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Objective and design: We investigated the effects of the thromboxane (TX) A2 antagonist seratrodast, the peptide leukotriene (p-LT) antagonist pranlukast, the antihistaminic drug terfenadine and the glucocorticoid dexamethasone on antigen-induced sneezing, biphasic nasal blockage and nasal hyperresponsiveness to histamine using a guinea pig model of allergic rhinitis.¶ Subjects: Male Hartley guinea pigs were used.¶ Treatment: Intranasally sensitized guinea pigs were challenged once every week for 13 weeks by inhalation of Japanese cedar pollen as the antigen. Dexamethasone and other agents were administered orally 3 and 1 h, respectively, before the 4th, 6th and 13th challenge.¶ Methods: Sneezing frequency and the change in specific airway resistance (sRaw) were measured at these challenges. Two days after the 13th challenge, nasal responsiveness to histamine was evaluated by measuring sRaw after intranasal instillation of increasing doses of histamine. Moreover, the levels of TXB2, p-LTs and histamine were estimated in nasal cavity lavage fluid (NCLF) collected at the 13th challenge.¶ Results: Only terfenadine (10 mg/kg) significantly inhibited sneezing at any challenge time. Seratrodast (3 and 10 mg/ kg), pranlukast (30 mg/kg) and dexamethasone (10 mg/kg), but not terfenadine, suppressed both the early and late phase elevation of sRaw (biphasic nasal blockage), although the degree of inhibition on the early phase response varied with the challenge time. In contrast, the development of nasal hyperresponsiveness to histamine was inhibited by only dexamethasone. Furthermore, biphasic increases in TXB2, p-LTs and histamine in NCLF were observed after the challenge in sensitized animals.¶ Conclusions: These results suggest that TXA2 and p-LTs, but not histamine, play important roles in both the early and the late phase nasal blockage in this model of allergic rhinitis. [ABSTRACT FROM AUTHOR]

Objective and Design: We previously demonstrated that oral l-ephedrine exerts an extremely rapid (within 20 s) inhibition of 48-h passive cutaneous anaphylaxis reaction (PCA) in rats by a possibly unidentified mode of action. In the present experiments, we elucidated the mechanism of the PCA inhibition by l-ephedrine using adrenoceptor agonists and antagonists.¶ Materials: Rat antiserum was prepared with dinitrophenylated Ascaris suum extract + Bordetella pertussis.¶ Treatment: Passively skin-sensitised Wistar rats were mainly used. l-Ephedrine, and adrenoceptor agonists and antagonists were orally administered immediately before PCA provocation. Catecholamine depleting (6-hydroxydopamine, 6-OH-DA), amine depleting (reserpine) or ganglion blocking (hexamethonium) agent was intraperitoneally or intravenously administered before the provocation.¶ Methods: The effects of the drugs on PCA were assessed by inhibition of the dye leakage.¶ Results: β-(propranolol) and β 2-(butoxamine) blocking agents reduced the inhibition of PCA by l-ephedrine, while the inhibition was not altered by either an α-blocking agent (phentolamine) or a β 1-(atenolol) selective antagonist. On the other hand, β-(isoproterenol) and β 2-selective (salbutamol) agonists showed extremely rapid inhibition of PCA. However, the β 1-selective agonist (dobutamine) had no effect on the reaction. The pretreatment with hexamethonium, reserpine or 6-OH-DA substantially attenuated the inhibitory effect of l-ephedrine on PCA.¶ Conclusions: The results strongly suggest that β 2-adrenoceptors locate in the stomach and that their receptor excitement finally may lead to the inhibition of PCA via the stimulation of the central and peripheral nervous systems. [ABSTRACT FROM AUTHOR]

No abstract available Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 μg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s., (© 2024 John Wiley & Sons Ltd.)

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-β-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-β production in the lungs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Regulated necrosis, termed necroptosis, represents a potential therapeutic target for refractory cancer. Ceramide nanoliposomes (CNLs), considered potential chemotherapeutic agents, induce necroptosis by targeting the activating protein mixed lineage kinase domain-like protein (MLKL). In the present study, we examined the potential of pronecroptotic therapy using CNLs for refractory triple-negative breast cancer (TNBC), for which there is a lack of definite and effective therapeutic targets among the various immunohistological subtypes of breast cancer. MLKL mRNA expression in tumor tissues was significantly higher in TNBC patients than in those with non-TNBC subtypes. Similarly, among the 50 breast cancer cell lines examined, MLKL expression was higher in TNBC-classified cell lines. TNBC cell lines were more susceptible to the therapeutic effects of CNLs than the non-TNBC subtypes of breast cancer cell lines. In TNBC-classified MDA-MB-231 cells, the knockdown of MLKL suppressed cell death induced by CNLs or the active substance short-chain C 6 -ceramide. Accordingly, TNBC cells were prone to CNL-evoked necroptotic cell death. These results will contribute to the development of CNL-based pronecroptotic therapy for TNBC.

