CD203c Expression Research Articles

Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood–membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.

Serum induced CD63 and CD203c activation tests in chronic urticaria

Abstract

Anaphylactic transfusion reaction in homozygous haptoglobin deficiency detected by CD203c expression on basophils

To the Editor: It has been reported that the incidence of anhaptoglobinemia who are homozygous for Hpdel is very rare in Africans or European-Africans. However, it is relatively high incidence in East Asian; 1/4,000 in Japan, 1/1,500 in Korea, and 1/1,000 in China 1. Some patients with anhaptoglobinemia develop anaphylactic or allergic transfusion reactions related to antihaptoglobin (Hp) antibodies 2. However, if anti-Hp IgE antibody is negative, it is difficult to elucidate an anaphylactic reaction. Flow cytometric basophil activating test (BAT) based on upregulation of the cell degradation/activation marker CD203c has been developed for monitoring the clinical course of patients with allergic diseases 3, 4. Here, we describe that BAT was a useful tool for diagnosing anaphylactic transfusion reaction associated with anti-Hp antibody. This case was an 8-year-old Japanese boy with acute lymphoblastic leukemia receiving intensive chemotherapy required transfusion. He was transfused with 14 units of leukocyte-reduced red cell concentrates (RCCs) and 150 units of leukocyte-reduced platelet concentrates for chemotherapy-induced anemia and thrombocytopenia without any transfusion-related reactions. However, immediately after starting the next infusion of RCCs, he developed dyspnea, urticaria, and hypotension. Screening tests for nonhemolytic transfusion reactions (NHTRs) were performed at the Japanese Red Cross. Serum analysis by nephelometry revealed Hp deficiency and their PCR method 1 showed complete absence of the Hp gene. He was diagnosed as having anhaptoglobinemia due to homozygous allelic Hpdel. Although anti-Hp IgG antibody was detected by ELISA and Western blotting, IgE antibody against Hp was not detected by ELISA or the IgE crosslinking-induced luciferase expression (EXiLE) test 5. To address the mechanism of this anaphylactic reaction, we performed BAT. As a control, a healthy adult and five age-matched children with severe egg allergy who had histories of food-induced anaphylaxis were examined. A commercial kit (Allergenicity Kit; Beckman Coulter, Fullerton, CA) was used for quantifying basophil CD203c expression 4. Upregulation of CD203c on basophils was determined using a threshold that was defined by the fluorescence of unstimulated cells and expressed as CD203c high % 6. CD203c high % was 23% for the patient and 4.38 ± 0.53% for the control, respectively (Table I). Haptoglobin induced enhancement of CD203c expression in the patient, but not in the control. Since basophils express high-affinity IgE receptors, it is reasonable to assume that the haptoblobin-induced basophil activation observed in the patient was mediated by haptoglobin-specific IgE bound to basophils. We did not detect antihaptoglobin IgE by the ELISA or EXiLE methods, but it is possible that the serum IgE concentration was too low to be detected, whereas the amount of cell-bound IgE was sufficient to induce anaphylaxis. Another possibility is that haptoglobin-specific IgG induced basophil activation. An IgG-mediated systemic anaphylactic reaction involving FcγRs, basophils, and platelet-activating factor was reported in a murine system 7, although it remains unclear whether such an IgG-mediated pathway also exists in humans. Our findings indicate that BAT may be useful for assessing the cause of NHTRs, especially anaphylactic reactions via high-affinity IgE receptors on basophils, leading to development of type I hypersensitivity response when no IgE can be detected in serum. The authors thank Yumi Tanaka, MSc (Blood Transfusion Service, Mie University Hospital) for kindly blood supplies. This work was supported by a Research Grant for Allergic Disease and Immunology (H20-015) from the Japanese Ministry of Health, Labor, and Welfare. Shotaro IwamotoMD, PHD, Takahiro YonekawaMD, Eiichi AzumaMD, PHD Pediatrics Unit, Mie University School of Medicine, Mie, Japan Takao FujisawaMD, PHD, Mizuho NagaoMD, PHD Clinical Research Unit, Mie National Hospital, Mie, Japan Eiko ShimadaMSC Central Blood Institute Unit, Blood Service Headquarters Japanese Red Cross Society, Tokyo, Japan Ryosuke NakamuraPHD, Reiko TeshimaPHD, Novel Foods and Immunochemistry Unit, National Institute of Health Sciences, Mie, Japan Kohshi OhishiMD, PHD Blood Transfusion Service Unit, Mie University Hospital, Mie, Japan Hidemi ToyodaMD, PHD, Yoshihiro KomadaMD, PHD Pediatrics Unit, Mie University School of Medicine, Mie, Japan

