A case of glucocorticoid-induced diabetes in which the efficacy between sitagliptin and metformin was compared.

To date, promising strategies for treating glucocorticoid (GC)-induced diabetes with antidiabetic drugs have not been established. We herein report the case of a woman with GC-induced diabetes in which we compared the efficacy of two kinds of orally administered antidiabetic drugs sitagliptin and metformin by continuous glucose monitoring (CGM) and meal-challenge test (MCT). As a result, CGM showed that daily fluctuation of blood glucose levels was reduced during administration of metformin but not during administration of sitagliptin. On the other hand, MCT showed that administration of metformin reduced plasma glucose levels accompanied by the decrease of plasma insulin levels and the increase of plasma glucagon levels, whereas administration of sitagliptin had little effects on these parameters. This case is the first report to compare the efficacy between sitagliptin and metformin in glucose homeostasis by CGM and MCT in a patient with GC-induced diabetes.

Metformin decreases glycated albumin to glycated haemoglobin ratio in patients with newly diagnosed type 2 diabetes.

BACKGROUND: To know whether metformin improves postprandial hyperglycaemia, we examined the effect of metformin on the glycated albumin (GA) to glycated haemoglobin (HbA1c) ratio (GA/HbA1c ratio) in patients with newly diagnosed type 2 diabetes. METHODS: Metformin and lifestyle interventions were initiated in 18 patients with newly diagnosed type 2 diabetes. Metformin was titrated to 1500 mg/day or maximum-tolerated dose. HbA1c and GA were measured every four weeks up to 24 weeks. RESULTS: HbA1c decreased significantly from 9.0 ± 2.1% at baseline to 6.5 ± 0.9% at week 24, and GA decreased significantly from 24.3 ± 8.2% to 16.2 ± 3.1%. The GA/HbA1c ratio decreased significantly from 2.66 ± 0.37 at baseline to 2.47 ± 0.29 at week 24 (P<0.01), despite that the GA/HbA1c ratio reached a plateau value at week 16. The change in the GA/HbA1c ratio during 24 weeks (ΔGA/HbA1c ratio) was significantly correlated with both baseline HbA1c and GA. Moreover, the ΔGA/HbA1c ratio was significantly correlated with the change in GA during 24 weeks but not with the change in HbA1c. CONCLUSIONS: Metformin decreased the GA/HbA1c ratio in patients with newly diagnosed type 2 diabetes. This suggests that metformin improves postprandial hyperglycaemia in patients with newly diagnosed type 2 diabetes.

Macrophage tolerance induced by stimulation with Toll-like receptor 7/8 ligands.

The engagement of Toll-like receptors (TLRs) results in resistance to subsequent challenge with respective ligands in macrophages. Studies have shown that stimulation by ligands for TLR2, TLR4, TLR5 and TLR9 induces this state of hypo-responsiveness (homo-tolerance) towards subsequent stimulation with the same ligands. However, whether homo-tolerance is induced by the ligands of TLR7/8 has not been previously determined. We found that ligands for TLR7/8, namely ss-RNA from HIV and an imidazoquinoline compound, R848, induced macrophage tolerance, as judged by the production of the chemokine MIP-1beta. IRAK-1 phosphorylation was also inhibited in the tolerant cells after subsequent stimulation with R848, although no significant differences were observed in the protein levels of TLR7 between tolerant and non-tolerant cells. These results indicate that macrophage tolerance induced by TLR7/8 ligands is regulated at least at the level of IRAK-1 activation.

Trans-basement membrane migration of human basophils: role of matrix metalloproteinase-9.

In allergic disorders, basophils migrate from the blood stream to inflamed tissue sites. Since trans-basement membrane migration is an important step for local basophil accumulation, we performed a human basophil transmigration assay using a model basement membrane, Matrigel. IL-3 in the upper chamber was critical for basophil trans-basement membrane migration over baseline levels, since none of the chemoattractants placed in the lower chambers induced migration. RANTES, IL-8, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) and platelet-activating factor (PAF) significantly up-regulated the transmigration of IL-3-treated basophils. Neutralizing experiments indicated the involvement of beta2 integrin and matrix metalloproteinase (MMP)-2/9 in basophil transmigration. Real-time quantitative PCR revealed that basophils constitutively expressed transcripts for MMP-9, and at lower levels, MMP-2, but cell-surface expression was only detected for MMP-9. MMP-9 was also detected in the cytoplasm and culture supernatant of the basophils. Treatment with IL-3 up-regulated the surface level of MMP-9 on the basophils. Our results suggest that basophils possess a unique regulatory mechanism for trans-basement membrane migration which is affected by cytokines, chemoattractants, beta2 integrin and MMPs, especially MMP-9. MMP-9 may be critically involved in the pathogenesis of local basophil influx in allergic diseases.

