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IgE and mast cells play a pivotal role in various allergic diseases, including asthma, allergic rhinitis, and urticaria. Treatment with omalizumab, a monoclonal anti-IgE antibody, has significantly improved control of these allergic diseases and introduced a new era for the management of severe allergic conditions. About 10 years of experience with omalizumab treatment for severe allergic asthma confirmed its effectiveness and safety, reducing symptoms, frequency of reliever use, and severe exacerbations in patients with intractable conditions. Omalizumab is particularly useful in childhood asthma, where atopic conditions often determine clinical courses of asthma. Recently, omalizumab is approved for the treatment of chronic spontaneous urticaria (CSU) with the fixed dose of 300 mg. Although the mechanisms underlying the actions of omalizumab in CSU are not fully clarified, nearly 90% of patients with CSU showed a complete or a partial response to omalizumab treatment. Furthermore, omalizumab is just approved for the treatment of severe Japanese cedar pollinosis (JC) based on the successful results of an add-on study of omalizumab for inadequately controlled severe pollinosis despite antihistamines and nasal corticosteroids. For proper use of omalizumab to treat severe JC, co-administration of antihistamines is necessary, while patients should meet the criteria including strong sensitization to Japanese cedar pollen (≥class 3) and poor control under standard treatment. In the management of severe allergic diseases using omalizumab, issues including cost and concerns about relapse after its discontinuation should be overcome. At the same time, possibilities for application to other intractable allergic diseases should be considered., (Copyright © 2020 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Basophils and mast cells have high affinity IgE receptors (FcεRI) on their plasma membrane and play important roles in FcεRI-associated allergic diseases, such as pollen allergy, food allergy, chronic spontaneous urticarial (CSU), and atopic dermatitis (AD). To date, several reports have revealed that high IgE antibody concentrations activate mast cells-which reside in tissue-in the absence of any antigens (allergens). However, IgE antibody-induced activation of basophils-which circulate in blood-has not been reported. Here, we investigated whether IgE antibodies may regulate functions of human peripheral basophils without antigens in vitro. We successfully removed IgE antibodies bound to FcεRI on the surface of human peripheral basophils by treating with 0.1% lactic acid. We also demonstrated that high IgE antibody concentrations (>1 μM) induced histamine release, polarization, and CD203c upregulation of IgE antibody-stripped basophils. Thus, high IgE antibody concentrations directly activate basophils, which express IgE-free FcεRI on the cell surface. This mechanism may contribute to the pathogenesis of patients with AD and CSU who have higher serum IgE concentrations compared to healthy donors.

MGL_1304, a major allergen in human sweat for patients with atopic dermatitis and/or cholinergic urticaria, is secreted from Malassezia globosa on human skin. The amounts of MGL_1304 and IgE against MGL_1304 are evaluated by the histamine release test using basophils or mast cells sensitized with serum containing IgE against MGL_1304, and enzyme linked sorbent assay (ELISA) using MGL_1304 and anti-MGL_1304 antibodies. Here, we identified a human monoclonal IgE (ABS-IgE) that binds to the high affinity IgE receptor (FcεRI) and MGL_1304 with high affinity (KD = 1.99 nM) but does not release histamine from basophils and mast cells. An ELISA using ABS-IgE as a standard IgE revealed that the amount of IgE against MGL_1304 (1000 U/ml) in the standard sera of patients with AD, employed in our previous report, is 32 ng/ml. A sandwich ELISA using ABS-IgE as a detection antibody showed approximately 10 times lower detection limit for MGL_1304 than ELISA in which MGL_1304 is directly bound to an ELISA plate. Moreover, ABS-IgE prevented histamine release from mast cells and basophils by neutralizing MGL_1304 not only in a free form in solution, but also on FcεRI expressed on the cell surface without cell activation. ABS-IgE may be used both to quantify the amount of MGL_1304 and anti-MGL_1304 IgE, and possibly for the treatment of diseases caused/aggravated by type I allergy to MGL_1304., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Immediate-type wheat allergy caused by a specific hydrolyzed wheat protein (HWP-IWA), Glupearl 19S (GP19S), typically develops food-dependent exercise-induced anaphylaxis (FDEIA), but is different from conventional FDEIA, or simple wheat allergy in many aspects. The skin prick test (SPT) is considered to be the most effective method for diagnosis of HWP-IWA. As SPT is a relatively qualitative method, we developed quantitative and high-throughput test method for HWP-IWA., Methods: An enzyme-linked immunosorbent assay (ELISA)-based GP19S-specific IgE assay was tested using sera from 14 HWP-IWA and five conventional wheat-dependent exercise-induced anaphylaxis (CO-WDEIA) patients, as well as five healthy subjects. Then a validation study at five different institutions was carried out using sera from 10 HWP-IWA and five CO-WDEIA patients, as well as five healthy subjects different from the previous studies., Results: The mean unit values converted from measured absorbance of ELISA were 68.3, 1.3 and 1.1 respectively. Furthermore, the validation study revealed reproducible results across all five institutions, with the standard deviation (SD) being 0.3-0.4 for the healthy group, 0.2-0.6 for the CO-WDEIA group, and 3.8-9.6 for HWP-IWA group except for one case. One case of HWP-IWA was excluded from analysis due to the high SD of 53.3 units, indicating that samples with a unit value > 100.0 will affect inter-laboratory reproducibility., Conclusions: Our findings suggest that the ELISA-based GP19S-specific IgE assay can be used to test HWP-IWA using venous blood samples, except for those with a unit value > 100.0.

