Importance of IgE-Induced Unique Plasma Leakage in the Skin for Urticaria-Like Symptoms in an Anaphylaxis-Dependent Spotted Distribution of Immune Complex in Skin (ASDIS) Mouse Model.

INTRODUCTION: Allergic diseases, such as anaphylaxis and urticaria, pose significant health concerns. The quest for improved prognostic outcomes in these diseases necessitates the exploration of novel therapeutic avenues. To address this need, we have developed a novel mouse model of anaphylaxis, denoted as anaphylaxis-dependent spotted distribution of immune complex in skin (ASDIS). ASDIS manifests as distinct dotted symptoms in the skin, detectable through in vivo imaging, resembling urticarial symptoms. In this study, we investigated the potential underlying mechanisms giving rise to these dotted symptoms, exploring the role of vascular permeability and characterizing the ASDIS model as a new urticaria model. METHODS: We employed haired and hairless HR mice (BALB/c background) and hairless HR-1 mice (a commercially available hairless strain with an unidentified genetic background). ASDIS was induced by the simultaneous intravenous injection of anti-ovalbumin IgE and fluorescein isothiocyanate (FITC)-ovalbumin, along with Evans blue - a recognized vascular permeability indicator. Anaphylaxis and scratching behavior were monitored through rectal temperature decrease and optical observation, respectively. Histamine, platelet-activating factor, and compound 48/80 were injected with or without FITC-ovalbumin for comparative analysis. The effects of an α1 adrenergic receptor agonist applied to the skin were also examined. RESULTS: In hairless mice, the simultaneous injection of histamine, compound 48/80, or IgE with FITC-ovalbumin induced comparable rectal temperature decreases and vascular permeability. However, only the combination of FITC-ovalbumin and IgE triggered ASDIS, specifically the dotted urticaria-like symptom. Evans blue visualization and optical observation of dotted swelling confirmed that the vascular permeability mediated the phenomenon. Hairless mice exhibited a more pronounced temperature decrease than their haired counterparts when exposed to histamine, platelet-activating factor, compound 48/80, and IgE with FITC-ovalbumin. The application of an α1 adrenergic receptor agonist to the skin attenuated the topical urticaria-like symptom. CONCLUSION: Our experiments revealed four findings. The first is that ASDIS mirrors urticaria-like symptoms resulting from increased vascular permeability, akin to human urticaria. The second finding is that the development of dotted symptoms involves an IgE-induced, yet unidentified, mechanism not triggered by histamine or compound 48/80 alone. The third finding highlights the heightened susceptibility of hairless mice to ASDIS induction. The fourth finding demonstrates that the inhibition of ASDIS by the topical application of an α1 adrenergic receptor agonist hints at a potential anti-urticarial application for this vasoconstrictor. Further elucidation of these unidentified IgE-dependent mechanisms and the specific generation of dotted symptoms by IgE-immune complexes could provide novel insights into allergic response processes and therapeutic interventions for these conditions.

Development of a Novel IgG1 Anaphylaxis Mouse Model with Uniquely Characteristic Skin Manifestations Induced Through the FcγRIII-Histamine Pathway.

BACKGROUND: Studies of passive anaphylaxis, in which mouse immunoglobulin G (IgG) and its antigens are administered to mice, believe that platelet-activating factor (PAF) is more important than histamine and that basophils or macrophages are primarily involved. However, the full extent of IgG-dependent anaphylaxis is still unclear; that is, little agreement has been reached about the mechanism. METHODS: First, we established the novel model of IgG1 anaphylaxis induced by the intravenous administration of two types of IgG1 and a fluorescent dye-labeled antigen, as IgG1 immune complex in HR-1 hairless mice. Subsequently, pharmacological analysis was used to investigate the underlying mechanisms of IgG1 anaphylaxis in this established model. RESULTS: The novel IgG1 anaphylaxis model can induce the IgG-induced Anaphylaxis-dependent Spotted Distribution of fluorescently labeled Immune complexes in the Skin, named "G-ASDIS". Moreover, this model was triggered primarily by the FcγRIII-dependent histamine release, which is different from the conventional model in which PAF was involved in the development of IgG1 anaphylaxis. Basophils in the circulation and mast cells in the skin may participate in the development of IgG1 anaphylaxis and increased G-ASDIS. CONCLUSION: Our results propose that the novel axis, namely the FcγRIII-basophils and/or mast cell-histamine pathway, is important for IgG1 anaphylaxis. Further analysis of our model in addition to other models will lead to a broader analysis and understanding of the IgG1 anaphylaxis mechanism.

