MCAM+CD161- Th17 Subset Expressing CD83 Enhances Tc17 Response in Psoriasis.

Recent studies have highlighted the pathogenic roles of IL-17-producing CD8+ T cells (T-cytotoxic 17 [Tc17]) in psoriasis. However, the underlying mechanisms of Tc17 induction remain unclear. In this study, we focused on the pathogenic subsets of Th17 and their mechanism of promotion of Tc17 responses. We determined that the pathogenic Th17-enriched fraction expressed melanoma cell adhesion molecule (MCAM) and CCR6, but not CD161, because this subset produced IL-17A abundantly and the presence of these cells in the peripheral blood of patients has been correlated with the severity of psoriasis. Intriguingly, the serial analysis of gene expression revealed that CCR6+MCAM+CD161-CD4+ T cells displayed the gene profile for adaptive immune responses, including CD83, which is an activator for CD8+ T cells. Coculture assay with or without intercellular contact between CD4+ and CD8+ T cells showed that CCR6+MCAM+CD161-CD4+ T cells induced the proliferation of CD8+ T cells in a CD83-dependent manner. However, the production of IL-17A by CD8+ T cells required exogenous IL-17A, suggesting that intercellular contact via CD83 and the production of IL-17A from activated CD4+ T cells elicit Tc17 responses. Intriguingly, the CD83 expression was enhanced in the presence of IL-15, and CD83+ cells stimulated with IL-1ß, IL-23, IL-15, and IL-15Rα did not express FOXP3. Furthermore, CCR6+MCAM+CD161-CD4+ T cells expressing CD83 were increased in the peripheral blood of patients, and the CD83+ Th17-type cells accumulated in the lesional skin of psoriasis. In conclusion, pathogenic MCAM+CD161- Th17 cells may be involved in the Tc17 responses via IL-17A and CD83 in psoriasis.

Evaluation of salivary α-amylase and immunoglobulin A responses after endurance exercise in adolescent males and females with similar aerobic fitness.

The objective of this study was to clarify whether there are sex-specific differences in salivary α-amylase and immunoglobulin A responses following acute endurance exercise in adolescent males and females with equivalent cardiorespiratory fitness levels. Twenty-six aerobically trained adolescent males and females with similar training status were enrolled in this study. Each individual executed a 1-h prolonged cycling exercise corresponding to a constant power output at 65% of peak oxygen uptake. Unstimulated whole salivary samples were taken with the passive drooling method at the 10-min period before and after exercise for the determination of salivary responses [α-amylase, immunoglobulin A, total protein and flow rate]. Salivary α-amylase activity, immunoglobulin A concentration and total protein concentration were significantly augmented immediately after acute endurance exercise. Regarding sex differences, only the salivary flow rate was significantly lower in females than in males. The findings of the present study imply that adolescent males and females appear to have similar salivary responses after acute endurance exercise, as represented by salivary α-amylase activity and immunoglobulin A concentration in connection with total protein concentration and salivary flow rate, when they are matched for peak oxygen uptake relative to fat-free body mass.

Suppression of Lipopolysaccharide-Induced IL-1ß Gene Expression by High-Molecular-Weight Adiponectin in RAW264.7 Macrophages.

Adiponectin is an abundant adipocytokine secreted by adipocytes. It exists in the plasma in its trimeric, hexameric, high-molecular-weight (HMW), and globular (a proteolytic product) isoforms. Adiponectin's anti-inflammatory effects on macrophages remain controversial. We have previously reported a simple and effective method for purifying native HMW adiponectin from human plasma. Here, we investigated whether native HMW adiponectin from human plasma has anti-inflammatory effects on macrophages. Pretreatment with human native HMW adiponectin inhibited lipopolysaccharide (LPS)-induced interleukin-1ß (IL-1ß) gene expression, but not tumor necrosis factor (TNF)-α expression. However, simultaneous treatment with HMW adiponectin and LPS did not inhibit IL-1ß expression. Further, HMW adiponectin pretreatment decreases glycogen synthase kinase-3ß (GSK-3ß) inactivation by abrogating LPS-induced Akt (Ser473) phosphorylation, which subsequently suppresses LPS-induced CCAAT/enhancer binding protein ß (C/EBPß) protein translation and nuclear translocation. However, HMW adiponectin pretreatment did not affect LPS-induced nuclear factor-kappaB (NF-κB) activation. These results suggest that HMW adiponectin mediates potent anti-inflammatory activities in macrophages by inhibiting its Akt-C/EBPß signaling pathway, thereby suppressing IL-1ß gene expression.