Background: MGL_1304 secreted by Malassezia globosa is contained in human sweat and induces histamine release from basophils in patients with atopic dermatitis (AD) at a high positive rate. The aims of this study were to establish the enzyme-linked immunosorbent assay (ELISA) measuring specific immunoglobulins against MGL_1304 and to investigate the levels of these immunoglobulins in sera of patients with various allergic diseases., Methods: Purified MGL_1304 from human sweat (QRX) and recombinant MGL_1304 (rMGL_1304) were prepared for ELISA. To quantify the amount of MGL_1304-specific immunoglobulins, the standard serum was created by pooling sera of 20 patients with AD whose basophils released histamine in response to QRX. A monoclonal antibody which exhibited the highest neutralizing ability against QRX was established as Smith-2, and used as a capture antibody for the assay of QRX-specific IgE. A total of 156 subjects [normal controls (n = 23), AD (n = 63), cholinergic urticaria (CU) (n = 24), bronchial asthma (n = 32), and allergic rhinitis (n = 14)] were enrolled in this study., Results: ELISA methods to quantify the specific IgE, IgG and IgG4 against MGL_1304 in sera were successfully established. Levels of QRX-specific IgE in sera of patients with AD and CU were significantly higher than those of normal controls. Moreover, the levels of QRX-specific IgE and rMGL_1304-specific IgE in patients with AD were significantly correlated with their disease severities., Conclusions: These ELISA methods to quantify the specific immunoglobulins against MGL_1304 are easy and useful means to assess allergy to MGL_1304. MGL_1304 contained in sweat is an important antigen for patients with AD and CU.

Background: Recently, an increasing number of patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) have been reported in Japan. Most of them had developed this condition during or after using hydrolyzed wheat protein (HWP)-containing soap (HWP-WDEIA)., Methods: To clarify the relation between WDEIA and HWP-containing soap and their prognosis, we retrospectively studied the patients who visited Hiroshima University Hospital and were diagnosed as WDEIA from January 2010 to June 2011. We took detailed clinical histories, performed skin prick tests, serum immunoassays for antigen-specific IgE and basophil histamine release test, and followed up their clinical courses after the diagnosis., Results: Among 36 patients with WDEIA, 30 patients had used only one type of HWP-soap. The patients with HWP-WDEIA were mainly women and had developed facial symptoms and angioedema. They suffered from blood pressure reductions less frequently than patients with conventional WDEIA. The levels of gluten-specific IgE were higher than those of omega-5 gliadin in patients with HWP-WDEIA (P < 0.05, One-way ANOVA). All patients with HWP-WDEIA were positive against HWP in histamine release test. Among the conventional wheat antigens, glutenins induced the highest histamine release from basophils of patients with HWP-WDEIA. The sensitivities of patients against glutens and glutenins were reduced over months along with the discontinuance of HWP-soap., Conclusions: The development of HWP-WDEIA is associated with the use of HWP-soap. The sensitivity to HWP that cross reacts with non-processed wheat may be reduced or possibly cured after the discontinuation of HWP-soap.

