OVA-specific IgE Levels Research Articles

Downregulation of Orai1 expression in the airway alleviates murine allergic rhinitis

Orai1 is the key subunit of the Ca2+-release-activated Ca2+ channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.

Cigarette smoke exacerbates mouse allergic asthma through Smad proteins expressed in mast cells

BackgroundMany studies have found that smoking reduces lung function, but the relationship between cigarette smoke and allergic asthma has not been clearly elucidated, particularly the role of mast cells. This study aimed to investigate the effects of smoke exposure on allergic asthma and its association with mast cells.MethodsBALB/c mice were sensitized and challenged by OVA to induce asthma, and bone marrow-derived mast cells (BMMCs) were stimulated with antigen/antibody reaction. Mice or BMMCs were exposed to cigarette smoke or CSE solution for 1 mo or 6 h, respectively. The recruitment of inflammatory cells into BAL fluid or lung tissues was determined by Diff-Quik or H&E staining, collagen deposition by Sircol assay, penh values by a whole-body plethysmography, co-localization of tryptase and Smad3 by immunohistochemistry, IgE and TGF-β level by ELISA, expressions of Smads proteins, activities of signaling molecules, or TGF-β mRNA by immunoblotting and RT-PCR.ResultsCigarette smoke enhanced OVA-specific IgE levels, penh values, recruitment of inflammatory cells including mast cells, expressions of smad family, TGF-β mRNA and proteins, and cytokines, phosphorylations of Smad2 and 3, and MAP kinases, co-localization of tryptase and Smad3, and collagen deposition more than those of BAL cells and lung tissues of OVA-induced allergic mice. CSE solution pretreatment enhanced expressions of TGF-β, Smad3, activities of MAP kinases, NF-κB/AP-1 or PAI-1 more than those of activated-BMMCs.ConclusionsThe data suggest that smoke exposure enhances antigen-induced mast cell activation via TGF-β/Smad signaling pathways in mouse allergic asthma, and that it exacerbates airway inflammation and remodeling.

Elf3 Regulates Allergic Airway Inflammation by Controlling Dendritic Cell-Driven T Cell Differentiation

Elf3 belongs to the Ets family of transcription factors and has been implicated in inflammation. Elf3 is highly expressed in the lungs, and Elf3(-/-) mice are impaired in IL-6 production after intranasal LPS exposure. To identify the role of Elf3 in Th17-driven pulmonary inflammation, we have performed epicutaneous sensitization of Elf3(-/-) mice with OVA followed by airway OVA challenge and have identified Elf3(-/-) mice to be impaired in induction of Th17 response, attributable to impairment of IL-6 production by dendritic cells (DCs). However, increased serum levels of OVA-specific IgG1 and IgE were observed, pointing toward an exaggerated Th2 response. To study Th2 response, we performed i.p. sensitization of Elf3(-/-) mice with OVA and confirmed loss of Elf3 to result in an aggravated Th2 response, characterized by increased generation of IL-4-producing T cells, increased levels of OVA-specific IgE and IgG1 Ab titers, and increased serum levels of Th2 cytokines, together with extensive inflammation and mucus production in airways. Elf3(-/-) DCs were impaired in priming Th1 differentiation, which, in turn, promoted Th2 differentiation. This was mediated by the ability of Elf3(-/-) DCs to undergo hypermaturation but secrete significantly lower levels of IL-12 in response to inflammatory stimuli. The impairment of IL-12 production was due to impairment of IL-12p40 gene induction in Elf3(-/-) DCs in response to inflammatory stimuli. Taken together, our study identifies a novel function of Elf3 in regulating allergic airway inflammation by regulating DC-driven Th1, Th2, and Th17 differentiation.