Co-induced Allergic Response to an Unrelated Allergen Exacerbates Imiquimod-Induced Psoriasis in Mice.

Psoriasis is classically regarded as a T-helper 1 (Th1) response-dominant disease believed to be antagonized by the Th2 response, which is responsible for allergic diseases, such as atopic dermatitis. The roles of these responses in psoriasis and the relationship between psoriasis and atopic dermatitis have received increasing attention because it is estimated that more than one million patients are concomitantly affected by psoriasis and atopic dermatitis. To address this, we attempted to determine the characteristics of imiquimod-induced psoriasiform lesions in mice with a concomitant allergic response after co-application of the unrelated allergen ovalbumin onto the skin. Imiquimod cream containing ovalbumin was successively applied to the right back skin of hairless HR female mice. Psoriasiform scores were determined for 11 d, and then, the resected skin thickness, spleen weight, and serum antibody levels were examined. In some experiments, mice were allowed free access to ovalbumin-containing water for 10 d before skin application to induce oral tolerance. Imiquimod cream induced psoriasis, and its severity increased upon simultaneous ovalbumin treatment. Increases in anti-ovalbumin immunoglobulin G2a (IgG2a) levels, a Th1 response indicator, and IgG1 and IgE levels, Th2 response indicators, were mediated by ovalbumin addition. Oral tolerance against ovalbumin effectively decreased ovalbumin-exacerbated imiquimod-induced psoriasis, in parallel with a decrease in levels of anti-ovalbumin antibodies. These results suggest that the concomitant allergic response induced by ovalbumin application exacerbates imiquimod-induced psoriasis. This implies that allergic responses to unrelated allergens might exacerbate psoriasis in humans and that modulating such responses could be an effective new approach to treat psoriasis.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Purification of Emu IgY for Therapeutic and Diagnostic Use Based on Monoclonal Secondary Antibodies Specific to Emu IgY.

The emu is the second largest ratite; thus, their sera and egg yolks, obtained after immunization, could provide therapeutic and diagnostically important immunoglobulins with improved production efficiency. Reliable purification tools are required to establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to the immunoglobulin Y (IgY) class. Therefore, we generated a monoclonal secondary antibody specific to emu IgY. Initially, we immunized an emu with bovine serum albumin multiply haptenized with 2,4-dinitrophenyl (DNP) groups. Polyclonal emu anti-DNP antibodies were partially purified using conventional precipitation method and used as antigen for immunizing a BALB/c mouse. Splenocytes were fused with myeloma cells and a hybridoma clone secreting a desirable secondary antibody (mAb#2-16) was established. The secondary antibody bound specifically to emu-derived IgY, distinguishing IgYs from chicken, duck, ostrich, quail, and turkey, as well as human IgGs. Affinity columns immobilizing the mAb#2-16 antibodies enabled purification of emu IgY fractions from sera and egg yolks via simple protocols, with which we succeeded in producing IgYs specific to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein with a practical binding ability. We expect that the presented purification method, and the secondary antibody produced in this study, will facilitate the utilization of emus as a novel source of therapeutic and diagnostic antibodies.

Immunoglobulin a suppresses B cell receptor-mediated activation of mouse B cells with differential inhibition of signaling molecules.

