Biological Evaluation of Isosteric Applicability of 1,3-Substituted Cuneanes as m-Substituted Benzenes Enabled by Selective Isomerization of 1,4-Substituted Cubanes.

We herein evaluate a biological applicability of 1,3-substituted cuneanes as an isostere of m-substituted benzenes based on its structural similarity. An investigation of a method to obtain 1,3-substituted cuneanes by selective isomerization of 1,4-substituted cubanes enables this attempt by giving a key synthetic step to obtain a cuneane analogs of pharmaceuticals having m-substituted benzene moiety. Biological evaluation of the synthesized analogs and in silico study of the obtained result revealed a potential usage of cuneane skeleton in medicinal chemistry.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Functional Characterization of 29 Cytochrome P450 4F2 Variants Identified in a Population of 8380 Japanese Subjects and Assessment of Arachidonic Acid ω-Hydroxylation.

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [Vmax , substrate concentration producing half of Vmax (S50 ), and intrinsic clearance (CL int ) as Vmax /S50 ] of AA ω-hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω-hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function.

Inhibition of thymic stromal lymphopoietin production by FK3453.

To prevent the onset and aggravation of allergic diseases, it is necessary to modulate excessive Th2-type immune responses. It is well accepted that thymic stromal lymphopoietin (TSLP) plays important roles in the change of Th1/Th2 balance to Th2 dominance and would be a druggable target. In this study, using a drug repositioning strategy, we identified 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) as a novel inhibitor of TSLP production. FK3453 inhibited constitutive production of TSLP in the KCMH-1 mouse keratinocyte cell line and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced one in PAM212 cells. FK3453 also inhibited TSLP mRNA expression induced by a mixture of tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, fibroblast-stimulation lipopeptide-1, and protease activated-receptor agonist and TPA in normal human epidermal keratinocytes (NHEKs). Although FK3453 inhibited TPA-induced IL-33 expression in NHEKs in addition to TSLP, it did not inhibit TNF-α and IL-6 production. In addition, FK3453 did not inhibit MAP kinase (ERK) phosphorylation. We have confirmed that topical treatment with FK3453 inhibited TSLP production in the lipopolysaccharide-induced air pouch-type inflammation model. FK3453 could be a lead compound for a novel type of medicine which prevents the onset and aggravation of allergic diseases.

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6ß-Hydroxylation.

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants (K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.

All-Trans Retinoic Acid Enhances Antibody Production by Inducing the Expression of Thymic Stromal Lymphopoietin Protein.

Many classical vaccines contain whole pathogens and, thus, may occasionally induce adverse effects, such as inflammation. Vaccines containing purified rAgs resolved this problem, but, owing to their low antigenicity, they require adjuvants. Recently, the use of several cytokines, including thymic stromal lymphopoietin (TSLP), has been proposed for this purpose. However, it is difficult to use cytokines as vaccine adjuvants in clinical practice. In this study, we examined the effects of all-trans retinoic acid (atRA) on TSLP production and Ag-induced Ab production. Application of atRA onto the ear lobes of mice selectively induced TSLP production without inducing apparent inflammation. The effects appeared to be regulated via retinoic acid receptors γ and α. Treatment with atRA was observed to enhance OVA-induced specific Ab production; however, this effect was completely absent in TSLP receptor-knockout mice. An enhancement in Ab production was also observed when recombinant hemagglutinin was used as the Ag. In conclusion, atRA was an effective adjuvant through induction of TSLP production. Therefore, we propose that TSLP-inducing low m.w. compounds, such as atRA, may serve as effective adjuvants for next-generation vaccines.

Grifola frondosa extract and ergosterol reduce allergic reactions in an allergy mouse model by suppressing the degranulation of mast cells.

The increasing number of patients suffering from allergic diseases is a global health problem. Grifola frondosa is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that G. frondosa extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow-derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells in vivo. Our findings suggest that G. frondosa, including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.

Expression of Histidine Decarboxylase and Its Roles in Inflammation.