Background: Oral administration of pulverized Konjac glucomannan (KGM) reduces increased plasma IgE and the amount of epsilon-germline transcript (epsilonGT) in the spleen, as well as preventing the development of dermatitis in mice. To elucidate the mechanism of action of pulverized KGM, we solubilized KGM and studied its effect on IgE in vitro and in vivo., Methods: Solubilized KGM was prepared by acid hydrolysis, and we analyzed the effective molecular size for the suppression of IgE production and epsilonGT in vitro and the level of plasma IgE induced by immunization with ovalbumin in BALB/c mice., Results: The production of IgE and epsilonGT in splenic cells, but not purified B cells, was inhibited by hydrolyzed KGM (KGM hydrolyzed with 0.25 N HCl; H-KGM) at the optimal size of between 10 and 500 kDa. However, no effect was observed when H-KGM was substituted with unhydrolyzed KGM in vitro. IgE production from purified B cells cocultured with purified monocytes, but not with purified T cells, was inhibited by H-KGM. The release of IFNgamma in cultures of monocytes but in purified B cells with or without T cells was enhanced in the presence of H-KGM. Injection of mice with H-KGM also suppressed the production of plasma IgE and IgG1 but not IgG2a in vivo., Conclusion: KGM at an optimal size prevents germline class-switching and IgE production both in vitro and in vivo. H-KGM may be useful as a tool to study the mechanism of action of KGM and as a dietary supplement to prevent atopic diseases., (Copyright 2009 S. Karger AG, Basel.)

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.

Background: The histamine releasing test which detects histamine released from basophils in vitro is safe, sensitive and widely used for clinical examination in the field of allergy. However, basophils of certain individuals do not release histamine, because of dysfunctions in their intracellular signal transduction (non-responder). To overcome potential shortcomings of the histamine releasing test, we applied surface plasmon resonance (SPR) to detect the activation of basophils., Methods: Basophils of patients with allergy, and those of non-allergic volunteers were isolated from peripheral blood. A batch of basophils obtained from a healthy volunteer was treated with lactic acid and IgE of a patient with atopic dermatitis in order to replace their endogenous IgE. They were fixed on the sensor chip of the SPR apparatus, pretreated with or without various inhibitors for intracellular signal transduction, and exposed to the antigens or anti-IgE antibody., Results: When basophils were sensitized with antigen specific IgE, they immediately caused the increase of resonance angle (AR) in response to either anti-IgE antibody or corresponding antigens, even when they did not release histamine. Moreover, the dose dependent reactions of basophils were reflected by the increase of AR as well as the release of histamine. The increase of AR in response to anti-IgE antibody was reduced by pre-treatment of basophils with inhibitors for intracellular signal transduction, but not more than the level for histamine release., Conclusions: SPR biosensors may be superior to the histamine release test for studying functions of human basophils including those not releasing histamine.

Konjac is a traditional Japanese food with a peculiar texture, and it has been suggested that its main ingredient, konjac glucomannan (KGM), ameliorates metabolic disorders such as diabetes and hypercholesteremia. We have found that feeding with pulverized KGM (PKGM) prevents skin inflammation in a murine model of atopic dermatitis. Here, we show that dietary PKGM suppresses allergic rhinitis-like symptoms in mice upon immunization and nasal sensitization with ovalbumin (OVA). The PKGM-fed mice showed a much lower frequency of sneezing than in control animals. We also found that PKGM supplementation exclusively suppressed OVA-specific IgE response without affecting IgG1/IgG2a responses as well as systemic Th1/Th2 cytokine production. These results suggest that PKGM can be a beneficial foodstuff in preventing nasal allergy such as seasonal pollinosis.

Fucoidan, a dietary fiber contained in seaweed, reduces the increase of antigen-specific IgE in mice exposed to ovalbumin. In this study, we investigated the effect of fucoidan on IgE production and intracellular events in B cells in vitro. Fucoidan inhibited the production of IgE and C epsilon germline transcription in murine B cells induced by IL-4 (100 ng/ml) and anti-CD40 antibodies (10 microg/ml), whereas it stimulated cell proliferation. A significant effect of fucoidan on IgE production was observed when B cells were stimulated with a higher dose (5 microg/ml) of anti-CD40 antibodies, but not when stimulated with lower doses (1.25, 2.5 microg/ml), regardless of the IL-4 concentrations. Moreover, nuclear translocation of NFkappaB p52, but neither that of NFkappaB p65, nor the phosphorylation of JAK1 and STAT6 was reduced by fucoidan. These results suggest that fucoidan inhibited IgE production by preventing the NFkappaB p52-mediated pathways activated by CD40.