Between 5 and 10% of asthma patients do not respond to glucocorticoid therapy. Experimental animal models are indispensable for investigating the pathogenesis of steroid-resistant asthma; however, the majority of murine asthma models respond well to glucocorticoids. We previously reported that multiple intratracheal administration of ovalbumin (OVA) at a high dose (500 µg/animal) induced steroid-insensitive airway eosinophilia and remodeling with lung fibrosis, whereas a low dose (5 µg/animal) caused steroid-sensitive responses. The aims of the present study were as follows: 1) to clarify whether airway hyperresponsiveness (AHR) in the two models is also insensitive and sensitive to a glucocorticoid, respectively, and 2) to identify steroid-insensitive genes encoding extracellular matrix (ECM) components and pro-fibrotic factors in the lung. In comparisons with non-challenged group, the 5- and 500-µg OVA groups both exhibited AHR to methacholine. Daily intraperitoneal treatment with dexamethasone (1 mg/kg) significantly suppressed the development of AHR in the 5-µg OVA group, but not in the 500-µg OVA group. Among genes encoding ECM components and pro-fibrotic factors, increased gene expressions of fibronectin and collagen types I, III, and IV as ECM components as well as 7 matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, transforming growth factor-β1, and activin A/B as pro-fibrotic factors were insensitive to dexamethasone in the 500-µg OVA group, but were sensitive in the 5-µg OVA group. In conclusion, steroid-insensitive AHR developed in the 500-µg OVA group and steroid-insensitive genes encoding ECM components and pro-fibrotic factors were identified. Drugs targeting these molecules have potential in the treatment of steroid-resistant asthma.

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.

Allergen immunotherapy (AIT), such as subcutaneous immunotherapy (SCIT), is a treatment targeting the causes of allergic diseases. The roles of extracellular vesicles (EVs), bilayer lipid membrane blebs released from all types of cells, in AIT have not been clarified. To examine the roles of EVs in SCIT, it was analyzed whether (1) EVs are phenotypically changed by treatment with SCIT, and (2) EVs derived from SCIT treatment suppress the function of group 2 innate lymphoid cells (ILC2s), which are major cells contributing to type 2 allergic inflammation. As a result, (1) expression of CD9, a canonical EV marker, was highly up-regulated by SCIT in a murine model of asthma; and (2) IL-5 production from ILC2s in vitro was significantly decreased by the addition of serum EVs derived from SCIT-treated but not non-SCIT-treated mice. In conclusion, it was indicated that EVs were transformed by SCIT, changing to a suppressive phenotype of type 2 allergic inflammation.