The Ang III/AT2R Pathway Enhances Glucose Uptake by Improving GLUT1 Expression in 3T3-L1 Adipocytes.

Angiotensin III (Ang III) is a heptapeptide derived from Ang II that has been confirmed as the preferred agonist of angiotensin II type 2 receptor (AT2R). Recent studies have revealed AT2R mainly exerts anti-inflammation effects. However, the effects of the Ang III/AT2R pathway on adipocytes remain unknown. Here, the effects of Ang III on glucose uptake were examined. The results showed that AT2R expression was upregulated during adipogenesis in 3T3-L1 preadipocytes, whereas AT1R expression was diminished. Also, Ang III (10 nM) significantly increased glucose uptake by 3T3-L1 adipocytes, which was blocked by PD123319, an AT2R blocker, but not by irbesartan, an AT1R blocker. Ang III also induced the expression of glucose transporter type 1 (GLUT1). These stimulatory effects were inhibited by pretreatment with PD123319, but not with irbesartan. Together, these results indicate that Ang III enhances glucose uptake by upregulating GLUT1 expression via AT2R.

Protective effects of a nicotinamide derivative, isonicotinamide, against streptozotocin-induced ß-cell damage and diabetes in mice.

OBJECTIVE: Nicotinamide rescues ß-cell damage and diabetes in rodents, but a large-scale clinical trial failed to show the benefit of nicotinamide in the prevention of type 1 diabetes. Recent studies have shown that Sirt1 deacetylase, a putative protector of ß-cells, is inhibited by nicotinamide. We investigated the effects of isonicotinamide, which is a derivative of nicotinamide and does not inhibit Sirt1, on streptozotocin (STZ)-induced diabetes in mice. RESEARCH DESIGN AND METHODS: Male C57BL/6 mice were administered with three different doses of STZ (65, 75, and 100 mg/kg BW) alone or in combination with subsequent high-fat feeding. The mice were treated with isonicotinamide (250 mg/kg BW/day) or phosphate-buffered saline for 10 days. The effects of isonicotinamide on STZ-induced diabetes were assessed by blood glucose levels, glucose tolerance test, and immunohistochemistry. RESULTS: Isonicotinamide effectively prevented hyperglycemia induced by higher doses of STZ (75 and 100mg/kg BW) alone and low-dose STZ (65 mg/kg BW) followed by 6-week high-fat diet in mice. The protective effects of isonicotinamide were associated with decreased apoptosis of ß-cells and reductions in both insulin content and insulin-positive area in the pancreas of STZ-administered mice. In addition, isonicotinamide inhibited STZ-induced apoptosis in cultured isolated islets. CONCLUSIONS: These data clearly demonstrate that isonicotinamide exerts anti-diabetogenic effects by preventing ß-cell damage after STZ administration. These findings warrant further investigations on the protective effects of isonicotinamide and related compounds against ß-cell damage in diabetes.

Inducible nitric-oxide synthase and nitric oxide donor decrease insulin receptor substrate-2 protein expression by promoting proteasome-dependent degradation in pancreatic beta-cells: involvement of glycogen synthase kinase-3beta.

Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic ß-cells. Gene disruption of IRS-2 results in failure of the ß-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in ß-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in ß-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in ß-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1ß (IL-1ß), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3ß (GSK-3ß) and c-Jun N-terminal kinase (JNK/SAPK) in ß-cells. Inhibition of GSK-3ß by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in ß-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated ß-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3ß-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expression may contribute to the progression and/or exacerbation of ß-cell failure in diabetes.

Assessment of whole-body DNA oxidation following prolonged exercise in adolescent males and females matched for aerobic capacity.