The increase of serum IgE levels is closely associated with atopic dermatitis. We have previously revealed that cellular extract of PAM212 cells (PAM-extract), BALB/c mouse keratinocyte cell line, induced a remarkable increase of serum IgE levels, in vivo, when subcutaneously injected into BALB/c mice. However, precise mechanism of IgE-increasing activity was unclear. To elucidate the mechanism of IgE-increase in sera of BALB/c mice induced by PAM-extract, we explored the direct influence of PAM-extract on immunoglobulin production and class-switching in the culture of splenic lymphocytes and purified B-cells, in vitro. Splenic lymphocytes or purified B-cells obtained from BALB/c mice were cultured with various combinations of IL-4, anti-CD40 antibody, and PAM-extract for 7 days. IgE and IgG concentrations of culture supernatants were measured by ELISA. Epsilon germ-line transcriptions were assessed by RT-PCR from the cultured cells. IgE and IgG concentrations in culture supernatant of splenic lymphocytes were increased by an addition of PAM-extract in the presence of both IL-4 and anti-CD40 antibody. Epsilon germ-line transcript was also induced in parallel to the increase of IgE production. Similar results were obtained when purified B-cells were employed in stead of whole splenic lymphocytes. The enhancement of IgE production in vitro was also observed, when splenic lymphocytes of CBAj mouse were cultured with cellular extract of KCMH-1 cells, CBAj mouse keratinocyte cell line. The cellular extract of keratinocyte promotes immunoglobulin class-switching to IgE and IgE production from mouse splenic B-cells in an IL-4- and CD40-stimuli-dependent manner. Such enhancement may account for the increase of serum IgE in patients with dermatitis in association with a Th2 microenvironment.

Increased serum IgE levels are found in atopic diseases, especially atopic dermatitis. It is generally accepted that the high serum IgE levels are increased by exposure to exogenous antigens. However, exposure to antigens in the absence of proper adjuvants does not usually increase serum IgE. Here, we studied the involvement of skin factor(s) in increasing serum IgE. The soluble fractions were obtained from two keratinocyte cell lines, PAM 212 and KCMH-1, derived from BALB/c and CBA/J mice, respectively. These were injected subcutaneously into parental mice strains at 2-week intervals. The levels of serum immunoglobulins and the release of IgE in vitro from spleen cells were measured by ELISA. The biological activity of the increased serum IgE was assessed by sensitization of RBL-2H3 mast cells with serum. Six injections of PAM 212-derived fraction (PAM extract) consistently increased the serum levels of IgE, IgG1 and IgG2b, but not those of IgM, IgG2a, IgG3 or IgA in BALB/c mice. The high serum levels of IgE were sustained for at least 10 weeks after one or two injections of PAM extract. When RBL-2H3 cells were sensitized with the serum from mice injected with PAM extract, they released histamine in response to anti-IgE. Moreover, spleen cells of these mice produced significantly higher amounts of IgE than those of uninjected mice in the presence of IL-4 and anti-CD40 antibody. On the other hand, no significant increase in immunoglobulins was observed following injection of the soluble fraction from KCMH-1 cells in CBA/J mice. These results suggest that the release of the soluble fraction of keratinocytes and its exposure to immunologically competent cells may induce an increase in serum IgE levels by a Th2-dominant mechanism.