CONTEXT: We previously reported that monoclonal mouse immunoglobulin (Ig) A, OA-4, attenuates sensitization in mice by suppressing B cell activation. OBJECTIVE: Here, it is demonstrated for the first time that mouse IgA inhibits mouse B cell activation in vitro under natural conditions (i.e. in the absence of chemical, physical, and genetic modifications of IgA and B cells). MATERIALS AND METHODS: Mouse splenocytes were stimulated with anti-B cell receptor (BCR) antibody or lipopolysaccharide (LPS) in the presence or absence of OA-4. Splenic B cell proliferation and the activation of several intracellular signaling molecules were measured. RESULTS: Anti-BCR antibody-induced proliferation was markedly inhibited by OA-4 or the commercially available mouse IgA S107, whereas LPS-induced proliferation was weakly attenuated by a high concentration of OA-4. Moreover, OA-4 markedly decreased the anti-BCR antibody-induced phosphorylation of p44/42 mitogen-activated protein kinase (ERK) and CD22 and decreased phosphorylated phospholipase (PLC) γ2 and intracellular Ca2+ levels moderately, whereas protein kinase B (Akt) phosphorylation was not affected by OA-4. The MAPK/ERK kinase-ERK and phosphoinositide 3-kinase-Akt pathways were found to play a role in the proliferation of splenocytes induced by anti-BCR antibody based on experiments with their inhibitors. In contrast to that in splenic B cells, ERK phosphorylation induced by anti-BCR antibody in A20 cells was not inhibited by OA-4. The modulatory effects of IgA were different among the cell types and signaling pathways. CONCLUSION: IgA is a potential immunoregulatory drug utilizing new mechanisms that affect splenic B cells but not A20 lymphomas.

IgA directly inhibits antigen-dependent B cell activation following distinctive distribution of the antigen in mice.

CONTEXT: Serum IgA suppresses immune responses when exposed to antigens recognized by the antibody; however, the underlying mechanism remains unclear. OBJECTIVE: We herein clarified the relationships between changes in antigen distribution and antigen-dependent B cell activation in the presence or absence of IgA against the antigen in mice. MATERIALS AND METHODS: DBA/1J and HR-1 mice were intravenously injected with ovalbumin (OVA) and anti-OVA monoclonal IgA OA-4. The distribution of the antigen and B cell responses were measured. RESULTS: B cell activation by injected OVA, namely, increases in anti-OVA IgG production and the populations of B220(+)GL7(+) and B220(+)CD69(high) splenocytes, was diminished by the co-injection of OA-4. Co-injected OA-4 increased OVA in the serum as well as in the bile and gut. This was coincident with its decrease in the urine due to the inhibition of OVA monomer secretion through the formation of immune complexes. The apparent similarities in the association between fluorescein isothiocyanate (FITC)-OVA and splenic B cells in the presence and absence of OA-4 in vivo appeared to be attributed to compensation between the two effects of OA-4; an increase in serum OVA in vivo and inhibition of the association between OVA and B cells, as suggested by in vitro experiments. DISCUSSION: Based on these results, the stimulation of B cells by OVA may be directly reduced, at least partly, by the neutralization of OVA by OA-4. CONCLUSION: IgA may be an effective drug for the treatment of immune disorders due to its ability to blunt antigen-specific B cell activation.

Intravenous IgA complexed with antigen reduces primary antibody response to the antigen and anaphylaxis upon antigen re-exposure by inhibiting Th1 and Th2 activation in mice.

CONTEXT: Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown. OBJECTIVE: We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice. MATERIALS AND METHODS: DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured. RESULTS: IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA. CONCLUSION: These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Derivatization-assisted immunoassays: application for group-specific detection of potent methamphetamine and amphetamine enantiomers.

Reliable and feasible tools for detecting (S)-methamphetamine [(S)-MAP] and (S)-amphetamine [(S)-AP] are required for regulating their illicit circulation. Antibodies that react equally to these stimulants are desirable for this purpose, but have been difficult to generate because of the crucial difference between their characteristic structures: i.e., N-methylamino (MAP) and amino (AP) groups. Furthermore, their small molecular masses (Mr < 150) have hampered the generation of high-affinity antibodies. To overcome these problems, we converted (S)-MAP and -AP into their 2-(trimethylsilyl)ethyl carbamate forms, Teoc-(S)-MAP and -AP, respectively, as surrogate analytes. The Teoc-derivatization not only increases their molecular masses, but also masks their structural differences. We generated a novel monoclonal antibody that showed a satisfactory affinity to Teoc-(S)-MAP residues (Kd = 13 nM as the IgG form) and developed a competitive enzyme-linked immunosorbent assay (ELISA) using microplates containing immobilized Teoc-(S)-MAP residues. Almost overlapping dose-response curves were obtained for Teoc-(S)-MAP and -AP, with the limit of detection of 0.078 and 0.10 ng per assay, respectively. A fixed amount of test powder sample (1 mg) was derivatized with Teoc-O-succinimidyl for 5 min, and subjected to ELISA using Teoc-(S)-MAP as the calibration standard. Under this protocol, (S)-MAP and -AP were converted to their Teoc derivatives with 30% and 34% yield, respectively, determined using ELISA as "Teoc-(S)-MAP equivalent," being distinguished from the derivatization products of (R)-MAP, (R)-AP, ephedrine, (S)-methylenedioxymethamphetamine, tyramine, dopamine, and ß-alanine. This ELISA detected as little as 10 µg of (S)-MAP and -AP, and (S)-MAP in urine obtained from (S)-MAP-administered rats. Immunochromatography devices were also developed using gold nanoparticles coated with the monoclonal antibody, with which 0.10 mg of (S)-MAP and -AP was detected by the naked eye. We conclude that the present derivatization-assisted immunoassays may be useful for the detection of (S)-MAP and/or -AP in early stage screening of suspicious substances.