Basophil activation test in the diagnosis and monitoring of mastocytosis patients with wasp venom allergy on immunotherapy

There is need for an accurate diagnostic test in mastocytosis patients with wasp venom allergy (WVA) and monitoring of these patients during immunotherapy (IT). In this study, we aimed to evaluate sensitivity and specificity of the Basophil Activation Test (BAT) as a diagnostic and monitoring test in patients with mastocytosis and WVA. Seventeen patients with mastocytosis and WVA and six mastocytosis patients without WVA were included. BAT was performed before the start of IT (first visit) and at 6 weeks (second visit) and 1 year (third visit), after reaching the maintenance dose. Of 17 patients included, 11 completed the third visit. In mastocytosis patients with WVA, dose-dependent wasp-venom induced upregulation of CD63 and CD203c expression on basophils was observed compared with mastocytosis patients without WVA. Serum specific IgE, IgG4, and tryptase levels were measured in all patients. BAT had a sensitivity of 87% and specificity of 100% in diagnosing WVA in mastocytosis patients. Basophil allergen threshold sensitivity with respect to CD63 and CD203c was significantly decreased in the second visit compared with the first visit and increased significantly in the third visit compared with the second visit. Specific IgE levels increased significantly in the second visit compared with first and decreased significantly in the third visit compared with the second. Specific IgG4 levels rose significantly in the second visit compared with the first and on the third visit compared with the second. Tryptase levels did not change significantly during the study. BAT represents a diagnostic test with 100% specificity in allergic patients with mastocytosis and these patients are better to be monitored for a longer period during IT.

Increased Level of Basophil CD203c Expression Predicts Severe Chronic Urticaria

Increased FcεR1α expression with upregulated CD203c expression on peripheral basophils is seen in patients with chronic urticaria (CU). However, there has been no published report on the association between CD203c expression level and clinical disease activity in CU patients. To investigate whether the increase of basophil activation is associated with the disease activity of CU, we measured basophil CD203c expression using a tricolor flow cytometric method in 82 CU patients and 21 normal controls. The relationship between the percentage of CD203c-expressing basophils and clinical parameters was analyzed. The mean basophil CD203c expression was significantly higher in CU patients than in healthy controls (57.5% vs 11.6%, P < 0.001). The basophil CD203c expression in severe CU patients was significantly higher than in non-severe CU (66.5% ± 23.3% vs 54.0% ± 23.3%, P = 0.033). Multiple logistic regression analysis indicated that both ≥ 72% basophil CD203c expression and urticaria activity score (UAS)≥ 13 were significant predictors of severe CU (P = 0.005 and P = 0.032, respectively). These findings suggest that the quantification of basophil activation with CD203c at baseline may be used as a potential predictor of severe CU requiring another treatment option beyond antihistamines.

Basophil Activation Test in the diagnosis and monitoring of mastocytosis patients with wasp venom allergy.