Staphylococcus Aureus Enterotoxin B Contributes to Induction of Nasal Polypoid Lesions in an Allergic Rhinosinusitis Murine Model

Studies on the pathophysiology of nasal polyps in human subjects have been limited; thus an animal model is needed. There is increasing evidence supporting the role of Staphylococcus aureus enterotoxin B (SEB) in the pathogenesis of nasal polyposis. The aim of this study was to investigate the histological and immunologic effects of SEB on the formation of nasal polypoid lesions in an allergic rhinosinusitis murine model. After induction of an ovalbumin (OVA)-induced allergic rhinosinusitis, OVA with SEB (5 or 500 ng) was instilled into the nasal cavity of mice for 8 weeks. Control mice did not receive SEB or OVA instillation. Histopathological changes were observed using hematoxylin and eosin, Sirius red, Giemsa, Masson's trichrome, and Alcian blue stains. The levels of interleukin (IL)-4, IL-5, IL-8, IL-13, eotaxin, interferon gamma, total IgE, and OVA-specific IgE from serum or nasal lavage fluid were measured using enzyme-linked immunosorbent assay. The group treated with OVA plus 5 ng of SEB had significantly more mucosal lesions with epithelial disruption and nasal polypoid lesions than mice treated with OVA only, showing a significant increase in the infiltration of total inflammatory cells, eosinophils, and lymphocytes than the other groups. Levels of IL-5, eotaxin, and OVA-specific IgE in nasal lavage fluid were increased in the group treated with OVA plus 5 ng of SEB than in the other groups. A higher number of secretory cells in the groups treated with OVA plus SEB was observed than in other groups. Low-dose SEB induced nasal polypoid lesions with an increased eosinophilic infiltration in an allergic rhinosinusitis murine model.

Effects of simulated downwind coal combustion emissions on pre-existing allergic airway responses in mice

Context: Coal-fired power plant emissions can contribute a significant portion of the ambient air pollution in many parts of the world.Objective: We hypothesized that exposure to simulated downwind coal combustion emissions (SDCCE) may exacerbate pre-existing allergic airway responses.Methods: Mice were sensitized and challenged with ovalbumin (OVA). Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day for 3 days to air (control, C) or SDCCE containing particulate matter (PM) at low (L; 100 μg/m3), medium (M; 300 μg/m3), or high (H; 1000 μg/m3) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after SDCCE exposure, mice received another OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air/SDCCE. Measurement of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ~24 h after the last exposure.Results: SDCCE significantly increased BAL macrophages and eosinophils in OVA-sensitized mice from the post-OVA protocol. However, there was no effect of SDCCE on BAL macrophages or eosinophils in OVA-sensitized mice from the pre-OVA protocol. BAL neutrophils were elevated following SDCCE in both protocols in nonsensitized mice. These changes were not altered by filtering out the PM. In the post-OVA protocol, SDCCE decreased OVA-specific IgG1 in OVA-sensitized mice but increased levels of total IgE, OVA-specific IgE and OVA-specific IgG1 and IgG2a in non-sensitized animals. In the pre-OVA protocol, SDCCE increased OVA-specific IgE in both sensitized and non-sensitized animals. Additionally, BAL IL-4, IL-13, and IFN-γ levels were elevated in sensitized mice.Conclusion: These results suggest that acute exposure to either the particulate or gaseous phase of SDCCE can exacerbate various features of allergic airway responses depending on the timing of exposure in relation to allergen challenge.

The central role of CD30L/CD30 interactions in allergic rhinitis pathogenesis in mice

CD30 ligand (CD30L) plays an important role in the amplification and/or activation of effector CD4(+) T cells, irrespective of Th cell subset. To examine the role of CD30L in allergic rhinitis, we evaluated an OVA model of allergic rhinitis in CD30L knock out (KO) mice on a BALB/c background sensitized with OVA. Symptoms of allergic rhinitis such as eosinophil infiltration into the nasal mucosa were drastically diminished in OVA-sensitized CD30L KO mice following intranasal challenge with OVA. The levels of OVA-specific IgE in the sera and the Th2 response in nasopharynx-associated lymphoid tissues and cervical LNs of CD30L KO mice were significantly lower than those of WT mice following intranasal challenge with OVA. Intranasal administration of CD30-Ig during the effector phase with OVA significantly prevented the development of allergic rhinitis in WT mice. These results suggest that CD30L plays an important role in allergic rhinitis and that the inhibition of CD30L/CD30 signaling might be useful as a novel biological therapy for allergic rhinitis.