Expression and function of toll-like receptors in human basophils.

We investigated the expression and function of a panel of Toll-like receptors (TLRs) in human basophils. Basophil preparations constitutively expressed TLR2, TLR4, TLR9 and TLR10 mRNAs (TLR4 > TLR2 >> TLR9, TLR10). Although TLR mRNA expression in basophils was generally less prominent compared with those in neutrophils and monocytes, basophils expressed significantly higher levels of TLR2 and TLR4 mRNA than eosinophils. Various TLR ligands (Pam3Cys-Ser-Lys4, poly I:C, lipopolysaccharide, R-848, CpG DNA) were tested, but none affected the expression level of adhesion molecule CD11b or the viability of freshly purified basophils. On the other hand, when basophils were pretreated with interferon-gamma before stimulation with TLR ligands, only the TLR4 ligand, lipopolysaccharide, upregulated CD11b expression. However, the surface levels of TLR2 and TLR4 on the interferon-gamma-treated basophils showed no obvious changes. These results suggest that TLR4 on basophils may be involved in the pathogenesis of infection-induced exacerbation of allergic inflammation by modulating basophil functions.

5-Lipoxygenase products regulate basophil functions: 5-Oxo-ETE elicits migration, and leukotriene B(4) induces degranulation.

BACKGROUND: 5-Lipoxygenase (5-LO) products have been strongly implicated in the pathogenesis of allergic diseases. In addition to their physiologic effects on residential cells, 5-LO products are capable of stimulating various eosinophil functions. However, little is known regarding the effects of 5-LO products on basophil functions. OBJECTIVE: This study was designed to elucidate the effects of the main 5-LO products (ie, leukotriene [LT] B(4), LTD(4), and 5-oxo-6,8,11,14-eicosatetraenoic acid [5-oxo-ETE]), as well as their receptor expression on human basophils. METHODS: We studied the effects of 5-LO products on Ca(2+) mobilization, migration, CD 11b expression, and degranulation of human basophils. Expression of the receptors for LTC(4)/D(4)/E(4) (cysteinyl leukotriene 1 [CysLT(1)] and CysLT(2)), LTB4 (BLT(1) and BLT(2)), and 5-oxo-ETE (oxoeicosanoid [OXE]) was assessed by means of real-time PCR and flow cytometry. RESULTS: At the mRNA level, basophils strongly expressed OXE and predominantly expressed CysLT(1) and BLT(2). The expression level of OXE mRNA in basophils was approximately 20-fold higher than in neutrophils and similar to that in eosinophils. At the protein level, basophils expressed CysLT(1), CysLT(2), BLT(1), and OXE, but not BLT(2). All products elicited a transient increase of cytosolic calcium, with the order of magnitude being LTB(4)>5-oxo-ETE>LTD(4). 5-Oxo-ETE induced a strong basophil migratory response that was almost equivalent to that of prostaglandin D(2). LTB(4) elicited significant degranulation of IL-3-primed basophils. In contrast, no functional significance was observed for LTD(4). CONCLUSION: Among 5-LO products, 5-oxo-ETE induces a potent basophil migratory response, and LTB(4) elicits degranulation under certain conditions. Our results strongly suggest that 5-oxo-ETE might afford opportunities for therapeutic targeting in allergic inflammation.

IgE- and FcepsilonRI-mediated migration of human basophils.

