Immunosuppressive functions of hepatic myeloid-derived suppressor cells of normal mice and in a murine model of chronic hepatitis B virus.

The immunosuppressive state of tumour-bearing hosts is attributable, at least in part, to myeloid-derived suppressor cells (MDSC). However, the role of MDSC in physiological conditions and diseases other than cancer has not been addressed. As the liver is a tolerogenic organ, the present study attempted to localize and assess functions of hepatic MDSC in a normal liver and in a murine model of chronic hepatitis B virus (HBV) infection. MDSC was identified in the liver of normal mice and HBV transgenic mice (TM) as CD11b(+) Gr1(+) cells by dual-colour flow cytometry. Highly purified populations of MDSC and their subtypes were isolated by fluorescence-activated cell sorting. The functions of MDSC and their subtypes were evaluated in allogenic mixed lymphocyte reaction (MLR) and hepatitis B surface antigen (HBsAg)-specific T cell proliferation assays. Normal mice-derived liver MDSC, but not other myeloid cells (CD11b(+) Gr1(-) ), suppressed T cell proliferation in allogenic MLR in a dose-dependent manner. Alteration of T cell antigens and impaired interferon-γ production seems to be related to MDSC-induced immunosuppression. In HBV TM, the frequencies of liver MDSC were about twice those of normal mice liver (13·6±3·2% versus 6·05±1·21%, n=5, P<0·05). Liver-derived MDSC from HBV TM also suppressed proliferative capacities of allogenic T cells and HBsAg-specific lymphocytes. Liver MDSC may have a critical role in maintaining homeostasis during physiological conditions. As liver MDSC had immunosuppressive functions in HBV TM, they may be a target of immune therapy in chronic HBV infection.

Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B.

The immune modulator capacity of antigen-pulsed dendritic cells (DC) has been documented in patients with cancers and in animal models of chronic viral infections. Cancer antigen-pulsed DC are now used for treating patients with cancer. But viral antigen-pulsed DC are not used in chronic viral-infected patients because safety of antigen-pulsed DC has not been evaluated in these patients. DC were isolated from human peripheral blood mononuclear cells by culturing with human-grade granulocyte-macrophage colony stimulating factor and interleukin-4. Human blood DC were cultured with hepatitis B surface antigen (HBsAg) for 8h to prepare HBsAg-pulsed DC. After immunogenicity assessment of HBsAg-pulsed DC in vitro, five million HBsAg-pulsed DC were administered intradermally to five patients with chronic hepatitis B (CHB) 1-3 times. HBsAg-pulsed DC were immunogenic in nature because they produced significantly higher levels of interleukin-12 and interferon-γ compared to unpulsed DC (P<0.05). Also, HBsAg-pulsed DC induced proliferation of HBsAg-specific T lymphocytes in vitro. CHB patients injected with HBsAg-pulsed DC did not exhibit generalized inflammation, exacerbation of liver damage, abnormal kidney function, or features of autoimmunity. Administration of HBsAg-pulsed DC induced anti-HBs in two patients and HBsAg-specific cellular immunity in 1 patient. This is the first study about preparation of antigen-pulsed DC using human consumable materials for treating patients with CHB. Because HBsAg-pulsed DC were safe for all patients with CHB and had immune modulation capacity in some patients, phase I and phase II clinical trials with antigen-pulsed DC in CHB and other chronic infections are warranted.

Mechanism of restoration of immune responses of patients with chronic hepatitis B during lamivudine therapy: increased antigen processing and presentation by dendritic cells.