Background: There is need for an accurate diagnostic test in mastocytosis patients with wasp venom allergy (WVA) and monitoring of these patients during immunotherapy (IT). In this study, we aimed to evaluate sensitivity and specificity of the Basophil Activation Test (BAT) as a diagnostic and monitoring test in patients with mastocytosis and WVA. Methods: Seventeen patients with mastocytosis and WVA and 6 mastocytosis patients without WVA were included. BAT was performed before the start of IT (1st visit) and at 6 weeks (2nd visit) and 1 year (3rd visit), after reaching the maintenance dose. Of 17 patients included, 11 complerted the 3rd visit.In mastocytosis patients with WVA, dose-dependent wasp-venom induced upregulation of CD63 and CD203c expression on basophils was observed compared to mastocytosis patients without WVA. Serum specific IgE, IgG4 and tryptase levels were measured in all patients. Results: BAT had a sensitivity of 87% and specificity of 100% in diagnosing WVA in mastocytosis patients. Basophil allergen threshold sensitivity with respect to CD63 and CD203c was significantly decreased in the second visit compared to the first visit and increased significantly in the third visit compared to the second visit. Specific IgE levels increased significantly in the 2nd visit compared to first and decreased significantly in the third visti compared to the second. Specific IgG4 levels rose significantly in the 2nd visit compared to the 1st and on the 3rd visit compared to the 2nd . Tryptase levels did not change significantly during the study. Conclusions: BAT can represent a diagnostic test in allergic patients with mastocytosis and these patients are better to be monitored for a longer period during IT. © 2013 Clinical Cytometry Society.

An in vitro study on the risk of non-allergic type-I like hypersensitivity to Momordica charantia

BackgroundMomordica charantia (MC) is a tropical plant that is extensively used in folk medicine. However, the knowledge about side effects of this plant is relatively little according to knowledge about its therapeutic effects. The aim of this study is to reveal the effects of non-allergic type-I like hypersensitivity to MC by an experiment which was designed in vitro.MethodsIn the present study, the expression of CD63 and CD203c on peripheral blood basophils against different dilutions of MC extracts was measured using flow cytometry and compared with one another. In addition to this, intra-assay CV’s of testing extracts were calculated for precision on reproducibility of test results.ResultsIt was observed that the fruit extract of MC at 1/100 and 1/1000 dilutions significantly increased active basophils compared to same extract at 1/10000 dilution.ConclusionsIn conclusion, Momordica charantia may elicit a non-allergic type-I like hypersensitivity reaction in especially susceptible individuals.

A case of anaphylaxis to erythritol diagnosed by CD203c expression–based basophil activation test

A case of anaphylaxis to erythritol diagnosed by CD203c expression–based basophil activation test

AB0094 Monocytes expressing fc gamma receptor 3b (cd16b) is significantly increased in patients with active rheumatoid arthritis.

BackgroundWe have previously demonstrated that high affinity polymorphism of Fc gamma receptor 3b (NA1/NA1), but not Fc gamma receptor 3a, is associated with infusion reaction to IgG1 monoclonal antibodies, such...

Expression of CD203c on basophils as a marker of immunoglobulin E-mediated l-asparaginase allergy

Immediate allergy to l-asparaginase (ASP) is a major obstacle in treating lymphoid malignancies. ASP-specific immunoglobulin G (ASP-IgG) has been used as a surrogate marker. Recently, the CD203c-basophil activation test (BAT) was found to be useful in diagnosing IgE-mediated allergies. We compared the diagnostic utility of the CD203c-BAT to that of ASP-IgG levels in determining ASP allergies in children. Eight ASP allergic reactions occurred over 75 ASP treatment courses. The sensitivity, specificity and area under the receiver operating characteristic curve of CD203c-BAT were similar to the ASP-IgG levels (0.75 vs. 0.85, 0.82 vs. 0.78 and 0.81 vs. 0.85, respectively). Positive skin prick test results in patients with ASP allergy suggested that ASP-IgE was one of the key players in ASP allergy. A combination of the BAT with the ASP-IgG level had the highest specificity (0.95) and positive predictive value (0.62), which permitted us to identify ASP allergy more effectively.