Local accumulation of basophils at inflammatory sites is observed in experimental antigen challenge and in allergic diseases. It is not fully known what factor(s) regulates local basophil influx in tissues, and it has not been determined whether antigens belong in a panel of basophil chemoattractants. This study was designed to elucidate whether IgE- and high-affinity receptor for IgE (FcepsilonRI)-mediated stimulation can induce human basophil migration. The migration-inducing potency of an anti-FcepsilonRI alpha-chain mAb, CRA-1, was examined on human basophils. CRA-1 mAb elicited significant migration of basophils. The migration-inducing potency of this mAb was maximal at 100 ng ml-1, and CRA-1 mAb at 100 ng ml-1 attracted approximately 10% of total inoculated basophils above baseline levels after incubation for 2.5 h. Checkerboard analysis indicated that basophil migration induced by this mAb was mainly chemotactic and partially chemokinetic. An antigen, Der f 2, also induced migration of basophils from Der f-sensitive subjects. Basophils mixed with 1 ng ml-1 of CRA-1 mAb showed an exaggerated migration response to eotaxin, indicating that FcepsilonRI cross-linkage enhances basophil migration to other chemoattractants. Induction of basophil migration by IgE- and FcepsilonRI-cross-linking stimulation may, at least in part, explain the pathogenesis of local basophil accumulation clinically observed in allergic diseases such as asthma.

Hange-koboku-to, a Kampo medicine, modulates cerebral levels of 5-HT (5-hydroxytryptamine), NA (noradrenaline) and DA (dopamine) in mice.

Cerebral monoamine systems play important pathogenic roles in various psychiatric and neurologic diseases, such as depression, anxiety and swallowing disturbance. Hange-koboku-to, a Kampo (Japanese herbal) medicine, has been successfully used for the treatment of these disorders. To elucidate the mechanisms underlying its clinical efficacy for these disorders, the effects of Hange-koboku-to (500 mg/kg, p.o.) on the cerebral monoamine systems were examined. Regional levels of 5-HT (5-hydroxytryptamine), NA (noradrenaline), DA (dopamine) and their metabolites in mouse brain were measured using a high-performance liquid chromatography system. Hange-koboku-to increased the 5-HT and NA levels and decreased 5-HIAA (5-hydroxyindole-3-acetic acid), thus decreasing 5-HT and NA turnover (metabolites/monoamine ratio) in the hypothalamus. The levels of DA, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (4-hydroxy-3-methoxy-phenylacetic acid) were all increased, resulting in a decreased DA turnover in the striatum. Since decreased 5-HT turnover has been observed after administration of various antidepressants, Hange-koboku-to-mediated reduction of 5-HT turnover may be related to the clinical efficacy of this Kampo medicine on certain psychiatric disorders. Furthermore, the beneficial therapeutic effects of Hange-koboku-to on swallowing disturbance may be related to the increased cerebral DA level brought about by this Kampo medicine.

The -431C>T polymorphism of thymus and activation-regulated chemokine increases the promoter activity but is not associated with susceptibility to atopic dermatitis in Japanese patients.

BACKGROUND: Thymus and activation-regulated chemokine (TARC) plays an important role in the pathogenesis of atopic dermatitis (AD). We recently detected the single nucleotide polymorphism (SNP) (-431C>T) in the 5'-flanking region of TARC gene. OBJECTIVES: To examine whether the -431C>T SNP of the TARC gene is associated with susceptibility to AD and whether it affects the promoter activity of the TARC gene. METHODS: We investigated the genotype and allele frequencies of the SNP in 193 AD patients and 158 healthy controls by polymerase chain reaction-restriction fragment length polymorphism method. We compared the promoter activities between TARC promoter carrying 431C and that carrying -431T by transient-transfection assay in DJM-1 cell line. RESULTS: There were no significant differences in genotype or allele frequencies between AD patients and controls (genotype: P = 0.38, allele: P = 0.22). Luciferase activity was higher in -431T constructs than in -431C constructs (2.3-fold, P = 9.5 x 10(-6)). CONCLUSION: These results suggest that the -431C>T SNP of the TARC gene enhances the promoter activity of TARC gene but is not associated with susceptibility to AD in Japanese population.

Transendothelial migration of human basophils.

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.

Lack of association of CCR4 single nucleotide polymorphism with atopic dermatitis in Japanese patients.

