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.

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.

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.

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).

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.

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.

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.

Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine.

Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APC(Min-/+), in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4(+) T cells, not CD8(+) T cells, by the intraperitoneal administration of corresponding mAb's decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.

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.

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.

Are bile duct lesions of primary biliary cirrhosis distinguishable from those of autoimmune hepatitis and chronic viral hepatitis? Interobserver histological agreement on trimmed bile ducts.

BACKGROUND: Primary biliary cirrhosis (PBC) is histopathologically characterized by chronic nonsuppurative destructive cholangitis and ductopenia of interlobular bile ducts. Bile duct injury is also often encountered in chronic viral hepatitis (CVH) and in autoimmune hepatitis (AIH). METHODS: In this study, we performed interobserver agreement analysis on 90 injured bile ducts from liver specimens of PBC (17 cases), CVH (26 cases), and AIH (18 cases), with 30 bile ducts chosen from each disease group. Digital images of bile ducts with minimal periductal elements were recorded in CD-ROM format and sent to 14 observers (six special hepatopathologists, four local hepatopathologists, and four general pathologists). We analyzed the following issues: (1) diagnostic accuracy of PBC, based only on bile duct lesions; (2) classification of bile duct lesions in AIH cases as destructive cholangitis equivalent to PBC-associated injury, or not. RESULTS: The diagnostic accuracy of PBC cases with severe bile duct injuries was very high (over 80%), although the accuracy in cases with only mild bile duct injuries was low (50% or less). For AIH, each observer classified 9 of the 30 bile ducts, on average, as destructive cholangitis. CONCLUSIONS: This study revealed that 66.9% of PBC cases could be diagnosed based on trimmed bile ducts alone. Bile duct injury similar to that in PBC could be encountered in AIH.

Role of nitric oxide (NO) in interferon-alpha therapy for hepatitis C.

BACKGROUND AND AIM: The role of nitric oxide in infectious disease is gaining increased attention because antiviral effects of nitric oxide. In addition, there is evidence that nitric oxide synthase-2 expression was noted in chronic hepatitis C found within mononuclear cells. METHODS: We studied serum levels of nitrite and nitrate before and during interferon alpha therapy in 66 patients with chronic hepatitis C. RESULTS: There was no significant difference of their levels between the healthy control subjects and the patients before the treatment with interferon (55.9+/-21.8 microM vs. 60.9+/-30.0 microM). Their levels were determined at 2 weeks after the initiation of treatment with interferon and compared with those before the treatment in the patients with chronic hepatitis C. In the total patients treated, there was no significant difference between their levels before and at 2 weeks after the treatment (60.9+/-30.0 microM vs. 65.5+/-30.0 microM, P=0.14). However, when the levels were compared between sustained responders, in whom hepatitis C virus was eradicated, and non-responders, in whom the virus was not eradicated, the former had significantly higher levels of nitrite and nitrate than the latter at 2 weeks after the initiation of treatment (83.7+/-40.9 microM vs. 57.6+/-19.5 microM, P<0.01). The multivariate logistic regression analysis showed that the rise of nitrite and nitrate was an independent predictive factor for efficacy of interferon treatment. CONCLUSIONS: Nitric oxide may be an important factor for antiviral therapy by interferon treatment for chronic hepatitis C, which suggests an additional therapeutic pathway for further study.

[Edema in liver disease].

Patients with advanced cirrhosis show an abnormal regulation of extracellular fluid volume, resulting in the accumulation of fluid as ascites or edema. As portal hypertension develops, splanchnic arterial vasodilation also does due mainly to the production of nitric oxide (NO). Splanchnic arterial vasodilation decreases effective arterial blood volume, leading to fluid accumulation and renal function abnormalities which are a consequence of the homeostatic activation of vasoconstrictor and antinatriuretic factors. And the net effect is retention of sodium and water as well as renal vasoconstriction. The portal hypertension and splanchnic hyperdynamic circulation elevate the pressure of the splanchnic capillary circulation, leading to the accumulation of retained fluid as ascites.

Immunotherapy using fusions of autologous dendritic cells and tumor cells showed effective clinical response in a patient with advanced gastric carcinoma.