Immunostimulatory effects of collagen from jellyfish in vivo

We focused on the biological activity of the collagen extracts obtained from the giant edible jellyfish, Nemopilema nomurai. Jellyfish collagen extracts stimulates the production of immunoglobulins (Igs) and cytokines by human hybridoma cells and human peripheral blood lymphocytes. Therefore, we examined the immunoregulatory function of jellyfish collagen extracts in mice. Intake of jellyfish collagen extracts facilitated the Ig production activity of lymphocytes from spleen and Peyer's patch. Furthermore, the levels of Igs in the serum clearly increased after the administration of jellyfish collagen extracts. Intake of bovine collagen from Achilles' tendon also activated lymphocytes activity in mice. The activity of total and antigen-specific Ig production in splenocytes from OVA-challenged mice was also enhanced by collagen intake. However, the total and OVA-specific IgE levels in the serum were not affected by the collagen intake. These results suggested that jellyfish collagen extracts stimulates an immune response in vivo, without inducing allergic complications.

Acinetobacter baumannii Infection Inhibits Airway Eosinophilia and Lung Pathology in a Mouse Model of Allergic Asthma

Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen.

Suppression of ovalbumin-induced allergic diarrhea by diminished intestinal peristalsis in RAMP1-deficient mice

Recent studies have revealed that various neurotransmitters regulate the immune system via their receptors expressed on the immune cells. Calcitonin gene-related peptide (CGRP), a sensory nerve C-fiber neuropeptide, is also known to have the ability to modulate the functions of immune cells in vitro. However, the contribution of CGRP to the immune regulation in vivo remains to be fully elucidated. Here we report that mice deficient in receptor activity-modifying protein 1 (RAMP1), which is a subunit of the CGRP receptor, showed a significantly lower incidence of diarrhea compared with wild-type (WT) mice in the ovalbumin (OVA)-induced food allergic model. Serum OVA-specific IgE levels and the differentiation of T helper cells was comparable in WT mice and RAMP1-deficient mice. Moreover, there were no significant differences between recruitment and degranulation of mast cells in the small intestine of these mice. In contrast, significantly diminished intestinal peristalsis was observed by the allergy induction in RAMP1-deficient mice compared with WT mice. These results suggest that this suppression of allergic diarrhea is due to the diminished intestinal peristalsis in RAMP1-deficient mice.

Protective Role of 14-Deoxy-11,12-didehydroandrographolide, a Noncytotoxic Analogue of Andrographolide, in Allergic Airway Inflammation

Our group recently reported novel anti-inflammatory effects of andrographolide (2), a bioactive molecule isolated from Andrographis paniculata, in a mouse asthma model. However, 2 has been shown to possess cytotoxic activity. 14-Deoxy-11,12-didehydroandrographolide (1) is an analogue of 2 that can be isolated from A. paniculata. We hypothesized that 1 retains the anti-inflammatory effects for asthma but is devoid of cytotoxicity. In contrast to 2, 1 did not elicit any cytotoxic activity in A549 and BEAS-2B human lung epithelial cells and rat basophilic leukemia (RBL)-2H3 cells using a MTS assay. Compound 1 dose-dependently inhibited ovalbumin (OVA)-induced increases in total and eosinophil counts, IL-4, IL-5, and IL-13 levels in lavage fluid, and serum OVA-specific IgE level in a mouse asthma model. Compound 1 attenuated OVA-induced airway eosinophilia, mucus production, mast cell degranulation, pro-inflammatory biomarker expression in lung tissues, and airway hyper-responsiveness. This substance also blocked p65 nuclear translocation and DNA-binding activity in the OVA-challenged lung and in TNF-α-stimulated human lung epithelial cells. The present findings reveal for the first time that 1 retains the anti-inflammatory activities of 2 for asthma probably through the inhibition of NF-κB. 14-Deoxy-11,12-didehydroandrographolide (1) may be considered as a safer analogue of 2 for the potential treatment of asthma.