Restoration of host immunity has been reported in patients with chronic hepatitis B (CHB) after treatment with lamivudine; however, the underlying mechanisms of this treatment have not been determined. This study examined the role of antigen-presenting dendritic cells (DC) in restoration of host immunity. Circulating DC were isolated from peripheral blood of 23 patients with CHB before and 1, 3, and 12 months after starting lamivudine therapy. The non-antigen-specific proliferation of DC was assessed in allogenic mixed leucocyte reaction. Dendritic cells were cultured with hepatitis B surface antigen (HBsAg) to prepare HBsAg-pulsed DC. Proliferative capacity and production of interleukin (IL)-12 and interferon (IFN)-γ of HBsAg-pulsed DC were evaluated. Circulating unpulsed DC and HBsAg-pulsed DC showed significantly higher levels of T-cell proliferation capacities 1 month after lamivudine therapy compared to proliferation levels before therapy (P<0.05). HBsAg-pulsed DC also produced significantly higher levels of IL-12 and IFN-γ with lamivudine therapy compared to levels before therapy (P<0.05). HBsAg-pulsed DC from lamivudine-treated patients induced proliferation of T cells of patients with CHB in an antigen-specific manner (P<0.05). However, T-cell stimulatory capacity of DC did not increase significantly 3 and 12 months after lamivudine therapy compared to 1 month after lamivudine therapy. Immune restoration as a result of lamivudine therapy is regulated at least in part by activation of DC. However, progressive activation of DC was not seen as treatment duration progressed, indicating the limitations of this mechanism of viral clearance.

Impaired dendritic cell functions because of depletion of natural killer cells disrupt antigen-specific immune responses in mice: restoration of adaptive immunity in natural killer-depleted mice by antigen-pulsed dendritic cell.

The primary aim of this study was to evaluate the role of natural killer (NK) cells on antigen-specific adaptive immune responses. After analysing the mechanism of impaired adaptive immune responses of NK-depleted mice, an immune interventional approach was developed to restore adaptive immunity in NK-depleted mice. NK cells were depleted from mice by administration of anti-asialo GM1 antibody (100 mul/mouse), twice, at an interval of 48 h. Hepatitis B surface antigen (HBsAg) was administered intraperitoneally to normal C57BL/6 mice (control mice) and NK-depleted mice. The levels of antibody to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen were assessed. The functions of T lymphocytes, B lymphocytes and dendritic cells (DCs) were evaluated in vitro. HBsAg-pulsed DCs were prepared by culturing spleen DCs with HBsAg for 48 h and administered once to NK-depleted mice. The levels of anti-HBs in the sera and HBsAg-specific lymphocytes were significantly lower in NK-depleted mice compared with control mice (P < 0.05). The functions of T and B lymphocytes were similar between control mice and NK-depleted mice. However, the functions of spleen DC and liver DC were significantly lower in NK-depleted mice compared with control mice (P < 0.05). Administration of HBsAg-pulsed DCs, but not HBsAg, induced HBsAg-specific humoral and cellular immune responses in NK-depleted mice. Our study suggests that cross-talk between NK cells and DCs regulates the magnitude of adaptive immunity. In addition, antigen-pulsed immunogenic DCs represent potent immune modulator even if subjects with diminished innate immunity.

Diabetic peripheral neuropathy and prevalence of erectile dysfunction in Japanese patients aged <65 years with type 2 diabetes mellitus: The Dogo Study.

Only limited epidemiological evidence exists regarding the relationship between diabetic neuropathy and erectile dysfunction (ED) among Japanese patients with type 2 diabetes mellitus. To investigate the relationship between diabetic neuropathy and ED among Japanese patients with type 2 diabetes mellitus, a multicenter cross-sectional study was conducted in 287 male Japanese patients with type 2 diabetes mellitus, age (19-65 years). Diabetic neuropathy was diagnosed if the patients showed two or more of the following three characteristics: neuropathic symptoms, decreased or disappeared Achilles tendon reflex and/or abnormal vibration perception. ED, moderate to severe ED, and severe ED were defined as present when a subject had a Sexual Health Inventory for Men score <22, <12 and <8, respectively. The prevalence values of diabetic neuropathy and severe ED were 47.0 and 39.0%, respectively. Diabetic neuropathy was independently positively associated with severe ED, but not ED and moderate ED: the adjusted odds ratio was 1.90 (95% confidence interval: 1.08-3.38). No relationships were found between diabetic retinopathy or diabetic nephropathy and ED. Diabetic neuropathy is positively associated with severe erectile dysfunction among Japanese type 2 diabetes mellitus patients aged <65 years.