Immunotherapy using tumor antigen-loaded dendritic cells is a new approach for the treatment of various types of malignant tumors. Here, we describe a patient with advanced gastric carcinoma who received immunotherapy using fused autologous dendritic cells and carcinoma cells (fusions) and showed effective clinical responses to the treatment. A 74-year-old man showed massive ascitic effusion due to peritonitis carcinomatosa after surgical operation for gastric carcinoma. A gastric carcinoma cell line was established from the patient's tumor tissue. Dendritic cells were obtained by cultivation of the adherent cell fraction of the patient's peripheral blood mononuclear cells (PBMCs) with granulocyte macrophage-colony stimulating factor, interleukin-4, and tumor necrosis factor-alpha. The cells were mixed with irradiated tumor cells and treated with 50% polyethyleneglycol (PEG) for the generation of fusions, as described previously. The PEG-treated cells were injected subcutaneously every 2 weeks. Low-grade fever was observed after the first and second treatments. After the third treatment, ascitic effusion and leg edema decreased markedly, without any other treatments. Serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 decreased to levels lower than those at the initiation of treatment. PBMCs collected after the fifth treatment elicited cytotoxic activity against autologous tumor cells. Although treatment was continued in the same way, recurrence of the disease was observed about 5 months after the start of the treatment. This is the first report of immunotherapy utilizing fusions of autologous dendritic cells and tumor cells resulting in effective clinical responses in advanced gastric carcinoma, without severe adverse effects.

Potential role of Helicobacter pylori in hepatocarcinogenesis.

Helicobacter species can induce carcinoma in the liver of certain mice. Furthermore, Helicobacter pylori (H. pylori) exhibits hepatotoxicity in vitro. These reports indicate that H. pylori may play a role in hepatocarcinogenesis. The aim of this study was to assess the presence of H. pylori in human hepatocellular carcinoma (HCC) to determine if H. pylori may affect the development of this disease. Liver specimens from 15 HCC patients dissected into tumor and non-tumor tissues were examined for H. pylori by PCR using two sets of primers for 16S rRNA and urease B. DNA sequencing analysis was performed to confirm that PCR products with 16S rRNA primers were derived from H. pylori DNA. The specimens were also examined for H. pylori by immunohistochemistry using anti-H. pylori antibody. H. pylori was found in 13 of 15 tumor tissues, not in the non-tumor tissues. By contrast, Escherichia coli and Bacteroides fragilis, frequent colonizers of gut, were not detected by PCR in the HCC tumors. Ten cirrhotic liver tissue specimens and seven normal liver tissue specimens were also negative for H. pylori DNA by PCR. The nucleotide sequence of the amplified fragment shared 100% identity with the 16S rRNA gene of H. pylori. H. pylori was also detected in HCC tissue by immunohistochemical analysis. The presence of H. pylori in human HCC tissue was demonstrated by PCR and immunohistochemical analysis. These findings suggest that H. pylori might contribute to the development of HCC. Further study is needed to prove the pathogenetic role of H. pylori in the development of human HCC.

Effect of oral branched chain amino acid supplementation in the late evening on the nutritional state of patients with liver cirrhosis.

Previous studies have shown that nocturnal glucose supplementation and a late evening meal reduced raised protein turnover rates and led to a better nitrogen balance in patients with cirrhosis. In this study, we investigated whether or not oral branched chain amino acid (BCAA) supplementation in the late evening would improve the nutritional state of patients with liver cirrhosis. Fourteen patients with liver cirrhosis spent two 14 day periods in the ward. All the patients received three meals a day and two doses of BCAA supplementation. Meals were given at 0800, 1200, and 1830. BCAA supplementation was given at 0830 and 1900 (after dinner) and at 0830 and 2230 (late evening), respectively. The daily excretion of 3-methylhistidine, the ratio of 3-methyhistidine to creatinine, and serum free fatty acid in the late evening treatment group were significantly lower as compared to the usual treatment group. These results suggest that oral BCAA supplementation in the late evening also may be useful in improving protein catabolism and lypolysis in cirrhotic patients.