Histamine is a well-known mediator of inflammation that is released from mast cells and basophils. To date, many studies using histamine receptor antagonists have shown that histamine acts through four types of receptors: H1, H2, H3, and H4. Thus, histamine plays more roles in various diseases than had been predicted. However, our knowledge about histamine-producing cells and the molecular mechanisms underlying histamine production at inflammatory sites is still incomplete. The histamine producing enzyme, histidine decarboxylase (HDC), is commonly induced at inflammatory sites during the late and chronic phases of both allergic and non-allergic inflammation. Thus, histamine levels in tissues are maintained at effective concentrations for hours, enabling the regulation of various functions through the production of cytokines/chemokines/growth factors. Understanding the regulation of histamine production will allow the development of a new strategy of using histamine antagonists to treat inflammatory diseases.

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the DPYD gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, DPYD mutations, including DPYD*2A, DPYD*13, c.2846A>T, and c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these DPYD polymorphisms has been identified in the Asian population. Recently, 21 DPYD allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue-native PAGE, respectively. Additionally, the values of three kinetic parameters-the Michaelis constant (Km ), maximum velocity (Vmax ), and intrinsic clearance (CLint = Vmax/Km )-were determined for the reduction of 5-FU. Eleven variants exhibited significantly decreased intrinsic clearance compared with wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue-native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare DPYD variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.

Ergosterol and its derivatives from Grifola frondosa inhibit antigen-induced degranulation of RBL-2H3 cells by suppressing the aggregation of high affinity IgE receptors.

Grifola frondosa is an edible mushroom consumed as a health food and/or traditional medicine in Asia. However, the anti-allergic effects of G. frondosa are not yet understood. In this study, we demonstrated the effects of G. frondosa extract (GFE) on IgE-mediated allergic responses, using antigen-stimulated RBL-2H3 cells. Three active compounds: ergosterol, 6ß-methoxyergosta-7,22-dien-3ß,5α-diol (MEDD), and 6-oxoergosta-7,22-dien-3ß-ol (6-OXO) were isolated from GFE and shown to inhibit the antigen-induced release of ß-hexosaminidase and histamine. Among the three active components, we focused on ergosterol because of its high content in GFE. Ergosterol inhibited the aggregation of high-affinity IgE receptor (FcεRI), which is the first step in the activation of mast cells and antigen-induced tyrosine phosphorylation. Furthermore, ergosterol suppressed antigen-increased IL-4 and TNF-α mRNA. Taken together, our findings suggest that G. frondosa, including ergosterol and its derivatives as active components, has the potential to be a novel functional food that prevents type I allergies.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Induced histamine regulates Ni elution from an implanted Ni wire in mice by downregulating neutrophil migration.

Histamine regulates various inflammatory reactions. We have reported that the expression of histidine decarboxylase (HDC) was induced by subcutaneous implantation of nickel (Ni) wire. However, the source and functions of histamine in Ni elution and Ni wire-induced inflammation have not been completely studied. We aimed to elucidate the effects of de novo synthesized histamine on leucocyte infiltration and Ni elution. Implantation of Ni wire induced an increase in the Ni ion content of the surrounding tissues and serum and in the mRNA levels of HDC, a histamine-producing enzyme, macrophage inflammatory protein-2 (MIP-2), a chemoattractant for neutrophils, and monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes. The Ni wire induced HDC expression even in mast cell-deficient WBB6F1-W/WV mice. In HDC knockout (HDC KO) mice, the Ni wire-induced increase in MIP-2 mRNA expression was significantly higher than that in wild-type mice but not MCP-1. MIP-2 expression was enhanced in histamine H2 receptor knockout (H2R KO) mice but not in WBB6F1-W/WV mice. Histamine inhibited NiCl2 -induced MIP-2 mRNA expression in mouse bone marrow-derived macrophages (BMDMs) obtained from wild-type mice; this inhibition was not observed in BMDMs from H2R KO mice. Ni elution increased in HDC KO mice, in which leucocyte infiltration also increased, and was suppressed in mice treated with neutrophil-specific antibody. These results suggest that the Ni wire induced HDC expression in non-mast cells and that, in the chronic phase of inflammation, endogenous histamine reduced Ni elution, probably through regulation of MIP-2 expression and neutrophil migration.

Influence of Japanese Regulatory Action on Denosumab-Related Hypocalcemia Using Japanese Adverse Drug Event Report Database.