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Subgroups of patients with severe asthma showing marked increases in sputum eosinophils and/or neutrophils are insensitive to corticosteroids. Previous reports have shown that exogenous administration of an anti-inflammatory cytokine, interleukin (IL)-10 negatively regulated both eosinophilic and neutrophilic migration into tissues. The objective of this study was to elucidate whether intratracheal IL-10 administration suppresses asthmatic responses in a steroid-insensitive model of mice. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 500 µg/animal four times. Dexamethasone (1 mg/kg, intraperitoneal) or IL-10 (25 ng/mouse, intratracheal) was administered during the multiple challenges. The number of leukocytes, expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-10 receptor in the lung, and the development of airway remodeling and hyperresponsiveness were evaluated after the fourth challenge. Consistent with our previous study, dexamethasone hardly suppressed the development of airway remodeling and hyperresponsiveness. Although intratracheal IL-10 administration did not affect the development of airway remodeling, the infiltration of eosinophils and neutrophils, and the development of airway hyperresponsiveness were significantly inhibited. Moreover, IL-10 administration significantly decreased the numbers of ICAM-1 + and VCAM-1 + pulmonary vascular endothelial cells, which express IL-10 receptor 1, even though neither production of eosinophilic nor neutrophilic cytokines in the lung was inhibited. Therefore, IL-10 can suppress eosinophil and neutrophil infiltration by inhibiting the proliferation of ICAM-1 + and VCAM-1 + pulmonary vascular endothelial cells, resulting in inhibition of airway hyperresponsiveness in steroid-insensitive asthmatic mice. IL-10 replacement therapy may be clinically useful for the treatment of steroid-insensitive asthma., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Allergen-specific immunotherapy (AIT) is the only causative treatment for allergic diseases by modification of the immune response to allergens. A key feature of AIT is to induce immunotolerance to allergens by generating antigen-specific regulatory T (Treg) cells in allergic patients. Type 1 regulatory T (Tr1) cells and forkhead box protein 3 (Foxp3)-expressing Treg cells are well known among Treg cell subsets. Foxp3 was identified as a master transcription factor of Treg cells, and its expression is necessary for their suppressive activity. In contrast to Foxp3 + Treg cells, the master transcription factor of Tr1 cells has not been elucidated. Nevertheless, Tr1 cells are generally considered as a distinct subset of Treg cells induced in the periphery during antigen exposure in tolerogenic conditions and can produce large amounts of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor-β, followed by down-regulation of the function of effector immune cells independently of Foxp3 expression. Since the discovery of Tr1 cells more than 20 years ago, research on Tr1 cells has expanded our understanding of the mechanism of AIT. Although the direct precursors and true identity of these cells continues to be disputed, we and others have demonstrated that Tr1 cells are induced in the periphery by AIT, and the induced cells are re-activated by antigens, followed by suppression of allergic symptoms. In this review, we discuss the immune mechanisms for the induction of Tr1 cells by AIT and the immune-suppressive roles of Tr1 cells in AIT., Competing Interests: Takeshi Nabe is an external evaluation committee of Ono Pharmaceutical Co., Ltd. (Osaka, Japan)., (© 2022 Matsuda, Terada, Kitatani, Kawata and Nabe.)

A certain population of asthma patients is resistant to steroid therapy, whereas the mechanisms remain unclear. One of characteristic features of steroid-resistant asthma patients is severe airway eosinophilia based on type-2 inflammation. Aims of this study were: 1) to develop a murine model of steroid-resistant asthma, 2) to elucidate that predominant cellular source of a type-2 cytokine, IL-5 was group 2 innate lymphoid cells (ILC2s), 3) to analyze pathogenic alteration of ILC2s in the severe asthma, and 4) to evaluate therapeutic potential of anti-IL-5 monoclonal antibody (mAb) on the steroid-resistant asthma. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 5 or 500 μg/animal 4 times. Development of airway eosinophilia and remodeling in 5-μg OVA model were significantly suppressed by 1 mg/kg dexamethasone, whereas those in 500-μg OVA model were relatively insensitive to the dose of dexamethasone. ILC2s isolated from the lung of the steroid-insensitive model (500-μg OVA) produced significantly larger amounts of IL-5 in response to IL-33/TSLP than ILC2s from the steroid-sensitive model (5-μg OVA). Interestingly, TSLP receptor expression on ILC2s was up-regulated in the steroid-insensitive model. Treatment with anti-IL-5 mAb in combination with dexamethasone significantly suppressed the airway remodeling of the steroid-insensitive model. In conclusion, multiple intratracheal administration of a high dose of antigen induced steroid-insensitive asthma in sensitized mice. IL-5 was mainly produced from ILC2s, phenotype of which had been pathogenically altered probably through the up-regulation of TSLP receptors. IL-5 blockage could be a useful therapeutic strategy for steroid-resistant asthma., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There is a certain population of intractable asthma patients, who can not be controlled by corticosteroid therapy. It has been suggested that 5-10% of asthma patients have been suffered from steroid resistance. Since it has been difficult to develop a steroid-resistant asthma model, the detailed mechanisms have been unclear. Recently, an intractable asthma model showing steroid insensitivity was developed by the author and colleagues. We found that pathogenic changes in type 2 innate lymphoid cells (ILC2) were induced in the intractable asthma. When ovalbumin (OVA) + Al(OH) 3 -sensitized BALB/c mice were intratracheally challenged with OVA at 5 μg/animal, development of airway remodeling as well as lung eosinophilia and neutrophilia were markedly suppressed by treatment with dexamethasone. In contrast, when increasing the dose of OVA for challenges to 500 μg/animal, those asthmatic responses turned to be steroid insensitive. When Th2 cells and ILC2 in the lung were stimulated in vitro, ILC2 produced larger amounts of type 2 cytokines than Th2 cells. Interestingly, amounts of type 2 cytokines produced by the steroid-insensitive model-derived ILC2 were significantly larger than those by the steroid-sensitive, and that the former ILC2 exhibited higher expression of thymic stromal lymphopoietin (TSLP) receptor and signal transducer and activator of transcription (STAT) 5a gene. Treatment with anti-IL-5 antibody improved the steroid sensitivity. Taken together, ILC2 have been transformed to be pathogenic in the intractable asthma. IL-5 hyper-produced from ILC2 may be involved in the development of steroid resistance. The molecules related to the above mentioned are expected to be targets for development of new therapeutic drugs for intractable asthma.