Sa1289 Non Celiac Gluten Sensitivity: Lack of Response to In Vitro Gliadin Challenge and Basophils Activation Assay

In recent years it has become more frequent for physicians to come across types of gluten sensitivity other than celiac disease (CD). Regardless of the absence of any serological CD markers or intestinal damage, gluten sensitive (GS) patients often report intestinal and extraintestinal symptoms shortly after the ingestion of gluten and the disappearance of such symptoms on a gluten free diet (GFD). Regrettably, at present, there is no evidence of any mucosal or serological modifications in GS patients, or that gluten is really the trigger factor in gluten sensitivity. We aimed at evaluating the impact of gliadin challenge on two experimental settings, i.e. in vitro gliadin challenge of duodenal mucosa and peripheral blood basophils in GS patients compared to CD patients and healthy controls. We consecutively enrolled patients referred to tertiary centers for food intolerance and celiac disease. Participants underwent a complete clinical screening in order to rule out CD (serum a-tTG, EMA, total IgA and HLA status, where appropriate), an interview for evaluation of diet and symptoms, a skin prick test to exclude wheat allergy, upper endoscopy for duodenal biopsies used for both histological assessment and for the in vitro evaluation of the gliadin-induced mucosal expression of early and late inflammatory markers: PY99, epithelial HLA-DR, ICAM1, crypt HLA-DR, CD3, CD25 and CD69. In addition, a basophils activation assay, in which patients' basophils were challenged with a gliadin extract, was performed. Basophils were analyzed for the expression of hematopoietic membrane antigens CD203c, CD63 and CD45 by flow cytometry looking at differences in antigen expression. 119 subjects were screened for CD and GS and, according to the results, four groups were obtained: GS patients (no.16), CD on gluten free diet (no.34), CD on gluten containing diet (no.35) and healthy controls (no.34). As for the in vitro gliadin challenge, all CD patients, regardless of their dietetic status, showed a clear mucosal activation (increased immunofluorescence intensity both for early and delayed inflammation markers) when stimulated with gliadin, while only 3 controls and 3 GS patients showed an weak response for some, not all, inflammation markers. Also, there were no significant differences in the skin prick test results or in the level of CD63 and CD203c basophils expression, used as a marker of cell activation, among the groups. The present study shows that in GS patients, gliadin challenge test did not show any significant modifications in the expression of those mucosal inflammation markers found increased in CD patients. Therefore, this tool should not be used as a diagnostic instrument in case of GS. Moreover, the lack of differences in basophils activation and skin prick test seems to exclude this form of cellular sensitivity in our GS series also.

Identification of Der p 23, a Peritrophin-like Protein, as a New Major Dermatophagoides pteronyssinus Allergen Associated with the Peritrophic Matrix of Mite Fecal Pellets

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

The basophil activation test identified carminic acid as an allergen inducing anaphylaxis