Nitrogen dioxide: no influence on allergic sensitization in an intranasal mouse model with ovalbumin and diesel exhaust particles

The role of traffic-related air pollution in the development of allergic diseases is still unclear. We therefore investigated if NO2, an important constituent of traffic-related air pollution, promotes allergic sensitization to the allergen ovalbumin (OVA). We also examined if NO2 influenced the allergy adjuvant activity of diesel exhaust particles (DEP). For this purpose, mice were exposed intranasally to OVA with or without DEP present, immediately followed by exposure to NO2 (5 or 25 parts per million [ppm]) or room air for 4 h in whole body exposure chambers. Eighteen hours after the last of three exposures, the lungs of half of the animals were lavaged with saline and markers of lung damage and lung inflammation in the bronchoalveolar lavage fluid (BALF) were measured. Three weeks later, after intranasal booster immunizations with OVA, the levels of OVA-specific IgE and IgG2a antibodies in serum were determined. Both NO2 (25 ppm) and DEP gave lung damage, measured as increased total protein concentration in BALF, whereas only NO2 seemed to stimulate release of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast, only DEP significantly increased the number of neutrophils. Furthermore, DEP in combination with OVA stimulated the production of serum allergen-specific IgE antibodies. NO2, however, neither increased the production of allergen-specific IgE antibodies, nor influenced the IgE adjuvant activity of DEP. Thus, based on our findings, NO2 seems to be of less importance than combustion particles in the development of allergic diseases after exposure to traffic-related air pollution.

Induction of Immune Tolerance in Asthmatic Mice by Vaccination with DNA Encoding an Allergen–Cytotoxic T Lymphocyte-Associated Antigen 4 Combination

Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA₃.₁) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed and transfected into COS-7 cells to obtain the fusion protein OVA-CTLA-4, which was able to bind the B7 ligand on dendritic cells (DCs), and induced CD25⁺ Foxp3⁺ regulatory T (Treg) cells by the coculture of naive CD4⁺ T cells with DCs in vitro. In an animal study, BALB/c mice were sensitized and challenged with OVA to establish the asthmatic model. Vaccination with a high dose of OVA-CTLA-4-pcDNA₃.₁ significantly decreased interleukin-4 (IL-4) and IL-5 levels and eosinophil counts and prevented OVA-induced reduction of the gamma interferon level in the bronchoalveolar lavage fluid. In addition, these mice suffered less severe airway inflammation and had lower levels of OVA-specific IgE and IgG1 titers in serum. Also, high-dose OVA-CTLA-4-pcDNA₃.₁ vaccination inhibited the development of airway hyperreactivity and prevented OVA-induced reduction of the percentages of Foxp3⁺ Treg cells in the spleen. Our results indicate that a high dose of allergen-CTLA-4-encoding DNA vaccine was more effective in preventing an allergen-induced Th2-skewed immune response through the induction of Treg cells and may be a new alternative therapy for asthma.

Increased susceptibility of 129SvEvBrd mice to IgE-Mast cell mediated anaphylaxis

BackgroundExperimental analyses have identified strain-dependent factors that regulate susceptibility to anaphylaxis in mice. We assessed the susceptibility of the widely used 129SvEvBrd (also known as 129S5) mouse strain to IgE/mast cell-mediated anaphylaxis as compared to BALB/c. Mice were subjected to passive and oral Ovalbumin [OVA]-induced active anaphylaxis. Tissue mast cell, plasma histamine, total IgE and OVA-specific IgE levels and susceptibility to histamine i.v infusion were assessed. Bone marrow mast cell (BMMC)s were examined for FcεRI, c-kit, degranulation efficiency, proliferation, apoptosis and cytokine profile.Results129S5 mice had significantly increased susceptibility to passive and oral OVA-induced active anaphylaxis. Increased susceptibility to anaphylaxis was associated with increased homeostatic mast cell levels but not OVA-specific IgE or IgG1 levels. In vitro analyses of BMMCs revealed no difference in FcεRI and c-Kit expression, however, 129S5 BMMCs possessed greater proliferative capacity and reduced caspase-3-mediated apoptosis. IgE-BMMC degranulation assays demonstrated no difference in degranulation efficiency. Furthermore, 129S5 mice possessed increased sensitivity to histamine-induced hypothermia.ConclusionsWe conclude that 129S5 mice have increased susceptibility to anaphylaxis as compared to BALB/c strain and their increased susceptibility was associated with altered mast cell proliferation and homeostatic tissue levels and responsiveness to histamine. Given the wide spread usage of the 129SvEvBrd strain of mice in experimental gene targeting methodology, these data have important implications for studying IgE-reactions in mouse systems.