The anti-receptor activator of nuclear factor kappa-B ligand (RANKL) antibody, Denosumab (DEN), was approved in April 2012 in Japan, but a Dear Healthcare Professional Letter of Rapid Safety Communication was released in September, 2012 by the regulatory authority because of the severe hypocalcemia risks. Currently, the effectiveness of this regulatory action has not been evaluated and, therefore, this study aimed to assess its impact on DEN-induced hypocalcemia using the Japanese Adverse Drug Event Report database (JADER). The case reports from April 2012 to September 2014 were collected from the JADER, which included 151642 adverse events for the primary suspected drugs. The reporting odds ratio (ROR) of hypocalcemia as a signal of the target adverse event was analyzed for DEN and zoledronic acid (ZOL, a reference drug). Changes in RORs were compared between the pre- (Pre, April 2012 to September 2012) and post- (Post 1, October 2012 to September 2013 and Post 2, October 2013 to September 2014) periods of the regulatory action. A decrease in the hypocalcemia ROR was observed for DEN in the post-periods, especially Post 2. Multivariate logistic regression analysis showed a significant decrease in hypocalcemia signal in Post 1 (p=0.0306 vs. Pre) and Post 2 (p=0.0054 vs. Pre). ZOL caused no significant changes in ROR of hypocalcemia, and none of the drugs caused ROR changes in jaw osteonecrosis (a reference adverse event). This study suggests that the regulatory action against hypocalcemia in DEN effectively decreased hypocalcemia signal. Further studies using medical information databases are needed to confirm this result.

Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes.

Thymic stromal lymphopoietin (TSLP) is regarded as the main factor responsible for the pathogenesis of atopic dermatitis (AD). Cigarette smoke is an aggravating factor for allergies, but has been reported to decrease the risk of AD. In the present study, we evaluated the role of nicotine, the main constituent in cigarette smoke extract, and its underlying mechanism of action in the regulation of TSLP expression. We found that nicotine significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced TSLP expression in BALB/c mice and the mouse keratinocyte cell line PAM212. Nicotine inhibition of TSLP production was abolished by pretreatments with α7 nicotinic acetylcholine receptor (α7 nAChR) antagonists, AMP-activated protein kinase (AMPK) inhibitor, and phosphoinositide 3-kinase (PI3K) inhibitors. The same inhibitors abolished inhibition of nuclear factor-κB (NF-κB) activation by nicotine. These results suggest that nicotine inhibits the expression of TSLP by suppressing the activation of NF-κB through the α7 nAChR-PI3K-AMPK signaling pathway.

Genetic Polymorphisms of CYP2A6 in a Case-Control Study on Bladder Cancer in Japanese Smokers.

Several of the procarcinogens inhaled in tobacco smoke, the primary risk factor for bladder cancer, are activated by CYP2A6. The association between the whole-gene deletion of CYP2A6 (CYP2A6*4) and a reduced risk of bladder cancer was suggested in Chinese Han smokers. However, there is no evidence for association between the risk of bladder cancer and CYP2A6 genotypes in the Japanese population. Using genomic DNA from smokers of the Japanese population (163 bladder cancer patients and 116 controls), we conducted a case-control study to assess the association between CYP2A6 polymorphisms and the risk of bladder cancer. Determination of CYP2A6 genotypes was carried out by amplifying each exon of CYP2A6 using polymerase chain reaction (PCR) and Sanger sequencing. The CYP2A6*4 allele was identified by an allele-specific PCR assay. Bladder cancer risk was evaluated using the activity score (AS) system based on CYP2A6 genotypes. The odds ratios (95% confidence interval) for the AS 0, AS 0.5, AS 1.0, and AS 1.5 groups were 0.46 (0.12-1.83), 0.43 (0.15-1.25), 0.86 (0.40-1.86), and 1.36 (0.60-3.06), respectively. In conclusion, although decreased CYP2A6 AS tended to reduce the risk of bladder cancer in Japanese smokers, no significant association was recognized in this population. However, given the relatively small size of the sample, further study is required to conclude the lack of a statistically significant association between CYP2A6 genotypes and the risk of bladder cancer.

CYP2A13 Genetic Polymorphisms in Relation to the Risk of Bladder Cancer in Japanese Smokers.