CCR4, a member of the CC chemokine receptor family, is believed to play an important role in the pathogenesis of atopic dermatitis. To examine whether CCR4 single nucleotide polymorphism (SNP) is associated with susceptibility to atopic dermatitis, we investigated the allele and genotype frequencies of C1014T SNP of CCR4 in 198 Japanese patients with atopic dermatitis and controls by a PCR-restriction fragment length polymorphism method. There was no significant difference in allele or genotype frequencies between patients with atopic dermatitis and controls. Serum IgE levels and peripheral blood eosinophil counts were not significantly different among genotypes. There was also no significant difference in allele or genotype frequencies between the patient subgroup with and without asthma, with mild or moderate disease, with and without family history of atopic dermatitis, or with and without family history of atopic disorders. C1014T SNP of CCR4 does not appear to be associated with susceptibility to atopic dermatitis in Japanese patients.

Identification of granulocyte subtype-selective receptors and ion channels by using a high-density oligonucleotide probe array.

BACKGROUND: During inflammation, neutrophils, basophils, and eosinophils release cell type-specific mediators and proteases through signaling molecules, such as G protein-coupled receptors and ion channels. As such, ion channels and receptors, including G protein-coupled receptors, are common drug targets. OBJECTIVE: We sought to identify, for the first time, ion channels and receptors preferentially expressed by each granulocyte subtype. METHODS: Using GeneChip, we compared approximately 20,000 transcripts present in 7 leukocyte types, platelets, mast cells, and fibroblasts to identify granulocyte subtype-selective transcripts for receptors and ion channels. Granulocyte subtype-selective transcripts were chosen on the basis of several conditions, such as the transcript having a 5-fold or greater expression level compared with the maximum level of other leukocytes. RESULTS: Fifty-one transcripts were chosen to be preferentially expressed by each granulocyte subtype. Seventeen of the 51 transcripts have not been previously reported as granulocyte subtype selective. Among the 17 receptors and ion channels, 6 were basophil selective, eosinophil selective, or both and were not highly expressed by other organs, indicating that they might be potential targets for antiallergy drugs. CONCLUSION: Use of this database of potential cell type-selective drug targets should minimize the efforts required for pharmaceutical development.

Involvement of IRAK-M in peptidoglycan-induced tolerance in macrophages.

The molecular mechanisms by which pathogen-associated molecular patterns recognized by TLR2, such as peptidoglycan (PGN), induce homotolerance are largely unknown. It was recently reported that IRAK-M negatively regulates TLR signaling. In this study, we elucidate the molecular mechanisms of tolerance induced by PGN, with a focus on the role of IRAK-M. We demonstrate that pretreatment of macrophage RAW264.7 cells with a high concentration (30 microg/ml) of PGN for 16 h effectively induces tolerance against following stimulation with 30 microg/ml of PGN; while pretreatment with a low concentration (1 microg/ml) of PGN does not. IRAK-M is induced in cells treated with the high concentration of PGN 4-24 h after PGN stimulation, but not in cells treated with the low concentration of PGN up to 24 h after stimulation. Phosphorylation of MAPKs and IkappaBalpha is inhibited after the second PGN stimulation in tolerant cells. Kinase activity of IRAK-1 and association between IRAK-1 and MyD88 are also suppressed in PGN-induced tolerant cells. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates the production of TNF-alpha after PGN restimulation. These results suggest that induction of IRAK-M and inhibition of kinase activity of IRAK-1 are crucial to PGN-induced tolerance in macrophages.

Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells.

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.

Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand.

We investigated the expression of a panel of Toll-like receptors (TLRs) and their functions in human eosinophils. Eosinophils constitutively expressed TLR1, TLR4, TLR7, TLR9, and TLR10 mRNAs (TLR4 greater than TLR1, TLR7, TLR9, and TLR10 greater than TLR6). In contrast, neutrophils expressed a larger variety of TLR mRNAs (TLR1, TLR2, TLR4, TLR6, TLR8 greater than TLR5, TLR9, and TLR10 greater than TLR7). Although the expression levels in eosinophils were generally less prominent compared with those in neutrophils, eosinophils expressed a higher level of TLR7. Furthermore, among various TLR ligands (S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-Cys-Ser-(Lys)(4), poly(I:C), LPS, R-848, and CpG DNA), only R-848, a ligand of TLR7 and TLR8, regulated adhesion molecule (CD11b and L-selectin) expression, prolonged survival, and induced superoxide generation in eosinophils. Stimulation of eosinophils by R-848 led to p38 mitogen-activated protein kinase activation, and SB203580, a p38 mitogen-activated protein kinase inhibitor, almost completely attenuated R-848-induced superoxide generation. Although TLR8 mRNA expression was hardly detectable in freshly isolated eosinophils, mRNA expression of TLR8 as well as TLR7 was exclusively up-regulated by IFN-gamma but not by either IL-4 or IL-5. The up-regulation of the TLRs by IFN-gamma had potentially functional significance: the extent of R-848-induced modulation of adhesion molecule expression was significantly greater in cells treated with IFN-gamma compared with untreated cells. Although the natural ligands for TLR7 and TLR8 have not yet been identified, our results suggest that eosinophil TLR7/8 systems represent a potentially important mechanism of a host-defensive role against viral infection and mechanism linking exacerbation of allergic inflammation and viral infection.

