136] and lower total bilirubin (below 3.0 mg/dl; OR=2.50, 1.60-3.89, P

Fulminant hepatitis and late onset hepatic failure in Japan.

AIM: A nationwide survey was performed to clarify the present state of fulminant hepatitis and late onset hepatic failure (LOHF) between 1998 and 2003 in Japan. METHODS: Three hundred and sixteen, 318 and 64 patients, respectively, with acute and subacute types of fulminant hepatitis and LOHF, in which grade II or more severe hepatic encephalopathy occurred within 10 days, between 11 days and 8 weeks and between 8 and 24 weeks, respectively, after the onset of disease symptoms, were analyzed. RESULTS: Complications such as metabolic syndrome were underlying in 41.5% of patients with subacute fulminant hepatitis and 51.6% of patients with LOHF, and most of such patients had received daily medications. The etiology of fulminant hepatitis was viral infection in 71.2% of the acute type and 31.8% in the subacute type. Hepatitis B virus (HBV) infection was found in most of these patients; transient infection prevailed in the acute type; and HBV carrier prevailed in the subacute type. The etiology was unknown in 42.8% and 53.1% of the subacute type and LOHF, respectively. Autoimmune hepatitis and drug allergy-induced liver injury were found in 10.7% and 11.3%, respectively, of the subacute type. Artificial liver support with plasma exchange and/or hemodiafiltration took place in more than 90% of all patients. The survival rates of the patients without liver transplantation were 53.7% in the acute and 24.4% in the subacute type, and 11.5% in LOHF. The prognosis was especially poor in HBV carriers and patients with autoimmune hepatitis. The survival rates of those who underwent liver transplantation were 56.3%, 39.3% and 23.4% in the acute type, subacute type and LOHF, respectively. CONCLUSION: The etiology and prognosis differed in patients with fulminant hepatitis and LOHF depending on the disease types in Japan, and liver transplantation improved the prognosis of the patients irrespective of the disease type and etiology.

A large-scale, multicentre, double-blind trial of ursodeoxycholic acid in patients with chronic hepatitis C.

BACKGROUND: Combined pegylated interferon and ribavirin has improved chronic hepatitis C (CH-C) therapy; however, sustained virological response is achieved in only about half of the patients with a 1b genotype infection. We assessed oral ursodeoxycholic acid (UDCA) on serum biomarkers as a possible treatment for interferon non-responders. METHODS: CH-C patients with elevated alanine aminotransferase (ALT) were assigned randomly to 150 (n = 199), 600 (n = 200) or 900 mg/day (n = 197) UDCA intake for 24 weeks. Changes in ALT, aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT) were assessed. This study is registered at ClinicalTrial.gov, identifier NCT00200343. RESULTS: ALT, AST and GGT decreased at week 4 and then remained constant during drug administration. The median changes (150, 600 and 900 mg/day, respectively) were: ALT, -15.3, -29.2 and -36.2%; AST, -13.6, -25.0 and -29.8%; GGT, -22.4, -41.0 and -50.0%. These biomarkers decreased significantly less in the 150 mg/day than in the other two groups. Although changes in ALT and AST did not differ between the 600 and 900 mg/day groups, GGT was significantly lower in the 900 mg/day group. In subgroup analysis, ALT decreased significantly in the 900 mg/day group when the baseline GGT exceeded 80 IU/l. Serum HCV-RNA did not change in any group. Adverse effects were reported by 19.1% of the patients, with no differences between groups. CONCLUSIONS: A 600 mg/day UDCA dose was optimal to decrease ALT and AST levels in CH-C patients. The 900 mg/day dose decreased GGT levels further, and may be preferable in patients with prevailing biliary injuries.

Interferon-gamma produced by interleukin-12-activated tumor infiltrating CD8+T cells directly induces apoptosis of mouse hepatocellular carcinoma.

BACKGROUND/AIMS: Interleukin-12 (IL-12), a cytokine with antitumor activity, was examined for the suppressive effect on hepatocellular carcinoma (HCC) in mouse model, and its mechanism of antitumor activity was analyzed. METHODS: Mice implanted with MIH-2 HCC cells were treated with recombinant mouse IL-12 (500 ng/mouse). Involvement of CD4(+), CD8(+), NK cells and interferon (IFN)-gamma on tumor suppression by IL-12 was examined by treatment of mice with each antibody. Interferon-gamma (IFN-gamma) production by tumor infiltrating cells was analyzed by immunofluorescence microscopy and flow cytometric analysis. Signal transduction for apoptosis induction was examined by immunoblot analysis. RESULTS: The growth of implanted MIH-2 tumors was significantly suppressed by IL-12 and the suppression was inhibited by depletion of CD8(+)T cells. IL-12 treatment caused numerous IFN-gamma-producing CD8(+)T cells to infiltrate into MIH-2 tumors. Antitumor activity of IL-12 was blocked by treating mice with anti-IFN-gamma mAb. CD8(+)T cells from IL-12-treated mice attached to MIH-2 cells and produced IFN-gamma in vitro. Cell attachment might be associated with intercellular adhesion molecule-1 induced by IFN-gamma. In vitro treatment with IFN-gamma induced apoptosis of MIH-2 cells via a mitochondria-dependent pathway. CONCLUSIONS: IL-12 suppressed HCC growth in mouse model. IFN-gamma produced by IL-12-activated tumor-infiltrating CD8(+)T cells directly induced apoptosis of HCC cells.