Hesperidin Suppresses Ovalbumin-Induced Airway Inflammation in a Mouse Allergic Asthma Model

Hesperidin, a flavanone glycoside comprised of the flavanone hesperetin and the disaccharide rutinose, is a plentiful and inexpensive by-product of citrus cultivation. It has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, and anticarcinogenic properties. In this study, we attempt to determine whether hesperidin inhibits inflammatory mediators in the mouse allergic asthma model. Mice were sensitized and challenged by ovalbumin (OVA) to induce chronic airway inflammation and airway remodeling. The administration of hesperidin significantly decreased the number of infiltrating inflammatory cells and Th2 cytokines in bronchoalveolar lavage (BAL) fluid compared with the OVA-induced group of mice. In addition, hesperidin reduced OVA-specific IgE levels in serum. Hesperidin markedly alleviated the OVA-induced airway hyperresponsiveness (AHR) to inhaled methacholine. Based on lung histopathological studies using hematoxylin and eosin and alcian blue-periodic acid-Schiff staining, hesperidin inhibited inflammatory cell infiltration and mucus hypersecretion compared with the OVA-induced group of mice. These findings provide new insight into the immunopharmacological role of hesperidin in terms of its effects in a murine model of asthma.

Immunomodulatory Role of Histamine H2 Receptor in Allergen‐Specific Immunotherapy

The purpose of this pilot study was to investigate the effects of HR2 on allergen-specific immunotherapy in a mouse model of allergic rhinitis. An in vivo study using an animal model. Catholic Research Institutes of Medical Science. Fifty mice were divided into 5 groups: control, allergic rhinitis (AR), immunotherapy (IT), immunotherapy with HR2 agonist (HI), and immunotherapy with HR2 antagonist (HB). All mice except for the control group were sensitized with ovalbumin (OVA). After 1 week, mice in the IT, HI, and HB groups underwent immunotherapy by intradermal injections of OVA. During immunotherapy, the HI group was injected with HR2 agonist, whereas the HB group was injected with HR2 antagonist. All sensitized mice were challenged with intranasal OVA. After the final challenge, allergic behavior was evaluated. Interleukin (IL)-13, interferon-γ, IL-10, and transforming growth factor (TGF)-β levels in nasal lavage fluid (NALF), as well as OVA-specific IgE levels in serum, were measured. The number of eosinophils in lamina propria was evaluated. The levels of serum OVA-specific IgE and IL-13 in NALF were significantly increased in the HB group compared with the IT group (P < .05). Also, the tissue eosinophil counts were higher in the HB group than in the IT group (P < .05). HR2 antagonist impaired OVA-specific immunotherapy in mice. Although confirmation of this preliminary result is needed, these findings suggest that HR2 receptors may have inhibitory effects on immune tolerance. The authors suggest that application of this property could enhance the efficiency of allergen-specific immunotherapy.

Cimetidine Enhances Antigen-Specific IgE and Th2 Cytokine Production

Treatment with anti-ulcer drugs has been shown to enhance IgE production against food antigens. However, little is known about the immunological effects of cimetidine, a histamine receptor type 2 (H2R) antagonist that is widely used as an anti-ulcer drug, in allergy. Therefore, the present study investigated the role of cimetidine in Th2 immune responses in mice. BALB/c mice were immunized intraperitoneally with ovalbumin (OVA) with and without cimetidine. The levels of cytokines in supernatants of spleen cells cultured in the presence of OVA for 4 days and the levels of total and OVA-specific IgG(1), IgG(2a) and/or IgE in sera from these mice were determined by ELISA. Administration of cimetidine to OVA-sensitized BALB/c mice promoted Th2 cytokine secretion by OVA-stimulated spleen cells in vitro and increased serum levels of OVA-specific IgE, IgG(1) and IgG(2a). These results indicate that cimetidine can enhance Th2 responses, suggesting that cimetidine may contribute to IgE production in allergies.