We investigate as yet an unidentified role of NOX1, a non-phagocytic isoform of the superoxide-generating NADPH oxidase, in immune responses using Nox1-knockout mice (Nox1-KO). The transcripts of NOX1 was expressed in lymphoid tissues, including the spleen, thymus, bone marrow, and inguinal lymphoid nodes. When antibody production after ovalbumin (OVA) immunization was examined, no significant differences were observed in serum anti-OVA IgG levels between wild-type mice (WT) and Nox1-KO. In the experimental asthma, the infiltration of eosinophils and the Th2 cytokine response after the induction of asthma with OVA were similar between the two genotypes. However, the severity and incidence of experimental collagen-induced arthritis (CIA) following the administration of a low dose of endotoxin (LPS) were significantly lower in Nox1-KO. While neither serum levels of autoantibodies nor in vitro cytokine responses were affected by Nox1 deficiency, NOX1 mRNA levels in the spleen significantly increased after the LPS challenge. Among the spleen cells, remarkable LPS-induced upregulation of NOX1 was demonstrated in both CD11b + monocytes/macrophages and CD11c + dendritic cells, suggesting that LPS-inducible NOX1 in monocytes/macrophages/dendritic cells may modulate the development of experimental CIA. Therapeutic targeting of NOX1 may therefore control the onset and/or severity of arthritis which is exacerbated by bacterial infection., Competing Interests: Declaration of competing interest C. Y.-N. is the cofounder of a startup company developing NOX inhibitors., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Objective: At least 3 years of sublingual immunotherapy (SLIT) is required to achieve long-term clinical tolerance for allergens. However, immunological changes with more than 3 years of SLIT have not yet been elucidated in detail. The present study investigated whether the numbers of regulatory T (Treg) cells and regulatory B (Breg) cells increased with 4 years of SLIT and if these increases correlated with clinical effects for pollinosis., Methods: Seven Japanese cedar pollinosis patients received SLIT in 2014 or 2015 and continued treatment until May 2019. In May 2017 and May 2019, peripheral blood mononuclear cells (PBMCs) were collected from the patients, and analyzed by flow cytometer., Results: (1) The visual analogue scale (VAS) was significantly higher in 2019 than in 2017. (2) The percentages of Foxp3 + Treg cells, type 1 regulatory T (Tr1) cells, and Breg cells in PBMCs were significantly higher in 2019 than in 2017. (3) The percentage of Foxp3 + Treg cells in PBMCs positively correlated with VAS, whereas those of Tr1 cells and Breg cells did not., Conclusions: These results suggest that 4 years of SLIT is needed to achieve sustained increases in Foxp3 + Treg cells, which are closely associated with the efficacy of SLIT.

Juvenile hormone (JH) agonists constitute a subclass of insect growth regulators and play important roles in insect pest management. In this work, a multi-step virtual screening program was executed to find novel JH agonists. A database of 5 million purchasable compounds was sequentially processed with three computational filters: (i) shape and chemical similarity as compared to known JH-active compounds; (ii) molecular docking simulations against a Drosophila JH receptor, methoprene-tolerant; and (iii) free energy calculation of ligand-receptor binding using a modified MM/PBSA (molecular mechanics/Poisson-Boltzmann surface area) protocol. The 11 candidates that passed the three filters were evaluated in a luciferase reporter assay, leading to the identification of a hit compound that contains a piperazine ring system (EC 50 =870 nM). This compound is structurally dissimilar to known JH agonists and synthetically easy to access; therefore, it is a promising starting point for further structure optimization., (© Pesticide Science Society of Japan 2021. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C 24:1 -ceramide for the formation of lamellipodia. These results suggested that C 24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C 24:1 -ceramide. Collectively, the CerS2-C 24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer., (© 2021 Federation of American Societies for Experimental Biology.)