Antimitochondrial antibody negative primary biliary cirrhosis in Japan: utilization of clinical data when patients applied to receive public financial aid.

BACKGROUND: We examined patients who showed laboratory and histological evidence of primary biliary cirrhosis (PBC) in the absence of antimitochondrial antibody (AMA) to elucidate the characteristics of AMA negative PBC. METHODS: From a total of 5,805 patients with symptomatic PBC, 2,419 cases (41.7%) were selected in the present study, who were diagnosed using the following criterion; chronic non-suppurative destructive cholangitis was histologically observed and laboratory data did not contradict PBC. The information collected from records included sex, age, symptoms, physical findings, and complicated autoimmune diseases. We then evaluated these data according to the positivity of AMA. RESULTS: Of the total subjects, 470 cases (19.4%) were found to be negative for AMA. The proportion of female patients was higher among the AMA negative group than among the AMA positive one. Pruritus was found less frequently among patients with AMA negative PBC than among those with AMA positive PBC. Levels of alkaline phosphatase,gamma-glutamyl transpeptidase, and IgM were significantly lower among patients with AMA negative PBC than among those with AMA positive PBC. Complications such as Sjögren's syndrome, rheumatoid arthritis, and scleroderma, including CREST syndrome, were found with significantly higher frequency among patients with AMA negative PBC than among those with AMA positive PBC. CONCLUSION: Considering serum level of IgM and frequencies of complicated autoimmune diseases, it is possible that Japanese patients with AMA negative PBC are consistent with the disease entity of autoimmune cholangitis reported in western countries.

Diagnosing clinical subsets of autoimmune liver diseases based on a multivariable model.

BACKGROUND: Diagnosing autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis, and other autoimmune liver diseases remains an imperfect process. We need a more accurate, evidence-based diagnostic system. METHODS: We conducted a national survey and identified 988 cases of liver disease which did not satisfy the inclusion criteria for any liver disease of known etiology. We expected these cases to include autoimmune liver disease (AILD) and its variant forms. We selected 269 prototype cases for which histological re-evaluation of liver biopsy by independent expert hepatopathologists and the original diagnosis coincided. We did a multiple logistic regression analysis to determine explanatory variables that would distinguish cases of AIH and PBC from those of non-AIH and non-PBC, respectively. We constructed a multivariable diagnostic formula that gave AIH and PBC disease probabilities and validated it in a study of an additional 371 cases (validation group). RESULTS: Based on the results of the statistical analysis, we selected three laboratory tests and four histological features as independent variables correlated to the diagnosis of both AIH and PBC. For the validation group, assuming that the original diagnosis was correct, the sensitivity and specificity for AIH were 86.3% and 92.4%, respectively. For PBC the sensitivity and specificity were 82.5% and 63.7%, respectively. A detailed analysis of inconsistent cases showed that the diagnosis based on the formula had given the correct diagnosis, for either AIH or PBC, except for 5 cases (1.3%) in which disease probability was low for both. CONCLUSIONS: A seven-variable formula based on three laboratory tests and four histological features gives significant information for the diagnosis of AILD.

Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancer-specific antigens and induce antitumor immunity against autologous tumor cells.

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2(-)/HLA-24(-)), carcinoembryonic antigen (CEA)(+), and MUC1(+) as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2(+) and/or HLA-A24(+)) and allogeneic COLM-6 resulted in MHC class I- and MHC class II-restricted proliferation of CD4(+) and CD8(+) T cells, high levels of IFN-gamma production in both CD4(+) and CD8(+) T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

Autoimmune hepatic inflammation by vaccination of mice with dendritic cells loaded with well-differentiated hepatocellular carcinoma cells and administration of interleukin-12.

Vaccination of mice with dendritic cells loaded with Hepa1-6, well-differentiated hepatocellular carcinoma cell line (DC/Hepa1-6), induced cytotoxic T lymphocytes against Hepa1-6. Liver-specific inflammation was generated by vaccination of mice with DC/Hepa1-6 and subsequent administration of interleukin (IL)-12. Vaccination with DCs loaded with MC38 or B16 and administration of IL-12 did not generate significant liver-specific inflammation. Splenic T cells from DC/Hepa1-6-vaccinated mice showed proliferative response by stimulation with S-100 protein of the liver and showed cytotoxic activity to hepatocytes. Hepatic mononuclear cells from DC/Hepa1-6 + IL-12-treated mice also showed cytotoxic activity to hepatocytes. Adoptive transfer of splenocytes from DC/Hepa1-6-vaccinated mice produced hepatic inflammation in recipient mice that had been pretreated with IL-12. IL-12 upregulated the expression of adhesion molecules and chemokines in the liver. In conclusion, CTLs responsive to hepatocytes induced by DC/Hepa1-6 and enhanced expression of adhesion molecules and chemokines in the liver by IL-12 would produce autoimmune hepatic inflammation.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12.

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.