Changing expression of IL-3 and IL-5 receptors in cultured human eosinophils.

IL-3, IL-5, and GM-CSF exert overlapping functions in eosinophils via a shared receptor beta-chain, and IL-3Ralpha transcript expression is the weakest in blood eosinophils. We investigated the long-term regulation of surface expression of IL-3Ralpha. IL-3 was the most potent inducer of CD69 expression after 24-h stimulation, but not after 1-h stimulation. Expression of IL-5Ralpha and GM-CSFRalpha was significantly downregulated by culturing with their respective ligands, while IL-3Ralpha expression was not. IL-3 at 30pM significantly increased IL-3Ralpha expression and IL-3Ralpha expression was also upregulated by both IL-5 and GM-CSF. In parallel with the surface protein expression, IL-3Ralpha mRNA was also upregulated by IL-3, IL-5, and GM-CSF. These results demonstrated that long-term culturing of eosinophils with CSFs induced a change in the potency order of CSFs, with IL-3 coming to exert the strongest effect. They thus suggest that IL-3 plays more important roles in local eosinophil activation than previously recognized.

Comparative effects of basophil-directed growth factors.

IL-3, IL-5, and GM-CSF exert various overlapping functions in basophils. We investigated the receptor expression profiles and concentration-dependent effects of IL-3, IL-5, and GM-CSF on several basophil functions in comparison with their effects on eosinophils. The order of the receptor expression levels was IL-3Ralpha>IL-5Ralpha>GM-CSFRalpha in basophils and IL-5Ralpha>or=GM-CSFRalpha>IL-3Ralpha in eosinophils. Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha and similar levels of IL-5Ralpha and GM-CSFRalpha. The order of potency was IL-3>IL-5=GM-CSF for degranulation, survival, and CD11b expression in basophils, and IL-5=GM-CSF>or=IL-3 for survival and CD11b expression in eosinophils. However, IL-3 induced CD69 expression preferentially in basophils. Our results indicate that IL-3 is the most potent activator of human basophils, and that the rank order of potency of hemopoietic growth factors virtually corresponded to their receptor expression levels in both cell types.

Induction of basophil desensitization in physiological medium: enhancement after IgE-dependent upregulation of surface IgE binding on basophils.

BACKGROUND: Although the ability of basophils to release mediators, called releasability, may be an important aspect which influences the proinflammatory role of these cells, clinical approaches aiming at the depletion of the releasability have not been established. We examined whether the desensitization procedure in Ca(2+)-containing physiological conditions can make basophils completely unresponsive to IgE-mediated stimulation, and whether basophil desensitization is affected by the surface IgE levels. METHODS: Human peripheral blood basophils were cultured with low concentrations of anti-IgE antibody or recombinant mite allergen. Following culture, cells were stimulated and their histamine release was measured. RESULTS: Culturing with mite allergen or anti-IgE antibody below threshold concentrations induced potent desensitization in basophils. The desensitizing effect of anti-IgE was dose- and time-dependent; IgE-dependent releasability was completely suppressed when basophils were incubated with a near-threshold concentration of anti-IgE for > or= 4 h. In the continuous presence of subthreshold doses of anti-IgE, basophils remained desensitized even after 3 days. Basophils which had undergone an increase in surface IgE levels after 24-hour culture with IgE demonstrated enhanced desensitization. CONCLUSIONS: Near-threshold stimulation in physiological medium can affect basophils, thereby inducing complete and sustained deprivation of releasability without triggering degranulation. Basophil desensitization is regulated by their surface IgE levels. Induction of full desensitization may represent a potentially important therapeutic strategy for IgE-mediated allergic diseases in which basophils play pathogenic roles.