Tobacco-specific nitrosamines including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), which can be activated by the metabolic enzyme CYP2A13, are potent procarcinogens. Smoking plays a role in carcinogenesis in the human bladder, which expresses CYP2A13 at a relatively high level. Numerous genetic polymorphisms of CYP2A13 causing amino acid substitution might reduce CYP2A13 metabolic activity toward NNK and NNN, resulting in decreased susceptibility to bladder cancer. The aim of this study was to reveal any association between bladder cancer development and CYP2A13 genetic polymorphisms in Japanese smokers. The CYP2A13 genotype of each subject (163 bladder cancer patients and 161 controls) was determined by next-generation sequencing (NGS) of the full CYP2A13 gene. All samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7). Based on biological logistic regression, the odds ratio (95% confidence interval) for the CYP2A13*1/*2 genotype was 0.34 (0.17-0.69). Thus, CYP2A13 genetic polymorphisms might play important roles in the development of bladder cancer in Japanese smokers.

Histamine synthesis is required for granule maturation in murine mast cells.

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator.

Exacerbation of allergic diseases by chemicals: role of TSLP.

Environmental chemicals, such as cigarette smoke and diesel exhaust, have been reported as risk factors that exacerbate allergic diseases. However, the exacerbation mechanisms induced by these chemicals are not yet fully understood. Thymic stromal lymphopoietin (TSLP) is produced mainly by epithelial cells and plays an important role as a master switch of allergic inflammation because it promotes Th2-type immune responses by inducing the activation of dendritic cells. Chemical compounds, such as formalin, have been shown to bind to proteins and form a new antigen that induces allergic responses. A second group of chemicals that enhance allergic responses to exogenous proteins have also been reported. We recently demonstrated that some of these chemicals induced TSLP production and may potentially augment Th2-type allergic responses. We proposed that TSLP-producing chemical compounds should be recognized as chemical allegro-accelerators.

The role of histamine H1 and H4 receptors in atopic dermatitis: from basic research to clinical study.

Histamine plays important roles in inflammation and nervous irritability in allergic disorders, including atopic dermatitis (AD). It has been shown to regulate the expression of pruritic factors, such as nerve growth factor and semaphorin 3A, in skin keratinocytes via histamine H1 receptor (H1R). Furthermore, H1R antagonist reduced the level of IL-31, a cytokine involving the skin barrier and pruritus, in chronic dermatitis lesions in NC/Nga mice and patients with AD. Histamine plays roles in the induction of allergic inflammation by activating eosinophils, mast cells, basophils, and Th2 cells via histamine H4 receptor (H4R). H4R, in addition to H1R, is expressed on sensory neurons, and a decrease in scratching behaviors was observed in H4R-deficient mice and mice treated with a H4R antagonist. We found that the combined administration of H1R and H4R antagonists inhibited the itch response and chronic allergic inflammation, and had a pharmacological effect similar to that of prednisolone. Although the oral administration of H1R antagonists is widely used to treat AD, it is not very effective. In contrast, JNJ39758979, a novel H4R antagonist, had marked effects against pruritus in Japanese patients with AD in a phase II clinical trial. Next generation antihistaminic agents possessing H1R and H4R antagonistic actions may be a potent therapeutic drug for AD.

Caspases downregulate nickel and hydrogen peroxide-induced IL-8 production via modification of c-Jun N-terminal kinases.

Nickel (Ni) is a typical hapten in allergic contact dermatitis. However, it has been used in various metal materials due to its usefulness. Although Ni ions induce apoptosis of inflammatory cells and the expression of inflammatory cytokines such as interleukin-8 (IL-8), the effects of the apoptotic pathway on the signaling that induces cytokine production have not been sufficiently clarified. Here, we found that NiCl2-induced IL-8 production was enhanced by the pan-caspase inhibitor Z-VAD-FMK in THP-1 cells. Moreover, Z-VAD-FMK enhanced H2O2-induced and NiCl2-induced IL-8 production, but not TNF-α-induced one. The analyses of signaling pathways apparently showed that NiCl2- and H2O2-induced phosphorylation of c-Jun, but not TNF-α-induced one were enhanced by Z-VAD-FMK. The cleavages of p54c-Jun N-terminal kinase (JNK) as well as PARP was induced by NiCl2 and H2O2 but not by TNF-α. Finally, a JNK inhibitor, SP600125, inhibited Z-VAD-FMK-induced enhancement of IL-8 production. In summary, we showed that caspase activation in the apoptotic pathway actively downregulates the JNK-mediated activation of inflammatory cells. This study highlighted the significance of apoptosis in inflammatory diseases, including Ni-induced dermatitis.