Depressive symptoms and prevalence of erectile dysfunction in Japanese patients with type 2 diabetes mellitus: the Dogo Study.

In several studies of patients with type 2 diabetes mellitus, a positive association between depressive symptoms and erectile dysfunction (ED) has been reported. No evidence exists, however, regarding the association between depressive symptoms and ED among Japanese patients with type 2 diabetes mellitus. Thus, we examined this issue among Japanese patients with type 2 diabetes mellitus. Study subjects were 469 male Japanese patients with type 2 diabetes mellitus, aged 19 years or over. ED, moderate to severe ED and severe ED were defined as present when a subject had a Sexual Health Inventory for Men score <22, <12 and <8, respectively. Depressive symptoms were defined as present when a subject had a Self-Rating Depression Scale (SDS) score >49. Adjustment was made for age, body mass index, waist, duration of type 2 diabetes, current smoking, current drinking, hypertension, dyslipidemia, coronary artery disease, stroke, glycated hemoglobin and diabetic neuropathy. The prevalence values of depressive symptoms, moderate to severe ED and severe ED were 15.1%, 64.2% and 51.0%, respectively. Depressive symptoms were independently positively associated with moderate to severe ED and severe ED (adjusted odds ratios were 2.23 (95% confidence interval (CI): 1.17-4.43) and 1.86 (95% CI: 1.04-3.41), respectively). In Japanese patients with type 2 diabetes mellitus, depressive symptoms may be associated with ED.

Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis.

Mature and activated dendritic cells (CD83-positive DCs) are essential for the recruitment and survival of activated tumor-specific lymphocytes during carcinogenesis. The frequencies of CD83 positive DCs were almost same in peripheral blood from patients with hepatocellular carcinoma (HCC) and cirrhosis of liver (LC). However, the numbers of CD83 positive DCs in liver tissues were significant lower in HCC compared with LC (6.6+/-10.9 vs. 33.3+/-24 DCs/specimen, P<0.05). Most importantly, there were no CD83-positive DCs at cancer nodules in HCC. A role of infiltration of activated DCs in liver during hepatocarcinogenesis is shown.

Macrophage migration inhibitory factor in hepatocellular carcinoma and liver cirrhosis; relevance to pathogenesis.

The levels of macrophage migration inhibitory factor (MIF), a proinflammatory and carcinogenic cytokine, were significantly higher in the sera from patients with hepatocellular carcinoma (HCC; 25.6+/-15.3 ng/ml, n=55) and liver cirrhosis (LC; 18.9+/-10.7 ng/ml, n=26) compared with sera from patients with gastrointestinal cancer (6.8+/-7.5 ng/ml, n=29) and normal controls (5.6+/-1.2 ng/ml, n=45; P<0.01). Hepatocytes from patients with LC and HCC, but not from chronic hepatitis, expressed very high levels of MIF. A possible association between overexpression of MIF and hepatocarcinogenesis is suggested.

Increased levels of serum interleukin-12 in Graves' disease.

We investigated serum total interleukin-12 (IL-12) levels in patients with Graves' disease and Hashimoto's thyroiditis. The serum IL-12 levels in Graves' disease were significantly increased in the hyperthyroid state, and were decreased during treatment with methimazole or propylthiouracil in accordance with the decline of free tri-iodothyronine (T(3)) levels, free thyroxine levels and thyroid-binding inhibiting immunoglobulin (TBII) levels. When T(3) was administered orally to normal subjects, serum IL-12 levels were slightly increased. These results suggest that IL-12 might be increased due to prolonged stimulation with thyroid hormone, and thyroid hormone by itself might be a self-perpetuating factor of Graves' disease via increased IL-12 production.