Sweating aggravates itch in atopic dermatitis, but the mechanism is unclear. In this study, we examined the involvement of type I hypersensitivity in the aggravation of atopic dermatitis by sweating. Skin tests with autologous sweat were positive in 56 of 66 patients (84.4%) with atopic dermatitis, but only in 3 of 27 healthy volunteers (11.1%). Sweat samples from both patients and healthy volunteers induced varying degrees of histamine release from basophils of patients with atopic dermatitis. However, the histamine release was impaired by removal of IgE on the basophils. Incubation of basophils with myeloma IgE before sensitization with serum of patients blocked the ability to release histamine-induced sweat. IgE antibody against antigen(s) in sweat may be present in serum of patients with atopic dermatitis. Key words:

Background: Ligelizumab, a next‐generation, humanized anti‐immunoglobulin E (IgE) monoclonal antibody is in development as a treatment for patients with chronic spontaneous urticaria, whose symptoms are inadequately controlled with standard‐of‐care therapy. Objective: To evaluate the long‐term safety and re‐treatment efficacy of ligelizumab 240 mg in patients who completed the core study and extension study. Methods: This open‐label, single‐arm, long‐term Phase 2b extension study was designed to assess patients who were previously administered various doses of ligelizumab, omalizumab or placebo in the Phase 2b, dose‐finding core study and who presented with active disease after Week 32. In the extension study, patients received ligelizumab 240 mg subcutaneously every 4 weeks, for 52 weeks and were monitored post‐treatment for 48 weeks. Results: Overall, ligelizumab was well‐tolerated with no newly identified safety signals. A total of 95.4% (226/237) screened patients received ligelizumab 240 mg in the extension study; 84.1% (190/226) of patients experienced at least one treatment‐emergent adverse event. Most reported events were mild (41.6%) or moderate (35.8%) and mostly unrelated to the study treatment. At Week 12, 46.5% of patients had a complete response increasing to 53.1% after 52 weeks. Following 52 weeks of extension study treatment, 75.8% (95% confidence interval, 69.9, 81.3) of patients had cumulative complete responses. The median time to relapse in complete responders was 38 weeks. Conclusion: The long‐term safety profile of ligelizumab 240 mg in patients with chronic spontaneous urticaria was consistent with the core study and re‐treatment efficacy was shown. Trial Registration: ClinicalTrials.gov Identifier: NCT02477332 and NCT02649218. [ABSTRACT FROM AUTHOR]

Background: Sweat aggravates atopic dermatitis (AD). In patients with AD, type-I hypersensitivity to sweat may be shown by histamine release from patients' basophils in response to the semi-purified sweat antigen (QR), and the presence of specific immunoglobulin E (IgE) binding to MGL_1304, the component of QR. However, there has been no information on the immunological changes of type-I hypersensitivity to the sweat antigen in patients with well-controlled AD using topical corticosteroids (TCSs) and/or biologics as treatments. Method: Histamine-releasing tests using patients' basophils and QR and the detection of serum IgE against MGL_1304 and mite allergen Der f 1 were performed in patients with AD who were well controlled by topical TCS with/without dupilumab for 53–96 weeks. Results: In total, 14 patients were enrolled. Seven patients received TCS therapy alone (TCS group), and seven patients received TCS with dupilumab therapy (dupilumab group). In all participants, the level of specific IgE against MGL_1304 decreased after treatments, but histamine release from basophils in response to QR did not show a statistically significant reduction; rather, it increased. In the dupilumab group, all changes in histamine release induced by QR (increase), the IgE level against MGL_1304 (decrease), and that against Der f 1 (decrease) were statistically significant, whereas the TCS group showed no significant change in any of them. Conclusion: The well-controlled condition for 53–96 weeks resulted in no reduction of the hyperreactivity of basophils against in patients with AD, even with the treatment with dupilumab. This study suggests persistent basophil hyperreactivity to sweat antigen over a year or longer. [ABSTRACT FROM AUTHOR]