A Novel Phycobiliprotein Alleviates Allergic Airway Inflammation by Modulating Immune Responses

it has been claimed that phycocyanin exhibits pharmaceutical functions in inhibiting histamine release and leukotriene biosynthesis. In allergic asthma, these inflammatory mediators are crucial for disease progression. the aim of this study was to evaluate the therapeutic potential of R-phycocyanin (R-PC) against allergic airway inflammation. mouse bone marrow-derived dendritic cells (BMDCs) were used to evaluate the immunomodulatory functions of R-PC. In addition, an airway inflammatory model was used to evaluate the therapeutic potential of R-PC. R-PC treatment resulted in a decrease of endocytosis, increase of costimulatory molecule expression, and enhancement of interleukin-12 production in mouse BMDCs. Moreover, R-PC-treated cultured dendritic cells were able to promote CD4(+) T-cell stimulatory capacity and increase interferon-γ expression in CD4(+) T cells. Intraperitoneal administration of R-PC suppressed ovalbumin (OVA)-induced airway hyperresponsiveness, serum levels of OVA-specific IgE and IgG1, eosinophil infiltration, Th2 cytokine levels, and eotaxin in bronchoalveolar lavage fluid of mice. Antibody against Toll-like receptor-4 was able to inhibit R-PC-induced IL-12 p70 production. Moreover, inhibition of nuclear factor-κB (NF-κB) by helenalin and inhibition of the JNK pathway by JNK inhibitor II inhibited R-PC-induced IL-12 p70 production. Western blotting and electrophoretic mobility shift assays showed that R-PC augmented phosphorylation of the inhibitors of NF-κB and inhibitors of NF-κB kinase and facilitated NF-κB activity. our data demonstrated that R-PC promoted activation and maturation of cultured dendritic cells and skewed the immunological function toward Th1 activity. Therefore, R-PC may have potential in regulating immune responses and application in reducing allergic asthma.

Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

Anti‐inflammatory effects of the R2 peptide, an inhibitor of transglutaminase 2, in a mouse model of allergic asthma, induced by ovalbumin

Transglutaminase 2 (TGase 2) expression is increased in inflammatory diseases, and TGase 2 inhibitors block these increases. We examined whether the R2 peptide inhibited the expression of TGase 2 in a mouse model of inflammatory allergic asthma. C57BL/6 mice were sensitized and challenged by ovalbumin (OVA) to induce asthma. OVA-specific serum IgE and leukotrienes (LTs) levels were measured by enzyme-linked immunosorbent assay. Recruitment of inflammatory cells into bronchoalveolar lavage (BAL) fluid or lung tissues and goblet cell hyperplasia were assessed histologically. Airway hyperresponsiveness was determined in a barometric plethysmographic chamber. Expression of TGase 2, eosinophil major basic protein (EMBP), the adhesion molecule vascular cell adhesion molecule-1, Muc5ac and phospholipase A(2) (PLA(2) ) protein were determined by Western blot. Expression of mRNAs of Muc5ac, cytokines, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were measured by reverse transcriptase-polymerase chain reaction and nuclear factor-κB (NF-κB) by electrophoretic mobility shift assay. R2 peptide reduced OVA-specific IgE levels; the number of total inflammatory cells, macrophages, neutrophils, lymphocytes and eosinophils in BAL fluid and the number of goblet cells. Airway hyperresponsiveness, TGase 2 and EMBP levels, mRNA levels of interleukin (IL)-4, IL-5, IL-6, IL-8, IL-13, RANTES, tumour necrosis factor-α, and MMP2/9, Muc5ac, NF-κB activity, PLA(2) activity and expressions, and LT levels in BAL cells and lung tissues were all reduced by R2 peptide. R2 peptide also restored expression of TIMP1/2. R2 peptide reduced allergic responses by regulating NF-κB/TGase 2 activity in a mouse model of allergic asthma. This peptide may be useful in the treatment of allergic asthma.

Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic-coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6.