OBJECTIVE: The purpose of this study was to investigate the effects of moderately extended cycling exercise on oxidative DNA damage (accounted for by urinary 8-hydroxy-2´-deoxyguanosine) in adolescent males and females matched for aerobic capacity. MATERIALS AND METHODS: Twenty-nine aerobically active adolescent males and females matched for peak oxygen uptake (VO2peak) relative to fat free mass (ml/kg FFM/min) participated in this study. Two-hour urinary samples were taken at three time points before (-2-0h), immediately (0-2h) after and 24-26 h after 60 min of cycling exercise at 65%VO2peak, followed by the analysis of urinary 8-OHdG (a potential marker of whole-body DNA damage and repair) determined with high performance liquid chromatography with electrochemical detection. RESULTS: The two-way (time x sex) analysis of variance demonstrated no significant main effects for time, sex or interaction regarding urinary 8-hydroxy-2´-deoxyguanosine level following moderate-intensity endurance exercise. CONCLUSIONS: These results of the present study suggest that no detrimental DNA damage can be observed after moderately prolonged exercise in aerobically fit males and females, potentially because of the enhanced antioxidant defense responses. Furthermore, the endurance-trained adolescent males and females appear to have similar DNA oxidation responses at the whole-body level when normalized to peak oxygen uptake relative to fat free mass.

[Examination of the Relationship between Worse Symptom and Differences Route during Administration of Argatroban in Acute Ischemic Stroke Patients].

The treatment of acute ischemic stroke usually involves argatroban administration by continuous infusion for 2 d and by intravenous infusion twice a day for 5 d after that. However, the appropriate dose of argatroban to be administered is not clear. Therefore, no studies have been reported a comparison of intravenous and continuous argatroban infusion after day 3 for acute ischemic stroke patients. We aimed to identify the connection between differences in argatroban administration and worsening of symptoms after day 3 in ischemic stroke patients. We retrospectively evaluated the data of 107 ischemic stroke patients who received treatment with argatroban. The study endpoint was defined as the worsening of symptoms from days 3 to 7. Logistic regression analysis was used to determine the risk factors that were significantly associated with worsening of symptoms. Patients were administered argatroban, with rates of 72.0%, and 28.0% for continuous, and intravenous infusion, respectively. A total of 10 (9.3%) patients experienced worsening of symptoms. In the single logistic regression analysis, carotid stenosis [non-adjusted odds ratio (OR) 5.775, 95% confidence interval (CI) 1.486-22.442, p=0.011] was only significantly associated with worsening of symptoms. Worsening of symptoms was not related to either intravenous or continuous infusion group (16.7% vs. 6.5%, p=0.104). Bleeding was also not associated with either group (6.7% vs. 3.9%, p=0.618). We suggest that the differences in the mode of argatroban administration were not related to the worsening of symptoms in ischemic stroke patients. We also found that safety was equivalent regardless of the administration route.

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2.

Math2 (NEX-1/NeuroD6) is a member of the basic helix-loop-helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. In the promoter region of rat Prg1, we identified four E-boxes [designated -E1 to -E4 (CANNTG)] recognized by the basic helix-loop-helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E-boxes. The Prg1 reporter assay showed that -E1 was critical for the regulation of Math2-mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F(2alpha)-synthetic activity.

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.

Regulation of the human leukocyte-derived arginine aminopeptidase/endoplasmic reticulum-aminopeptidase 2 gene by interferon-gamma.

The leukocyte-derived arginine aminopeptidase (L-RAP) is the second aminopeptidase localized in the endoplasmic reticulum (ER) processing antigenic peptides presented to major histocompatibility complex (MHC) class I molecules. In this study, the genomic organization of the gene encoding human L-RAP was determined and the regulatory mechanism of its expression was elucidated. The entire genomic structure of the L-RAP gene is similar to both placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP) genes, confirming the close relationship of these three enzymes. Interferon (IFN)-gamma up-regulates the expression of the L-RAP gene. Deletion and site-directed mutagenic analyses of the 5'-flanking region of the L-RAP gene and electrophoretic mobility shift assay indicated that while interferon regulatory factor (IRF)-2 is important in the basal condition, IRF-1 is the primary regulator of IFN-gamma-mediated augmentation of the gene expression. In addition, PU.1, a member of the E26 transformation-specific family of transcription factors, also plays a role in the regulation of gene expression. The maximum expression of the gene was achieved by coexpression of IRF-1 and PU.1 in HEK293 cells and IRF-2 suppressed the IRF-1-mediated enhancement of gene expression, suggesting that IFN-gamma-induced L-RAP gene expression is cooperatively regulated by IRFs and PU.1 transcription factors.