Background: Cholinergic urticaria (CholU) is a common type of chronic inducible urticaria, characterized by small itchy wheals that appear upon physical exercise or passive warming. Malassezia globosa, a skin resident fungus, has been identified as an antigen that induces mast cell/basophil degranulation and wheal formation through specific IgE, in Japanese patients with atopic dermatitis and CholU. In this study we aimed in assessing the rate of IgE sensitizations against skin resident fungi in European CholU patients. Methods: We assessed serum IgE levels to Malassezia furfur, Candida albicans and Trichophyton mentagrophytes using routine lab testing and Malassezia globosa using a newly established ELISA. We correlated the results to wheal formation and other clinical features. Results: Four patients (of 30 tested) had elevated levels of IgE against Malassezia furfur and Candida albicans and two had elevated levels of IgE against Trichophyton mentagrophytes. Four sera (of 25 tested) had elevated levels of IgE to the Malassezia globosa antigen supMGL_1304. Sensitization to one skin fungus was highly correlated with sensitization to the other tested fungi. We saw highly significant correlations of sensitization to supMGL_1304 with wheal size in the autologous sweat skin test (rs = 0.7, P = 0.002, n = 19), the Erlangen atopy score (rs = 0.5, P = 0.03, n = 19), total IgE serum levels (rs = 0.5, P = 0.04, n = 19) and a positive screen for IgE against common airborne/inhalant allergens s (sx1; rs = 0.54, P = 0.02, n = 19). Conclusions: Sensitization to skin resident fungi including Malassezia globosa is uncommon in European CholU patients, but is associated with atopy and pronounced wheal formation upon dermal contact with their own sweat. Trial registration German Clinical Trials Registry DRKS-ID: DRKS00004277 [ABSTRACT FROM AUTHOR]

Background Omalizumab, a humanized anti-IgE monoclonal antibody, proved efficacious and well tolerated in patients with chronic spontaneous urticaria (CSU) refractory to H 1 antihistamines (H 1 AH) in the POLARIS study ( NCT02329223 ), a randomized, double-blind, placebo-controlled trial in East Asian patients. However, data in Japanese patients, who have specific baseline characteristics (e.g., low angioedema incidence, different background medications) that may impact clinical outcomes, are lacking. This pre-specified analysis presents additional patient-level data over time, pharmacokinetic and pharmacodynamics data for omalizumab and IgE, and efficacy and safety data for omalizumab in Japanese patients. Methods Japanese patients (N = 105) were randomized 1:1:1 to omalizumab 300 mg, 150 mg, or placebo by subcutaneous injection every 4 weeks. Efficacy and safety were assessed primarily based on changes from baseline to Week 12 in weekly itch-severity scores (ISS7) and weekly urticaria activity scores (UAS7), and incidence of adverse events (AEs), respectively. Patient-level UAS7 data over time were also reviewed. Results At Week 12, least squares mean (LSM) changes from baseline in ISS7 were greater with omalizumab vs. placebo (−9.54 and −7.29 for omalizumab 300 mg and 150 mg, respectively, vs. placebo [−5.17]). Corresponding LSM changes from baseline in UAS7 were −21.61 and −15.59 (vs. placebo [−10.88]). Most responders in the omalizumab 300 mg group displayed improvement of disease activity within 2–4 weeks and had well-controlled symptoms during the treatment period. Overall AE incidence was similar across treatment arms. Conclusions This subgroup analysis demonstrated that omalizumab is a well-tolerated, beneficial option for treatment of CSU in H 1 AH-refractory Japanese patients. [ABSTRACT FROM AUTHOR]

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production in B cells from mice spleen in vitro and ovalbumin-sensitized mice in vivo. In this study, we examined the effect of fucoidan on IgE production in human peripheral blood mononuclear cells (PBMC) in vitro. PBMC, obtained from healthy donors or patients with atopic dermatitis (AD) with high levels of serum IgE, were cultured with IL-4 and anti-CD40 antibody in the presence or absence of fucoidan. Fucoidan significantly reduced IgE production in PBMC without affecting cell proliferation and IFN-γ production. Fucoidan also inhibited immunoglobulin germline transcripts of B cells in PBMC, and decreased the number of IgE-secreting cells. The inhibitory effects of fucoidan were similarly observed for both PBMC from patients with AD and those with healthy donors. Our findings indicate that fucoidan suppresses IgE induction by inhibiting immunoglobulin class-switching to IgE in human B cells, even after the onset of AD. [ABSTRACT FROM AUTHOR]