Atopic dermatitis (AD) is a common skin disease characterized by chronic inflammation and itchiness. Although skin barrier dysfunction and immune abnormalities are thought to contribute to the development of AD, the precise pathogenic mechanism remains to be elucidated. We have developed a unique, diet-induced AD mouse model based on the findings that deficiencies of certain polyunsaturated fatty acids and starches cause AD-like symptoms in hairless mice. Here, we present a protocol and tips for establishing an AD mouse model using a custom diet modified from a widely used standard diet (AIN-76A Rodent Diet). We also describe methods for evaluating skin barrier dysfunction and analyzing itch-related scratching behavior. This model can be used not only to investigate the complex pathogenic mechanism of human AD but also to study the puzzling relationship between nutrition and AD development.

Particularity of ocular immunity is manifested by "Immune privilege". For example, it has been generally known that corneal transplantation is a typically successful organ transplantation compared with other organs. This immune privilege can be explained by "immune-suppressive ocular microenvironment" and "anterior chamber-associated immune deviation, ACAID". This review focused on molecular mechanisms of the "immune-suppressive ocular microenvironment" and "ACAID", so that possible anti-inflammatory strategies could be raised. Especially, in murine ACAID model, anti-inflammatory actions were induced probably through induction of Treg cells. As an anti-inflammatory strategy, anti-inflammatory Treg cells could be induced in vitro. Treg cells that are specifically responsive for a specific antigen can be induced by culturing spleen cells with the antigen and transforming growth factor-β (TGF-β). The induced Treg cells were activated by stimulation with the specific antigen. When the induced Treg cells were adoptively transferred to recipient mice, antigen-induced inflammation was effectively suppressed. The Treg cells may be able to be efficiently induced by eye-based mechanisms. Further analyses of mechanisms underlying the ocular immune privilege can be useful for development of new anti-inflammatory strategies on the eye basis.

Herein, we describe the design and synthesis of cimetidine analogs, as well as their inhibitory activity toward the human multidrug and toxin extrusion transporter 1 (hMATE1), which is related to nephrotoxicity of drugs. Cimetidine is the histamine H 2 -receptor antagonist, but also inhibits hMATE1, which is known to cause renal impairment. We designed and synthesized cimetidine analogs to evaluate hMATE1 inhibitory activity to reveal whether the analogs could reduce the inhibition of hMATE1. The results showed that all analogs with an unsubstituted guanidino group exhibited hMATE1 inhibitory activity. On the other hand, there was a clear difference in the hMATE1 inhibitory activity for the other compounds. That is, compounds with a methylimidazole ring exhibited hMATE1 inhibition, while compounds with a phenyl ring did not. The results suggest that the ability to form hydrogen bonds at the azole moiety is strongly involved in the hMATE1 inhibition.

Background: Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors., Results: To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcription-inducing activity., Conclusion: Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides., (© 2020 Society of Chemical Industry.)

Several non-apoptotic regulated cell death pathways have been recently reported. Necroptosis, a form of necrotic-regulated cell death, is characterized by the involvement of receptor-interacting protein kinases and/or the pore-forming mixed lineage kinase domain-like protein. Recent evidence suggests a key role for lipidic molecules in the regulation of necroptosis. The purpose of this mini-review is to outline the regulation of necroptosis by sphingolipids and phospholipids.

Atopic dermatitis is a chronic inflammatory skin disease associated with CD4 + Th2 cell-shifted immune responses. Although the infiltration of skin lesions by CD8 + T cells has been recognized, their roles have not been fully defined. In this study, we examined the relationship between CD4 + and CD8 + cells in antigen-induced skin lesions of mice. BALB/c mice were repeatedly challenged with Dermatophagoides farinae (Der f) applied to the right ear nine times. Pre-treatment with anti-CD4 monoclonal antibody (mAb) during the third to sixth challenges, but not the post-treatment during the sixth to ninth challenges, exacerbated the development of Der f-induced ear swelling; pre-treatment with anti-CD25 mAb, which depletes regulatory T cells (Tregs), also exacerbated the lesions. Furthermore, the number of CD8 + T cells in lymph nodes was augmented by these pre-treatments. These findings prompted us to examine the effect of anti-CD8 mAb. Pre-treatment with anti-CD8 mAb, but not post-treatment, strongly inhibited the development of Der f-induced ear swelling; additionally, the epidermal hyperplasia and infiltration of mast cells were inhibited by the pre-treatment. Collectively, we revealed that CD8 + T cells regulated by CD4 + CD25 + Tregs in the early stage are key contributors to the development of Der f-induced skin lesions via increasing mast cell infiltration, indicating that CD8 + T and Tregs could be potential therapeutic targets for atopic dermatitis., (Copyright © 2019 Elsevier B.V. All rights reserved.)