Regulation of cytosolic prostaglandin E synthase by phosphorylation.

cPGES [cytosolic PG (prostaglandin) E synthase] is constitutively expressed in various cells and can regulate COX (cyclo-oxygenase)-1-dependent immediate PGE2 generation. In the present study, we found that cPGES underwent serine phosphorylation, which was accelerated transiently after cell activation. Several lines of evidence suggest that a cPGES-activating protein kinase is CK-II (casein kinase II). Recombinant cPGES was phosphorylated directly by and associated with CK-II in vitro, resulting in marked reduction of the K m for the substrate PGH2. In activated cells, cPGES phosphorylation occurred in parallel with increased cPGES enzymic activity and PGE2 production from exogenous and endogenous arachidonic acid, and these processes were facilitated by Hsp90 (heat-shock protein 90), a molecular chaperone that formed a tertiary complex with cPGES and CK-II. Treatment of cells with inhibitors of CK-II and Hsp90 and with a dominant-negative CK-II attenuated the formation of the cPGES-CK-II-Hsp90 complex and attendant cPGES phosphorylation and activation. Mutations of either of two predicted CK-II phosphorylation sites on cPGES (Ser113 and Ser118) abrogated its phosphorylation and activation both in vitro and in vivo. Moreover, the CK-II-Hsp90-mediated activation of cPGES was ameliorated by the p38 mitogen-activated protein kinase inhibitor SB20358 or by the anti-inflammatory glucocorticoid dexamethasone. Taken together, the results of the present study have provided the first evidence that the cellular function of this eicosanoid-biosynthetic enzyme is under the control of a molecular chaperone and its client protein kinase.

Prostaglandin E synthase.

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin (PG)H2 to PGE2, occurs in multiple forms with distinct enzymatic properties, modes of expression, cellular and subcellular localizations and intracellular functions. Cytosolic PGES (cPGES) is a cytosolic protein that is constitutively expressed in a wide variety of cells and tissues and is associated with heat shock protein 90 (Hsp90). Membrane-associated PGES (mPGES), the expression of which is stimulus-inducible and is downregulated by anti-inflammatory glucocorticoids, is a perinuclear protein belonging to the microsomal glutathione S-transferase (GST) family. These two PGESs display distinct functional coupling with upstream COXs in cells; cPGES is predominantly coupled with the constitutive COX-1, whereas mPGES is preferentially linked with the inducible COX-2. Several cytosolic GSTs also have the capacity to convert PGH2 to PGE2 in vitro. Accumulating evidence has suggested that mPGES participates in various pathophysiological states in which COX-2 is involved, implying that mPGES represents a potential novel target for drug development.

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic ß-cells.

Nitric oxide (NO) has been implicated in pancreatic ß-cell death in the development of diabetes. The mechanisms underlying NO-induced ß-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced ß-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated ß-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic ß-cell death independent of RIP1. Our findings raise the possibility that NO-mediated ß-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.

A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin.

Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).

Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children.

Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)-4 (C-590T), IL-4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL-10 (C-627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patient's attitude, indoor environment, and so on, were also investigated in this study. Our study was retrospective, and the correlation was analyzed by our defined clinical scores divided into three terms: worst symptoms, recent symptoms and general amelioration at the most recent examination during the disease course. Our results indicated that IL-10 AA, the genotype with lower IL-10 production, is associated with higher IgE levels in the serum (p < 0.0001, estimate; 0.912). Marginal liver abnormalities were observed in the subject group with both FA and AE (p < 0.1191, estimate; 0.1490). Our defined clinical scores enabled evaluation of various aspects of disease severity. Based on the scores, while no single SNP selected in this study determined severity, the combination of the SNP with laboratory data and environmental factors appeared to determine severity.

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases.

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.

Regulation of cytosolic prostaglandin E2 synthase by 90-kDa heat shock protein.

Cytosolic prostaglandin (PG) E(2) synthase (cPGES) is constitutively expressed in a wide variety of cells and converts cyclooxygenase (COX)-1-derived PGH(2) to PGE(2). Given the fact that cPGES is identical to p23, a heat shock protein 90 (Hsp90)-binding protein, we herein examined the effect of Hsp90 on PGE(2) generation by cPGES. Incubation of cPGES with Hsp90 resulted in a significant increase in PGES activity in vitro. Association of cPGES with Hsp90 was increased in cells stimulated with A23187 or bradykinin, accompanied by concomitant increases in cPGES activity and PGE(2) production. Moreover, treatment of cells with Hsp90 inhibitors, which destabilized the cPGES/Hsp90 complex, reduced cPGES activity and PGE(2) production to basal levels. These results suggest that the regulation of cPGES activity in cells depends on its association with Hsp90 and provide the first line of evidence that eicosanoid biosynthesis is under the control of the molecular chaperone.