In mast cells, crosslinking the high-affinity IgE receptor (FcɛRI) results in a dynamic reorganization of the actin cytoskeleton that is associated with membrane ruffling. Although the signaling involved in degranulation has been well described, it is less understood in morphological changes. In this study, we investigated the specific role of conventional protein kinase C (cPKC), a crucial signal for degranulation, in antigen-induced membrane ruffling of mast cells. In RBL-2H3 mast cells, antigen induced a long-lasting membrane ruffling, which was blocked with late-added Gö6976, a specific cPKC inhibitor, indicating that sustained activation of cPKC is required for maintaining the reaction. Immunofluorescence staining of endogenous PKCα/β and real-time imaging of transfected green fluorescent protein-tagged PKCα/β demonstrated that in response to antigen both PKCα and PKCβI quickly translocated to the plasma membrane and were colocalized with actin filaments at the ruffling sites. These reactions were blocked by expression of kinase-negative PKCβI, but not kinase-negative PKCα, and by treatment with a specific PKCβ inhibitor, LY333531. The adhesion, spreading and membrane ruffling of mouse bone marrow-derived mast cells (BMMCs), which are mostly nonadhesive, were promoted by both antigen and thymeleatoxin. Treatment with Gö6976 abolished all these reactions. Antigen-mediated migration of BMMC was also sensitive to Gö6076 and LY333531. In addition, BMMC adhesion by and migration toward stem cell factor were shown to be dependent on cPKC. Thus, cPKC, at least PKCβ subtype, may be critical for the dynamic morphological changes that lead to the migration of mast cells. [ABSTRACT FROM AUTHOR]

Background: Since the first report of a dog that developed severe systemic symptoms in response to a second injection of sea anemone toxin by Richet and Portier in 1902, no clear human cases of anaphylaxis related to exposure to sea anemones has been reported in the literature. Methods: A 24-year-old man with an episode of local urticaria on his first contact with a sea anemone (Stichodactyla haddoni), developed dyspnea, severe urticaria and hypotension on exposure to water containing the dead bodies of the organism. To study whether this reaction was mediated by antigen-specific IgE, we performed a histamine release test with blood, Western blotting with serum and lymphocyte proliferating test with peripheral blood mononuclear cells of the patient, for the homogenate of sea anemones. Results: The homogenate of sea anemones induced histamine release from the blood of the patient, but it also induced histamine release from the blood of control subjects. Moreover, it also caused hemolysis of blood of all donors. However, Western-blotting demonstrated the presence of an 86 kd protein-specific IgE in the serum of the patient. Conclusions: Protein antigen (s) in sea anemones may cause anaphylactic shock under the influence of the cytolytic effects and/or lymphocyte-stimulating activity elicited by the toxin of sea anemones. [ABSTRACT FROM AUTHOR]

Background: Most urticarias are induced by vasoactive mediators such as histamine released from mast cells. Although mast cells are activated by allergens through cross-linking of cell-surface-bound IgE, this mechanism does not appear to explain most cases of chronic urticaria, which, when allergic, infectious, drug-induced, or physical causes cannot be identified, are classified as idiopathic. Methods: We recruited 26 patients with chronic idiopathic urticaria, in whom intradermal injection of autologous serum caused a wheal-and-flare response. Serum from four patients that induced marked histamine release from basophils from a donor with very low serum IgE levels was studied with respect to the IgE dependence of the histamine release, the activity of the IgG fractions, and the neutralizing effect of a recombinant preparation of the soluble extracellular domain of the α subunit of the high-affinity IgE receptor (sFcεRIα). Results: The histamine-releasing activity of the serum was abolished by passive sensitization of basophils with myeloma IgE, enhanced after dissociation of IgE by treatment with lactic acid, and induced by IgG fractions from the serum of all four patients. Preincubation of the serum and isolated IgG with sFcεRIα resulted in almost complete neutralization. Conclusions: Histamine-releasing IgG autoantibodies against the α subunit of the high-affinity IgE receptor are present in the circulation of some patients with chronic urticaria. Autoantibody-induced cross-linking of IgE receptors may be an important mechanism in the pathogenesis of chronic urticaria and other diseases mediated by mast cells. (N Engl J Med 1993;328:1599-604.) [ABSTRACT FROM AUTHOR]