Dendritic cells produce interleukin-12 in hyperthyroid mice.

We previously reported that serum interleukin-12 (IL-12) levels were significantly increased in patients with hyperthyroid Graves' disease and in normal subjects after administration of thyroid hormone. In the present study, we investigated which cells produce IL-12 and the interactions between IL-12 and thyroid hormones, using a hyperthyroid mouse model. Thyroid hormones induced IL-12 production, and IL-12 was mainly produced by dendritic cells outside the thyroid glands in a hyperthyroid state.

In situ characterization of dendritic cells occurring in the islets of nonobese diabetic mice during the development of insulitis.

Type 1 diabetes mellitus in nonobese diabetic (NOD) mice, a well-known model of human type 1 diabetes, has been considered to be caused by the destruction of insulin-producing beta cells in the islets of the pancreas by self-reactive T cells. Antigen-presenting cells like dendritic cells (DCs) and macrophages are expected to be involved in the processes from their role in generating regulatory or effector T cells. These immunohistochemical studies revealed that CD11c-positive DCs already appeared in the islets of NOD mice as early as 4 weeks old when lymphocytes were not yet infiltrated in the islet, and thus insulitis was not developed. DCs were first observed to locate around swollen parainsular vessels. From age 7 weeks onward to age 13 weeks, more DCs were present in parainsular areas where lymphocytes had also accumulated, and the number of DCs in the islets as well as lymphocytes increased. However, at the end stage of insulitis from age approximately 17 weeks onward, the number of DCs in the islets decreased. In contrast, accumulation of DCs in the para- and periislets was not observed in 7- and 17-week-old ICR female mice that do not develop type 1 diabetes. Double-staining studies using confocal laser scanning microscopy showed that the CD11c-positive DCs coexpress both major histocompatibility (MHC) class II and costimulatory molecules, CD80 and CD86. Electron-microscopy studies further demonstrated that cell bodies and processes of the DCs make close contact with lymphocytes. These results suggest that DCs infiltrated into the pancreatic islets are capable of stimulating T cells by the MHC class II-antigenic peptide complex, together with costimulatory molecules, which eventually lead to the beta-cell destruction in NOD mice.

Effect of rabeprazole on histamine synthesis in enterochromaffin-like cells of mast cell-deficient (Ws/Ws) rats.

The effect of rabeprazole, the latest proton pump inhibitor, on the serum gastrin concentration, histidine decarboxylase activity and histamine content of the oxyntic mucosa in Wistar rats, mast cell-deficient (Ws/Ws) rats, and their normal type, +/+, rats was investigated. In Wistar rats, 2 weeks of treatment with rabeprazole (30 mg/kg/day, s.c.) induced a 1.8-fold increase in serum gastrin concentration and a 3.9-fold increase in histidine decarboxylase activity of the oxyntic mucosa over the control levels, whereas neither 2- nor 4-week treatment affected the histamine content of the oxyntic mucosa. In Ws/Ws and +/+ rats, the serum gastrin concentration, histidine decarboxylase activity and even histamine content of the oxyntic mucosa were increased significantly as compared with control levels after the 4-week treatment with rabeprazole. Immunohistochemistry using a histamine antibody confirmed the increase in the histamine content of the oxyntic mucosa after the 4-week treatment with rabeprazole. The finding that there were no differences in serum gastrin concentration and histidine decarboxylase activity between Ws/Ws and +/+ rats, both with and without the 4-week treatment, indicates that mast cells do not respond to endogenous hypergastrinemia elicited by acid-inhibitory treatment. Moreover, the present study clarified for the first time that enterochromaffin-like (ECL) cells in Ws/Ws rats synthesize and store histamine in response to gastrin.

Increased serum levels of macrophage migration inhibitory factor in alcoholic liver diseases and their expression in liver tissues.