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice.

BACKGROUND AND AIM: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice. MATERIAL AND METHODS: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-gamma as well as Th2 cytokines such as IL-4 and IL-5 were measured. RESULTS: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-gamma. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells. CONCLUSION: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation.

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO4). Degranulation of RBL2H3 was examined by the release of ß-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn2+, Ca2+, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of b-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO4, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn2+ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of b-hexosaminidase, ultrafine ZnO particles and ZnSO4, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn2+ easily and increase intracellular Zn2+ concentration efficiently, thus decreasing FceRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate.

OBJECTIVES: The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. METHODS: The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of beta-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. KEY FINDINGS: In the presence of ETBE, TBA or ethanol, IgE-induced release of beta-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca(2+), phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. CONCLUSIONS: ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.

Inhibition of the antigen-induced activation of RBL-2H3 cells by sinomenine.

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum, on the antigen-induced activation of RBL-2H3. For this investigation, the RBL-2H3 cells were sensitized with dinitrophenyl (DNP)-specific IgE overnight in 1.0 ml of Eagle's MEM (EMEM), and varying doses of SIN were added to the culture medium for 30 min and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. The effects of SIN on antigen-induced release of beta-hexosaminidase were measured by enzymatic assay, calcium influx by FACS, cytokines by ELISA, and signaling events by immunoblotting. The results showed that treatment with SIN was followed by a decrease in FcepsilonRI-mediated mast cell release of beta-hexosaminidase, production of IL-4 and TNF-alpha, phosphorylation of Gab2 (Scaffolding adapter Grb2-associated binder 2), Akt and p38 mitogen-activated protein kinase (MAPK). In addition, SIN had no effect on the phosphorylation of LAT and no significant difference on calcium mobilization was observed between control and SIN treated group. These results suggested that SIN might suppress the antigen-induced activation of RBL-2H3 cells via a Ca2+ independent pathway.

Improvement of an efficient separation method for chemicals in diesel exhaust particles: analysis for nitrophenols.

GOAL, SCOPE, AND BACKGROUND: Diesel exhaust is believed to consist of thousands of organic constituents and is a major cause of urban pollution. We recently reported that a systematic separation procedure involving successive solvent extractions, followed by repeated column chromatography, resulted in the isolation of vasodilatory active nitrophenols. These findings indicated that the estimation of the amount of nitrophenols in the environment is important to evaluate their effect on human health. The isolation procedure, however, involved successive solvent extractions followed by tedious, repeated chromatography, resulting in poor fractionation and in a significant loss of accuracy and reliability. Therefore, it was crucial to develop an alternative, efficient, and reliable analytical method. Here, we describe a facile and efficient acid-base extraction procedure for the analysis of nitrophenols. MATERIALS AND METHODS: Diesel exhaust particles (DEP) were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). RESULTS: A solution of DEP in 1-butanol was extracted with aqueous NaOH to afford a nitrophenol-rich oily extract. The resulting oil was methylated with trimethylsilyldiazomethane and subsequently subjected to GC-MS analysis, revealing that 4-nitrophenol, 3-methyl-4-nitrophenol, 2-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol were present in significantly higher concentrations than those reported previously. DISCUSSION: Simple acid-base extraction followed by the direct analysis of the resulting extract by GC-MS gave only broad peaks of nitrophenols with a poor detection limit, while the GC-MS analysis of the sample pretreated with (trimethylsilyl)diazomethane gave satisfactorily clear chromatograms with sharp peaks and with a significantly lowered detection limit (0.5 ng/ml, approximately 100 times). CONCLUSION: The present method involving an acid-base extraction, in situ derivatization, and GC-MS analysis has shown to be a simple, efficient, and reliable method for the isolation and identification of the chemical substances in DEP.