Autoantibodies (AAbs) against immunoglobulin E (IgE) antibodies (Abs) and their high-affinity receptor alpha subunits (FcεRIα) are key factors in the elicitation of type IIb autoimmune chronic spontaneous urticaria (type IIb aiCSU). In this study, we aimed to develop a new method to detect functional anti-FcεRIα and anti-IgE AAbs, which can crosslink the plural FcεRІα molecules and IgE Abs on the surface of mast cells and basophils, in sera from aiCSU patients using the amplified luminescence proximity homogeneous assay (Alpha). Sera were obtained from 14 aiCSU patients, as diagnosed by recurrent chronic spontaneous urticaria episodes and positive results for the autologous serum skin test and/or histamine release test (HRT). The AAbs to FcεRIα and IgE Abs were determined in sera from aiCSU patients using enzyme-linked immunosorbent assay (ELISA) and Alpha by cross-linking (AlphaCL) of IgE Abs and/or FcεRІα. Serum anti-FcεRIα and anti-IgE AAb levels were not significantly different between aiCSU patients and healthy subjects in ELISA. Anti-FcεRIα AAbs were detected in 10 of 14 aiCSU patients who displayed positive (5/5) and negative (5/9) results in the HRT for anti-FcεRIα AAbs by AlphaCL, whereas no signals were observed in healthy subjects. Additionally, anti-IgE AAbs were detected in two of four aiCSU patients who displayed positive results in the HRT for anti-IgE AAbs. A new assay method using AlphaCL can detect anti-FcεRIα and anti-IgE AAbs with FcεRIα- and IgE-crosslinking abilities in sera from aiCSU patients. This simple and practical assay method may be available as a diagnostic tool for urticaria patients. [Display omitted] [ABSTRACT FROM AUTHOR]

Fish roe allergy is a common health problem in countries where sea food is a major part of the diet, such as Japan. β′-component (β′-c) in fish roe has been identified as a major antigen for patients who show hypersensitivity to various fish roes. However, little is known about causative antigens for patients reactive to fish roe of specific species. Serum and basophils were obtained from patients who had reactivity to roes of Gadus chalcogrammus (GC) and/or other fish species. GC roe specific antigens were analyzed by immunoblotting, histamine release assay (HRA) and mass spectrometry. Recombinant-fragments of vitellogenin (Vg) were obtained by the Escherichia coli expression system. Serum IgE of a patient with specific reactions to GC roe bound to 15, 28, 40 and 70 kDa-proteins in GC roe extract. Mass spectrometry analysis revealed that proteins in these bands contained fragments corresponding to Vg. Immunoblotting of Vg immunoprecipitated by rabbit anti-Vg antiserum from the extract revealed 15, 28 and 54 kDa fragments bound by the patient's IgE. These bindings were inhibited by the pretreatment of recombinant phosvitin (rPv) and β′-c (rβ′-c). Fractions obtained by native gel electrophoresis containing 15, 28 and 54 kDa proteins, but not the other fractions, induced significant histamine release from the patient's basophils. Sera of the other patients with GC roe specific-IgE showed IgE binding to rPv and/or rβ′-c. The 15, 28 and 54 kDa-fragments of Vg which include structures of Pv and β′-c, could be antigens for GC roe specific type-I-hypersensitivity. [ABSTRACT FROM AUTHOR]

Abstract: Basophil activation in response to antigen may represent specificities of type I allergy of individuals and their reactions in the body. We previously demonstrated that surface plasmon resonance (SPR) sensor could detect the activation of human basophils in response to antigens. In this study, we further developed a technique based on SPR imaging (SPRI) system to detect reactions of individual basophils isolated from human blood, and investigated the potential of this sensor as a tool for diagnosis of type I allergy. To detect the change of refractive index (RI) in individual basophils, human basophils were isolated by negative selection with antibodies conjugated with magnetic beads, fixed on a gold film with anti-basophil antibody and stimulated with various antigens under the measurement of SPRI. The sensor could detect the reactions of individual basophils in response to specific antigens as well as non-specific activators. Moreover, the sensor well allocated two spots of basophils on a sensor chip and detected individual reactions to antigen. Thus, the technique developed in this study can visualize the effect of various stimuli or inhibitors on basophils as change of intracellular RI distribution at the single cell level. In combination with a device to rapidly isolate basophils from peripheral blood, this technique may be a useful tool as a high throughput screening system in clinical diagnosis for type I allergy. [Copyright &y& Elsevier]

Previous studies identified autoantibodies against the IgE high affinity receptor α-chain, FcεRIα, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-FcεRIα autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti- FcεRIα autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4- 34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG- mediated. Preincubation of sera or the IgG fraction with a recombinant α-chain of FcεRIα inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human FcεRIα antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of FcεRIα, with 0.01-1.0 μ/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-FcεRIα autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-FcεRIα autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients. [ABSTRACT FROM AUTHOR]