OBJECTIVES: To study the role of macrophage migration inhibitory factor (MIF) in the pathogenesis of alcoholic liver diseases. DESIGN AND METHODS: The levels of MIF in the sera were estimated by an enzyme-linked immunosorbent assay in 13 patients with alcoholic hepatitis (ALH), 9 patients with alcoholic cirrhosis (ALC) and 26 normal controls. MIF was localized in the liver specimens by immunohistochemistry. RESULTS: The mean levels of MIF in the sera were significantly higher in ALH and ALC compared with the normal controls (p < 0.05). Serial observations revealed a relationship between serum MIF levels and the serum transaminase levels. MIF was expressed by the hepatocytes and by the infiltrating cells around the site of accumulation of neutrophils and ballooned hepatocytes in ALH. CONCLUSIONS: This is the first report on MIF in human alcoholic liver diseases, and the data suggest that MIF may be related to abnormal cytokine homeostasis in ALH.

Soluble intercellular adhesion molecule-1 in serum in chronic hepatitis B and C.

Intercellular adhesion molecule-1 (ICAM-1) is expressed on the hepatocyte membrane in patients with chronic hepatitis B and C. We assayed levels of the soluble form of this molecule (sICAM-1) in serum by an enzyme-linked immunosorbent assay method; we then analyzed the results in relation to hepatitis activity. Fifty-one patients with chronic hepatitis (22 with type B and 29 with type C) and 10 normal controls were examined. The serum levels of sICAM-1 in hepatitis B and C were significantly higher (P < 0.01) than in normal controls. The serum level of sICAM-1 was correlated with serum glutamic-pyruvic transaminase level (P < 0.01), and the level of sICAM-1 in patients in whom exacerbation was seen was significantly higher (P < 0.05) than that in patients in a remission. There was no relationship between the serum level of sICAM-1 and the degree of ICAM-1 expression on the hepatocyte membrane. These results suggest that the serum level of sICAM-1 reflects the degree of activity of chronic hepatitis B and C.

Amyloidosis localized in the sigmoid colon.

A 79-year-old woman was admitted to our hospital presenting with bloody stool. Colonoscopic examination showed many irregularly-shaped discrete ulcerations in the sigmoid colon extending for a length of 6 cm, situated 16 cm proximal to the anal ring. Histopathology of the biopsy specimen in and around the ulcerations revealed the deposition of amyloid materials in the proper mucosa and the submucosa. Similar ulcerations were not detected by endoscopy in other parts of the gastrointestinal tract, i.e., esophagus, stomach, and duodenum. Multiple biopsy specimens taken from various parts of the gastrointestinal tract other than the sigmoid colon showed no amyloid deposition. Further investigations showed no particular abnormality in any organs and the patient was free of such systemic disease as myeloma or collagen disease. The findings were consistent with those of localized sigmoid amyloidosis. Localized colonic amyloidosis is rare, this being the fifth patient reported in Japan in the past 15 years.

Changes in gastrointestinal lymph and blood vessels in patients with cirrhotic portal hypertension.

BACKGROUND: The aim of this study was to characterize the lymph vessels in different parts of the gastrointestinal tract and also to evaluate morphometric changes in these vessels during cirrhotic portal hypertension. METHODS: Sixteen patients with cirrhotic portal hypertension and 18 control subjects without portal hypertension were enrolled in the study. Tissue specimens were collected at autopsy or surgery, and were stained enzyme histochemically, using 5'-nucleotidase and alkaline phosphatase to distinguish lymph vessels and blood vessels, respectively. The numbers of vessels and their luminal areas were estimated using computer graphics software (National Institutes of Health [NIH] image program). RESULTS: The numbers and luminal areas of the lymph vessels varied considerably among the different organs of the gastrointestinal tract, both in controls and in the patients with cirrhotic portal hypertension. There was no significant difference in the numbers of lymph vessels between controls and patients with cirrhotic portal hypertension. However, the luminal area of the lymph vessels in the esophagus and stomach was significantly greater in the patients with cirrhotic portal hypertension than in the controls. These differences in lymph vessels were not seen in the small intestine and colon. CONCLUSIONS: These data indicate that dilatation of lymph vessels may be related to the absorption of excess interstitial fluid, resulting from congestion, in cirrhotic portal hypertension.