T0 THE EDITOR: In the autumn of 2011, a 39-year-old Japanese woman was referred for evaluation of allergic reactions after ingestion of a commercial bottled supplement that contained vitamin Bs and C and fruit flavors. The reactions occurred 3 times, at 1-month intervals. The first 2 episodes were mild and consisted of an itchy throat and localized hives on her lower extremities. However, after drinking the aforementioned supplement every day for 3 months, she manifested more severe symptoms of anaphylactic shock, with an itchy throat, generalized urticaria, diarrhea, and low blood pressure. Interestingly, each of the allergic reactions was associated with her menstrual period; the episodes occurred within 3 days before or after the beginning day of menstruation. Skin prick tests were negative for the supplement. An intradermal test showed that a 10-fold dilution of the solution was an irritating concentration that elicited reactions in the patient and all 4 of the tested controls; lower, nonirritating concentrations showed negative intradermal results. We then tried the in vitro basophil activation test (BAT), which analyzes surface activation marker CD203c expression on basophils. Importantly, the drink itself and, among its constituents, only cochineal dye gave clearly positive results in BAT with the use of the patient’s blood, but not nonallergic subjects’ blood. BAT was highly sensitive and selective, because obvious induction of basophil surface CD203c was observed with 10to 640-fold dilutions of the supplement, the corresponding dose of cochineal dye and its main constituent, carminic acid (purity 97%), but not with a protein extracted from Coccus cacti (Figure 1). In vitro sensitization experiments showed that carminic acideinduced basophil activation was IgE-mediated, because passive sensitization of a normal donor’s basophils by the patient’s serum was completely blocked by the presence of an antiIgE antibody, omalizumab (Figure 2). On the basis of these results, the patient was strongly discouraged from consuming drinks and foods that contained cochineal dye. She has subsequently experienced no further anaphylactic episodes. Although confirmatory challenge was unable to be performed to prove causation because the patient refused to undergo this test, on the basis of her BAT and the absence of anaphylaxis when she avoided carminic acid, we believe that carminic acid, in association with her menstrual cycle, was responsible for her anaphylaxis. Cochineal dye is usually regarded as a highly safe material, and it is widely used as a food additive and cosmetic ingredient. To date, type I allergy due to the dye, including anaphylaxis, urticarial, and occupational asthma, has been reported only occasionally. Allergy to cochineal dye is generally because of a hypersensitivity reaction to a contaminating protein (ie, CC38K, a phospholipase), whereas allergy to the main compound, carminic acid, is rare. Carminic acid is a relatively small molecule (molecular weight 492) that is thought to exhibit allergenicity as a protein-bound hapten. Although skin tests are usually sensitive and useful for allergen determination, they sometimes cause nonspecific skin stimulation. This limits the reliability of the tests, as in our present patient. Here, we were impressed by the finding that BAT accurately identified the allergen. That is, the test clearly showed that the patient’s own basophils, but not those of nonallergic donors, responded to highly pure carminic acid but not to a protein extracted from Coccus cacti. Furthermore, the patient’s basophils showed an obvious response to the allergen in BAT during menstruation, compared with the rest of the menstrual cycle (Figure 1, E); this result was in clear contrast to the absence of any menstruation-related enhancement of a response to polyclonal anti-IgE antibody. On the basis of these results, we believe that an in vitro BAT with the use of peripheral blood would be highly useful for analyzing cochineal dye-induced allergy. At present, we do not know the exact mechanism(s) underlying menstruation-associated changes in the basophil response; overnight culture of our patient’s cells with estradiol or progesterone at 1 mmol/L failed to modulate the response to carminic acid. Recent progress in the pathogenesis of allergies has strongly implicated not only mast cells but also basophils as being critically involved in several allergic responses. Flow cytometric assessment of surface markers, including CD203c, has enabled precise determination of the activation status of basophils. In regard to carminic acid-related allergy, our present results suggest that basophils are highly sensitive to this allergen and that the cells might behave as a key initiator/effector in vivo. Thus, future analyses of the basophil activation status during clinical anaphylaxis will be important for elucidating the involvement of this cell type in this allergic reaction.