Enhancing Study Motivation and Efficacy among First-year Students Using Minute Papers in the Interdisciplinary Subject of Yakugaku Nyumon.

Active learning in higher education is important for learning efficacy and motivation. Accordingly, lectures that integrate strategies toward active learning, such as minute papers, debates, and collaborative learning, have become widely adopted. Minute papers facilitate communication among both teachers and students, and can be used as a tool for reviewing lectures. In the present study, we examined the effect of using minute papers on learning efficacy and motivation. To enhance the curriculum of the interdisciplinary course Yakugaku Nyumon, which consists of an omnibus lecture series and problem-based learning, minute papers with exercises were provided to applicants. In a follow-up questionnaire, students who used minute papers (S-USE) responded that they had a better understanding of the relationships, ranging from basic to clinical subject matter, than students who did not use such papers (S-NON). Using the Attention, Relevance, Confidence, and Satisfaction (ARCS) model questionnaire to measure study motivation, S-USE scored higher for some questionnaires than S-NON. This finding indicates that minute papers promoted learning motivation among students taking the Yakugaku Nyumon course. In regular examinations, the average score of S-USE was also statistically higher than that of S-NON. These results demonstrate that minute papers possibly encouraged students to actively review the lectures, thereby increasing both learning efficacy and motivation. This study shows that through promoting active, self-learning, minute papers are suitable for improving curricular strategies in subjects that rely on passive learning methods.

A new, rapid in vivo method to evaluate allergic responses through distinctive distribution of a fluorescent-labeled immune complex: Potential to investigate anti-allergic effects of compounds administered either systemically or topically to the skin.

We herein established a new method to evaluate allergic responses in mice rapidly and easily with ethical improvement by reducing the number of animals used. A single intravenous injection of a mixture of anti-OVA monoclonal IgE and fluorescein-ovalbumin (FITC-OVA) induced the distinctive spotted distribution of FITC-OVA in skin, named "ASDIS (Anaphylaxis-dependent Spotted Distribution of a fluorescent-labeled Immune complex in Skin)", and this was easily detected by in vivo imaging. The parallel induction of hypothermia, scratching, serum histamine increases, and ASDIS as well as the inhibition of ASDIS by either the systemic administration of a histamine H1 receptor antagonist or mast cell-depleting antibody suggested that our method, which only required 15 min, induced these allergic responses including ASDIS. Relatively mild but significant ASDIS was induced also in mice with passive systemic anaphylaxis by the method, requiring 2 separate days. The painting of anti-histamines on the skin markedly reduced ASDIS in the painted area only, suggesting the potential of this model to simultaneously compare the anti-allergic effects of several candidate compounds with control drugs in the same mice. ASDIS was suggested to originate from extravasated FITC-OVA/OE-1 immune complexes from blood to skin tissues other than mast cells. Our new method has the advantages of rapidity, easy method, and lower animal numbers to evaluate anti-allergic compounds as well as the characteristics of the used antibody, antigen, labeling molecules, additives, and other formulations. Our model for inducing ASDIS may contribute to the development of anti-allergic drugs, especially those intended for application to the skin.

Interdisciplinary Subject "Yakugaku Nyumon" for First-year Students Constructed with Lectures and Problem-based Learning.