Three patients with autoimmune cholangiopathy treated with prednisolone.

We describe three patients with autoimmune cholangiopathy, i.e., anti-mitochondrial antibody-negative and anti-nuclear antibody-positive primary biliary cirrhosis, who were treated with prednisolone. Serum anti-mitochondrial antibody and anti-pyruvate dehydrogenase-E2 component antibody were determined by immunofluorescence of frozen sections and enzyme-linked immunosorbent assay, respectively. Immunoblotting using mitochondria prepared from rat liver was performed to analyze anti-mitochondrial antibody in detail. Serum from one patient reacted with a 48-kilodalton protein, but sera from the other two patients failed to react with the mitochondrial proteins. There was a marked improvement in liver function test results after prednisolone treatment. Before treatment, liver biopsy in all three patients showed histological features of primary biliary cirrhosis with hepatocellular necrosis. Repeat biopsy during treatment showed marked amelioration of hepatocellular damage in all three patients, although bile duct involvement persisted in two patients. These findings suggest that prednisolone is an effective treatment for hepatocellular damage in patients with autoimmune cholangiopathy, but has little impact on the bile duct involvement.

Increased levels of serum interleukin-18 in Graves' disease.

We have previously reported that serum soluble interleukin-2 receptor (sIL-2R) and IL-12 levels were significantly increased in hyperthyroid Graves' disease. In this study, we investigated serum IL-18 levels in patients with either Graves' disease or Hashimoto's thyroiditis. The serum IL-18 levels in Graves' disease were significantly increased in the hyperthyroid state and were decreased during treatment with methimazole or propylthiouracil. On the other hand, the levels in Hashimoto's thyroiditis in the hypothyroid state showed no significant differences from those in healthy subjects. When liothyronine sodium was administered orally to healthy subjects, serum IL-18 levels were not changed. Positive correlations between serum IL-18 and IL-12, IL-12 and sIL-2R, and sIL-2R and IL-18 levels were noted in Graves' disease. These results suggest that Th1 cytokines might play an important regulatory role in Graves' disease.

The effectiveness of interferon therapy on occurrence of hepatocellular carcinoma in chronic hepatitis C.

To clarify the effect of interferon (IFN) therapy for chronic hepatitis C (CHC) on the occurrence of hepatocellular carcinoma (HCC), 149 patients who were observed over a period of five years (mean: 7.6 years) were studied. The C-1 group, 33 patients with complete response to IFN; the C-2 group, 55 patients with no response to IFN; and the C-3 group, 61 patients who did not receive IFN. The occurrence rate of HCC was 0.9%/year/person. In the C-1, C-2 and C-3 groups, the rates were 0%, 0.3%, and 1.6%, respectively. The rate in C-1 + C-2 groups was significantly lower than that of the C-3 group (P<0.05). These data suggest IFN may suppress the occurrence of HCC in CHC.

Immunohistochemical localization of metallothionein in hepatocellular carcinoma: preferential expression in non-cancerous cirrhotic nodules.

Metallothionein (MT), an oncofocal gene product was strongly expressed in 35%-95% of hepatocytes in hepatocellular carcinoma (HCC) and MT-positive hepatocytes were localized mainly in the non-cancerous cirrhotic nodules but not in malignant hepatocytes. On the other hand, <10% hepatocytes showed weak staining for MT in chronic hepatitis and cirrhosis of liver. Strong expressions of MT in non-cancerous cirrhotic nodule in HCC and low expressions in liver cirrhosis without HCC indicate a relationship between malignant transformation of hepatocytes and the expression of MT.