Issue highlights—March 2013

Issue highlights—March 2013

Positive Basophil Activation Test in a Patient with Exercise-Induced Anaphylaxis

RationaleExercise-induced anaphylaxis (EIA) is a disorder in which anaphylaxis is associated with physical exertion. The pathogenesis of this disorder is not completely understood but transient elevations of tryptase during attacks suggest mast cell activation. However, the specific mast cell triggering events that occur during exercise remain unknown. Here we present a patient with EIA found to have a positive basophil activation test using CD203c expression.MethodsIn vitro basophil activation test (BAT) using CD203c expression was performed by ARUP Laboratories.ResultsThis 36 year old female presented with a 2-year history of having exercise-induced pruritus. During her last 2 episodes of exercise she developed urticaria, facial swelling, rhinorrhea, sneezing and prolonged tachycardia requiring epinephrine injections. She was taking naproxen on both occasions, but there was no associated food ingestion. BAT was elevated at 1.43 Units (<1.33). Tryptase was < 2 ug/L and her total IgE 32 IU/ml. The patient was pretreated with 180 mg of fexofenadine before completing a 30 minute exercise challenge using a modified Bruce protocol, during which developed only mild rhinorrhea and sneezing.ConclusionsWe believe this to be the first reported case of a positive basophil activation test in a patient with EIA. This novel finding may suggest the presence of basophil stimulating autoantibodies that lead to mast cell activation in the presence of exercise, NSAIDS or other non-specific mast cell triggers. RationaleExercise-induced anaphylaxis (EIA) is a disorder in which anaphylaxis is associated with physical exertion. The pathogenesis of this disorder is not completely understood but transient elevations of tryptase during attacks suggest mast cell activation. However, the specific mast cell triggering events that occur during exercise remain unknown. Here we present a patient with EIA found to have a positive basophil activation test using CD203c expression. Exercise-induced anaphylaxis (EIA) is a disorder in which anaphylaxis is associated with physical exertion. The pathogenesis of this disorder is not completely understood but transient elevations of tryptase during attacks suggest mast cell activation. However, the specific mast cell triggering events that occur during exercise remain unknown. Here we present a patient with EIA found to have a positive basophil activation test using CD203c expression. MethodsIn vitro basophil activation test (BAT) using CD203c expression was performed by ARUP Laboratories. In vitro basophil activation test (BAT) using CD203c expression was performed by ARUP Laboratories. ResultsThis 36 year old female presented with a 2-year history of having exercise-induced pruritus. During her last 2 episodes of exercise she developed urticaria, facial swelling, rhinorrhea, sneezing and prolonged tachycardia requiring epinephrine injections. She was taking naproxen on both occasions, but there was no associated food ingestion. BAT was elevated at 1.43 Units (<1.33). Tryptase was < 2 ug/L and her total IgE 32 IU/ml. The patient was pretreated with 180 mg of fexofenadine before completing a 30 minute exercise challenge using a modified Bruce protocol, during which developed only mild rhinorrhea and sneezing. This 36 year old female presented with a 2-year history of having exercise-induced pruritus. During her last 2 episodes of exercise she developed urticaria, facial swelling, rhinorrhea, sneezing and prolonged tachycardia requiring epinephrine injections. She was taking naproxen on both occasions, but there was no associated food ingestion. BAT was elevated at 1.43 Units (<1.33). Tryptase was < 2 ug/L and her total IgE 32 IU/ml. The patient was pretreated with 180 mg of fexofenadine before completing a 30 minute exercise challenge using a modified Bruce protocol, during which developed only mild rhinorrhea and sneezing. ConclusionsWe believe this to be the first reported case of a positive basophil activation test in a patient with EIA. This novel finding may suggest the presence of basophil stimulating autoantibodies that lead to mast cell activation in the presence of exercise, NSAIDS or other non-specific mast cell triggers. We believe this to be the first reported case of a positive basophil activation test in a patient with EIA. This novel finding may suggest the presence of basophil stimulating autoantibodies that lead to mast cell activation in the presence of exercise, NSAIDS or other non-specific mast cell triggers.

A Patient with Carminic Acid-Induced Anaphylaxis; Usefulness of the Basophil Activation Test

T U E S D A Y 769 A Patient with Carminic Acid-Induced Anaphylaxis; Usefulness of the Basophil Activation Test Naoya Sugimoto, Masao Yamaguchi, Yuko Nakase, Yusuke Tanaka, Hiroyuki Tashimo, Hidenori Arai, Hiroshi Akiyama, Hiroyuki Nagase, MD, PhD, Ken Ohta; Teikyo University School of Medicine, Tokyo, Japan, National Institute of Health Sciences, Tokyo, Japan, National Hospital Organization Tokyo National Hospital, Tokyo, Japan. RATIONALE: In autumn, 2011, a 39-year-old female was referred for analysis of anaphylaxis that occurred following ingestion of a commercial bottled supplement. She had experienced anaphylactic shock with an itchy throat, generalized urticaria and low blood pressure after drinking the supplement every day for two months. Her anaphylaxis was associated with her menstrual period. METHODS: Skin tests and in vitro tests were performed. RESULTS: Her serum IgE concentration was slightly elevated (270 IU/ ml). An intradermal test showed a positive reaction to a 10-fold dilution of the drink. However, the solution also elicited skin reactions in four nonallergic volunteers, suggesting that the reaction was nonspecific. We then tried the in vitro basophil activation test (BAT), which analyzes activation marker CD203c expression on basophils. The drink itself and, among its constituents, only cochineal dye, gave positive results in BATusing the patient’s blood, but not nonallergic subjects’ blood. In addition, carminic acid (purity 97%), but not a protein extract of Coccus cacti, gave a positive BAT result. Furthermore, the patient’s basophils demonstrated a stronger BAT response to cochineal dye during the menstrual period, compared to the non-menstrual period. CONCLUSIONS: Cochineal dye is a highly safe material that is widely used as a food additive and cosmetic ingredient. To date, type I allergy due to the dye, including anaphylaxis, urticaria and occupational asthma, has been reported only occasionally. The present study shows the usefulness of in vitro BAT using peripheral blood for analyzing cochineal dye-induced allergy.