In 2013, Kobe Pharmaceutical University established "Yakugaku Nyumon", an interdisciplinary course, which consists of omnibus lectures and problem-based learning (PBL) on topics ranging from basic to clinical subjects. The themes of the PBL were original ones; "Study from package inserts of aspirin", which aimed to reinforce the contents of the interdisciplinary lectures, and "Let's think about aspirin derivatives (super-aspirin)", which aimed to engender an interest in studying pharmacy. The PBL featured questions from teachers to help with study and was therefore referred to as "question-led PBL" (Q-PBL). The Q-PBL regarding aspirin derivatives began with preparing answers to the questions for a small group discussion (SGD) as an assignment, followed by a SGD, a presentation, and peer-feedback. From an analysis of the questionnaire survey, it was found that students considered the Q-PBL satisfying and that they had achieved the 4 aims: (1) to increase the motivation to study, (2) to enhance an understanding of the relations and significance of basic and clinical sciences, (3) to comprehend the learning content, and (4) to recognize the importance of communication. The Q-PBL with assignments has two favorable points. One is that the first-year students can challenge difficult and high-level questions when they are given these as assignments. The other is that students, who are unfamiliar with SGD can engage in discussions with other students using the knowledge gained from the assignment. The introduction of omnibus lectures and Q-PBL, along with these improvements in theme, application, and review process, promises increased learning efficacy at the university.

Establishment of a Mouse Anti-ovalbumin IgE Monoclonal Antibody That Induces FcɛRII (CD23)-dependent Activation Without FcɛRI-Dependent Activation.

IgE mainly activates cells via two receptors, FcɛRI and FcɛRII. Blocking antibodies against and animals genetically targeted for these receptors have been successfully used to distinguish between these two activating pathways. In the present study, we investigated whether our newly established anti-ovalbumin (OVA) monoclonal IgE OE-2 induced FcɛRII-dependent activation, but not FcɛRI-dependent activation in vivo and in vitro, in contrast to the previously established anti-OVA IgE OE-1, which stimulated FcɛRI and FcɛRII. The FcɛRI-mediated degranulation of RBL2H3 cells and passive systemic anaphylaxis in mice were induced by OE-1 but not OE-2. On the other hand, the production of nitric oxide by rat peritoneal macrophages and the primary antibody response in mice against co-injected OVA, which were mediated through FcɛRII, were induced and enhanced by OE-1 and OE-2. Differences in the epitopes recognized by OE-1 and OE-2 may partially explain why OE-1, but not OE-2, triggered FcɛRI-dependent activation. OE-1 bridged FcɛRI through effective aggregation with OVA, whereas OE-2 crosslinked the receptor strongly and only moderately upon the addition of an anti-kappa antibody and polymerized OVA, namely, an OVA-conjugated resin, respectively, resulting in degranulation. Our results offer a novel approach for determining the relative importance of FcɛRI and FcɛRII in various IgE-dependent responses by using OE-1 and OE-2.

Therapeutic Potential of Monoclonal IgA Antibodies in Allergic Diseases: Suppressive Effect of IgA on Immune Responses Induced By Re-exposure to Antigen in Sensitized Mice by Monoclonal IgE Antibody That Binds to a Different Epitope of the Same Antigen.

Repeated exposure to an allergen induces allergic symptoms by activating mast cells that express anti-allergen IgE, which results in further sensitization to an allergen. Considering that additional sensitization elicits more severe allergic reactions upon the next allergen challenge, suppression of the boosting phase represents an efficacious way to prevent and ameliorate allergic diseases. In this study, we investigated the therapeutic potential of allergen-specific monoclonal IgA on allergic diseases. This antibody acts by decreasing immune responses upon exposure to allergens in mice previously sensitized by a monoclonal IgE that recognizes the allergen. The lack of inhibitory effects of anti-ovalbumin monoclonal IgA (OA-4) on either the binding of anti-ovalbumin monoclonal IgE (OE-1) to ovalbumin by ELISA or on ovalbumin-induced degranulation of rat basophilic leukemia RBL2H3 cells sensitized with OE-1 indicated that OA-4 and OE-1 recognized different epitopes on ovalbumin. Immune responses (anti-ovalbumin IgG1 production and cytokine release from splenocytes) induced by intravenous ovalbumin challenge in DBA/1J mice passively sensitized with OE-1 were inhibited by intravenous injection of OA-4 15 min before challenge without affecting anaphylaxis. Moreover, OA-4 injection 1 h after ovalbumin challenge also effectively suppressed immune responses. The achievement of immunosuppression by IgA injection occurred even after allergen challenge in mice in an epitope-independent fashion. These findings suggest that monoclonal IgA administered at the time of hospitalization of a patient with allergic symptoms, who was already exposed to the allergen in the presence of IgE recognizing an undefined epitope(s) on the allergen, should effectively relieve allergic disease through its immunosuppressive effects.