Higher allergenicity of high molecular weight hydrolysed wheat protein in cosmetics for percutaneous sensitization

Wheat protein derivatives are used in a variety of products worldwide. Gluten is commercially used 'as is' or with modifications such as hydrolysis, which is carried out to overcome its insolubility. Several cases of contact urticaria following exposure to hydrolysed wheat protein (HWP) in cosmetics or of anaphylaxis caused by deamidated gluten in food or non-food products have been described. To evaluate the types of HWP that have higher allergenicity for percutaneous sensitization. We enrolled 7 patients with wheat-dependent exercise-induced anaphylaxis who had been sensitized to HWP primarily through the percutaneous and/or the rhinoconjunctival route by using facial soap containing HWP. Reaction to wheat proteins was confirmed by IgE immunoblotting and basophil CD203c expression with six HWP variants. The IgE of all the patients reacted to HWPs composed of large polypeptide aggregates. High molecular weight (MW) HWPs were also found to induce significant enhancement of basophil CD203c expression. HWPs composed of large polypeptide aggregates possibly induce sensitization to a greater degree than lower-MW HWPs. Basophil surface CD203c expression is useful for evaluating the allergenicity of HWPs.

Human Cord Blood Derived Immature Basophils Show Dual Characteristics, Expressing Both Basophil and Eosinophil Associated Proteins

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.

Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance

In our previous study about 75% of children with cow's milk allergy tolerated baked milk products, which improved their prognosis and quality of life. We sought to identify biomarkers of varying degrees of clinical tolerance among a cohort of children with cow's milk allergy. One hundred thirty-two subjects were initially classified as baked milk-reactive, baked milk-tolerant, or having "outgrown milk allergy" based on the results of oral food challenges. The baked milk-tolerant group was then divided into 3 groups based on the amount and degree of heat-denatured milk protein that they could tolerate. Serum was analyzed for allergen-specific IgE and IgG(4) levels, basophil reactivity was assessed in whole blood stimulated with serial 10-fold dilutions of milk protein, and skin prick tests (SPTs) were performed to commercial milk extract. Activated basophils were defined by using flow cytometry as CD63(bright)CD203c(+)CD123(+)HLA-DR(dim/-)CD41a(-)lineage(-). Data were analyzed by using the Jonckheere-Terpstra test. Significant differences across the 5 clinical groups were seen for median casein- and milk-specific IgE levels, casein-specific IgG(4) levels, and casein IgE/IgG(4) ratios; milk-specific to nonspecific basophil activation ratio, median basophil reactivity, and spontaneous basophil activation (CD203c expression after stimulation with RPMI); and milk SPT wheal diameters. Casein- and milk-specific IgE level, milk-specific basophil reactivity, and milk SPT wheal diameter are all significantly greater among patients with milk allergy who react to baked milk than among those who tolerate it. The majority of patients with milk allergy are able to tolerate some forms of baked milk in their diets. Different phenotypes of children with cow's milk allergy can be distinguished by casein- and milk-specific IgE levels, milk-specific basophil reactivity, and milk SPT mean wheal diameters. Spontaneous basophil activation is greater among patients with more severe clinical milk reactivity.

Enhanced Basophil Reactivities during Severe Malaria and Their Relationship with the Plasmodium falciparum Histamine-Releasing Factor Translationally Controlled Tumor Protein

Recent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derived Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruited P. falciparum-infected patients in Senegal with mild malaria (MM; n = 19) or severe malaria (SM; n = 9) symptoms and healthy controls (HC; n = 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measured ex vivo using fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.